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Local anesthetics: New insights into risks and benefits

Lirk, P.

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Local anestheticsNew insights into risks and benefits

Philipp Lirk

Local anesthetics P

hilipp Lirk

Hierbij nodig ik u uitvoor het bijwonen

van de openbare verdedigingvan mijn proefschrift getiteld

Local anestheticsNew insights into risks

and benefits

De verdediging vindt plaats woensdag 11 juni 2014

om 14:00 in de

AgnietenkapelUniversiteit van AmsterdamOudezijds Voorburgwal 231

1012 EZ Amsterdam

Receptie na afloop

Philipp LirkVan Breestraat 91/21071ZH [email protected]

Local anesthetics

New insights into risks and benefits

Philipp Lirk

Local anesthetics: new insights into risks and benefits

Thesis, University of Amsterdam, The Netherlands

Copyright 2014 Philipp Lirk

All rights reserved. No part of this thesis may be reproduced, stored or transmitted in

any form by any means, without prior permission by the author.

ISBN Nr. 978-90-8891-900-8

Cover picture: Schematic representation of intraneural injection (left) and local

anesthetic diffusing into a peripheral nerve (right). Illustration reproduced with

grateful acknowledgment of permission by Leopold and Ferdinand Lirk to reproduce

their work on the cover of this Thesis. Copyright by the artists.

Cover layout: Petra and Philipp Lirk, Amsterdam, The Netherlands.

Printed by: Uitgeverij BOXpress, s’Hertogenbosch.

The research described in this manuscript was funded by

• Departmental funds of the Dept. of Anaesthesiology, Academic Medical

Center, University of Amsterdam and the Dept. of Anesthesiology and

Critical Care Medicine, Innsbruck Medical University, Austria; and

• Research Grants by the Dutch Association of Anesthesiology (Young

Investigator’s Award 2012), and the European Society of Regional

Anaesthesia and Pain Therapy (Research Grant 2013).

Local anesthetics

New insights into risks and benefits

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties ingestelde commissie,

in het openbaar te verdedigen in de Agnietenkapel

op woensdag 11 juni 2014, te 14:00 uur

door Philipp Lirk

geboren te Salzburg, Oostenrijk

Promotiecommissie

Promotor: prof. dr. dr. M.W. Hollmann

Co-promotores: dr. N.C. Hauck-Weber

dr. M.F. Stevens

Overige leden: prof. dr. W.S. Schlack

prof. dr. I.N. van Schaik

prof. dr. M.J. Schultz

prof. dr. J.P. Rathmell

prof. dr. P. Gerner

dr. G.J. van Geffen

dr. L. Kirchmair

Faculteit der Geneeskunde

Table of contents

Section 1 – General Introduction and Thesis Outline … 7

1.1. Outline of thesis … 9

1.2. Local anaesthetics – an introduction … 17

Section 2 – Clinical applications … 43

2.1. Spinal anaesthesia-induced hypotension (Survey) … 45

2.2. Pulmonary effects of spinal anaesthesia … 63

2.3. Failed epidural: causes and management (Review) … 79

Section 3 – Regional anesthesia and nerve damage … 109

3.1. Needle trauma and intraneural injection … 111

3.2. Regional anesthesia in neuropathic patients (Review) … 129

3.3. Regional anesthesia in subclinical neuropathy … 165

3.4. Regional anesthesia in early and late neuropathy … 185

3.5. Management of the neuropathic patient (Survey) … 209

Section 4 – Epigenetic effects of local anesthetics … 229

4.1. Lidocaine demethylates DNA in breast cancer cells … 231

4.2. Demethylating properties of local anesthetics … 251

Section 5 – Weighing risks and benefits of regional anesthesia … 269

Section 6 – Summary … 289

Section 7 – Appendices … 295

Appendix A Dutch summary / Samenvatting … 297

Appendix B Curriculum vitae of the candidate … 301

Appendix C List of publications of the candidate … 305

Appendix D Research portfolio of the candidate … 315

Appendix E Acknowledgment / Dankwoord … 319

7

Section 1

General Introduction and Thesis Outline

Outline of Thesis

9

Chapter 1.1

Outline of Thesis

Chapter 1.1

10

A brief history of Regional Anesthesia

Local anesthetics (LA) are indispensable in contemporary regional

anesthesia and pain management. Introduced in Western medicine in the late 19th

century, the prototype substance cocaine had been widely used by South American

cultures for thousands of years.1 However, these tribes were used to chewing coca

leaves, which dried out during shipping to Europe, such that the real breakthrough of

cocaine was made possible only by the isolation of cocaine in 1859.1 Cocaine was

used in oral form for various psychiatric diseases and fatigue by, among others,

Sigmund Freud. However, it was his colleague, Carl Koller, who is widely credited

for the first experimental topical application of cocaine.2 Koller was ophthalmologist

at the Vienna General Hospital, and had been looking for ways to alleviate the pain

of cataract surgery.1

The discovery of the topical numbing properties of cocaine led to the

development of regional anesthesia in its modern form, with the basic principle that

injection of cocaine next to nerves produces a transient and reversible interruption of

pain propagation, sensory and motor function. Over the following decades, many

types of nerve block and neuraxial (epidural and spinal) anesthesia and analgesia

were introduced. However, insufficient materials and medications with a narrow

therapeutic range hampered widespread acceptance. For example, the first published

administration of continuous epidural anesthesia in 1949 was achieved by an

urethral catheter introduced into the epidural space.3 Another anecdote is the first

performance, in 1889, of spinal anesthesia by Bier, which was successful from a

pharmacological point of view, but the large needle used led to days of severe post-

dural-puncture headache.

The introduction of the first short-acting amide-type local anesthetic,

lidocaine, in 1947, and the standard long-acting amide-type local anesthetic,

bupivacaine, were pharmacological milestones in regional anesthesia. In parallel,

improved needle and catheter equipment meant that regional anesthesia could be

applied in a more reliable and safe manner. In the 1990s, ultrasound-guided regional

anesthesia was coined by a group of physicians in the in the Vienna General

Hospital, the very workplace where hundred years earlier Carl Koller had made his

Outline of Thesis

11

ground-breaking discovery. Stephan Kapral and Peter Marhofer described the

guidance of the regional anesthesia needle by ultrasound, sparking renewed interest

in regional anesthesia techniques.4

If scientific publications can be taken as surrogate markers of clinical

relevance, regional anesthesia is experiencing a surge in technical advances. At the

same time, these publications reflect the quest to define evidence-based indications

for these blocks on a procedure- and patient-based foundation.

Contemporary focus

Regional anesthesia has become an integral component of the

anesthesiologist’s armamentarium. Perceived benefits include the reduction in

postoperative opioid demand, and attenuation of postoperative symptoms and

complications such as nausea and vomiting, and ileus.5 The endocrine stress

response to surgery can be substantially alleviated when regional anesthetic

techniques are employed.6 However, it is increasingly acknowledged that these

beneficial effects are not relevant in all types of surgery and patients. Many of the

beneficial effects of regional anaesthesia can be copied by the systemic

administration of local anesthetics.7 Current consensus is to consider epidural

anesthesia and analgesia in high risk patients, thoracotomy, and major upper

abdominal and vascular surgery, to treat most cases of lower abdominal and

laparoscopic surgery using multimodal analgesia, and to use peripheral regional

anesthesia for extremity surgery as targeted and distal as possible.8 Currently, a

large share of the current research efforts in Regional anesthesia is dedicated to

defining patient- and procedure-based indications for the different techniques.

Despite being accepted as a relatively safe anesthetic technique, toxic

effects of local anesthetics themselves can present a considerable risk. Importantly,

in a dose-dependent manner, every local anesthetic is potentially toxic to virtually

any kind of tissue.9 The question whether specific local anesthetics are more toxic

than others remains unanswered. For example, there are studies suggesting that

lidocaine is more toxic than bupivacaine in vivo 10

while other studies found no

Chapter 1.1

12

difference 11

and clinical evidence suggests that lidocaine is involved in nerve

damage more often than bupivacaine.12

Epidemiological studies suggest that after neuraxial (spinal, epidural)

anesthesia, neurological complications such as transient radicular irritation (back

pain with radiation down one or both buttocks or legs occurring within 24 h after

surgery 13

) may follow up to 30 % of spinal anesthetics 13 14

, and devastating

complications such as cauda equina syndrome (severe low back pain, lower

extremity motor weakness and sensory loss, bladder dysfunction, bowel

incontinence) affect roughly 1:8000 patients.12 15

This is in accordance with a recent

retrospective analysis of more than 100.000 neuraxial anesthetics, in which the

incidence of presumed neurological complication was 1:1000, but when imaging

techniques were used to validate these assumptions, the incidence was 0.07:1000

(with a 95% confidence interval of 0.02 – 0.13/1000).16

This may be of special importance in the collective of patients presenting

with diabetic neuropathy. Patients in the latter collective frequently feature relevant

cardiovascular or renal comorbidities that would predispose them to undergo many

types of surgery under regional anesthesia. On the other hand, limited

epidemiological and experimental evidence suggests that neuropathic nerves are

more susceptible to local anesthetic-induced neurotoxicity. 17 18

In the past, our group demonstrated involvement of the p38 Mitogen

Activated Protein Kinase (MAPK) (= MAPK 14) in local anesthetic-induced

neurotoxicity in vitro and in vivo.11 19-23

In particular, the p38 is specifically

activated in cell cultures incubated with lidocaine.23

In the Habilitationsschrift

(Innsbruck Medical University, 2010), we proposed a General Hypothesis that

exposure of primary sensory neurons to local anesthetics triggers opening of calcium

channels, resulting in an increase in cytosolic calcium, leading to a subsequent

activation of the p38 MAPK. Potential downstream modulators include the

lipoxygenase family of enymes. Furthermore, we postulated that when local

anesthetic is deposited at the axon, these events unfold at the neuron’s periphery,

without necessarily involving the cell body. We believe this causes not only an

Outline of Thesis

13

immediate neuronal injury apparent within hours, but also a prolonged inflammatory

response combined with sustained tissue damage still detectable after several days.22

Aims of this thesis

On the basis of these previous results, the investigations forming the basis

of the current thesis aimed to

- investigate and review the mechanism and management of side-

effects or failures of neuraxial anesthesia;

- investigate nerve injury related to the performance of regional

anesthesia, with special focus on patients with pre-existing

neuropathy;

- investigate potential novel actions of local anesthetics on the

epigenetic signature of tumour cells; and

- conclude with a weighing of risks and benefits of regional

anesthesia using local anesthetics.

References

1 Goerig M, Bacon D, Van Zundert A. Carl Koller, Cocaine, and Local Anesthesia.

Reg Anesth Pain Med 2012; 37: 318-21

2 Markel H. Ueber Coca: Sigmund Freud, Carl Koller and Cocaine. JAMA 2011;

305: 1360-1

3 Mulroy MF. The history of Regional Anesthesia. In: Eger E, Saidman LJ,

Westhorpe RN, eds. The Wondrous Story of Anesthesia. New York: Springer, 2014;

859-70

4 Kapral S, Krafft P, Eibenberger K, Fitzgerald R, Gosch M, Weinstabl C.

Ultrasound-guided supraclavicular approach for regional anesthesia of the brachial

plexus. Anesth Analg 1994; 78: 507-13

5 Bonnet F, Marret E. Influence of anaesthetic and analgesic techniques on outcome

after surgery. Br J Anaesth 2005; 95: 52-8

Chapter 1.1

14

6 Hahnenkamp K, Herroeder S, Hollmann MW. Regional anaesthesia, local

anaesthetics and the surgical stress response. Best Pract Res Clin Anaesthesiol 2004;

18: 509-27

7 Herroeder S, Pecher S, Schonherr ME, et al. Systemic lidocaine shortens length of

hospital stay after colorectal surgery: a double-blinded, randomized, placebo-

controlled trial. Ann Surg 2007; 246: 192-200

8 Lirk P, Hollmann M. Outcome after regional anesthesia: weighing risks and

benefits. Minerva Anestesiol 2013; epub ahead of print Nov 6

9 Werdehausen R, Braun S, Essmann F, et al. Lidocaine induces apoptosis via the

mitochondrial pathway independently of death receptor signaling. Anesthesiology

2007; 107: 136-43

10 Sakura S, Kirihara Y, Muguruma T, Kishimoto T, Saito Y. The comparative

neurotoxicity of intrathecal lidocaine and bupivacaine in rats. Anesth Analg 2005;

101: 541-7

11 Lirk P, Haller I, Colvin H, et al. In vitro, inhibition of Stress-Activated Protein

Kinase pathways protects against bupivacaine- and ropivacaine-induced

neurotoxicity. Anesth Analg 2008; 106: 1456-64

12 Auroy Y, Benhamou D, Bargues L, et al. Major complications of regional

anesthesia in France: The SOS Regional Anesthesia Hotline Service. Anesthesiology

2002; 97: 1274-80

13 Pollock JE, Neal JM, Stephenson CA, Wiley CE. Prospective study of the

incidence of transient radicular irritation in patients undergoing spinal anesthesia.

Anesthesiology 1996; 84: 1361-7

14 Pollock JE, Liu SS, Neal JM, Stephenson CA. Dilution of spinal lidocaine does

not alter the incidence of transient neurologic symptoms. Anesthesiology 1999; 90:

445-50

15 Auroy Y, Narchi P, Messiah A, Litt L, Rouvier B, Samii K. Serious

complications related to regional anesthesia: results of a prospective survey in

France. Anesthesiology 1997; 87: 479-86

Outline of Thesis

15

16 Pumberger M, Memtsoudis SG, Stundner O, et al. An analysis of the safety of

epidural and spinal neuraxial anesthesia in more than 100,000 consecutive major

lower extremity joint replacements. Reg Anesth Pain Med 2013; 38: 515-9

17 Hebl JR, Kopp SL, Schroeder DR, Horlocker TT. Neurologic complications

after neuraxial anesthesia or analgesia in patients with preexisting peripheral

sensorimotor neuropathy or diabetic polyneuropathy. Anesth Analg 2006; 103: 1294-

9

18 Kalichman MW, Calcutt NA. Local anesthetic-induced conduction block and

nerve fiber injury in streptozotocin-diabetic rats. Anesthesiology 1992; 77: 941-7

19 Lirk P, Haller I, Colvin HP, et al. In vitro, lidocaine-induced axonal injury is

prevented by peripheral inhibition of the p38 mitogen-activated protein kinase, but

not by inhibiting caspase activity. Anesth Analg 2007; 105: 1657-64

20 Lirk P, Haller I, Colvin HP, et al. In vitro, inhibition of mitogen-activated

protein kinase pathways protects against bupivacaine- and ropivacaine-induced


21 Lirk P, Haller I, Hausott B, et al. The neurotoxic effects of amitriptyline are

mediated by apoptosis and are effectively blocked by inhibition of caspase activity.

Anesth Analg 2006; 102: 1728-33

22 Lirk P, Haller I, Myers RR, et al. Mitigation of direct neurotoxic effects of

lidocaine and amitriptyline by inhibition of p38 mitogen-activated protein kinase in

vitro and in vivo. Anesthesiology 2006; 104: 1266-73

23 Haller I, Hausott B, Tomaselli B, et al. Neurotoxicity of Lidocaine Involves

Specific Activation of the p38 Mitogen-activated Protein Kinase, but Not

Extracellular Signal-regulated or c-jun N-Terminal Kinases, and Is Mediated by

Arachidonic Acid Metabolites. Anesthesiology 2006; 105: 1024-33

Local anesthetics – an introduction

17

Chapter 1.2

Local anesthetics: an introduction

Based on

Lirk P, Picardi S, Hollmann MW

Local anaesthetics: the essentials

Eur J Anaesthesiol 2014 (invited review, in press)

Chapter 1.2

18

This introductory section is based on an invited Review for the European

Journal of Anaesthesiology, and seeks to address 10 essential questions regarding

the clinical use of local anaesthetics. Local anaesthetics are a heterogeneous group

of compounds which block voltage-gated sodium channels. Each drug is

characterized by distinctive physicochemical properties. Sodium channel block is

caused by conformational change and the creation of a positive charge in the channel

lumen. Different local anaesthetics can reach the LA binding site from the

cytoplasmic compartment (“classic hydrophilic pathway”), directly via the lipid

membrane (“hydrophobic pathway”), or can enter via large-pore channels

(“alternative hydrophilic pathway”). Next to sodium channel blockade, LA exert

beneficial effects on pain, stabilize the inflammatory response and the haemostatic

system. Inflamed tissue is harder, but not impossible, to anesthetize. Increasing dose

or adding a vasoconstrictor, can lead to successful blockade. Systemic toxicity of

LA is potentially fatal. When preventative measures are taken, the incidence of

cardiac arrest is low. Intralipid has been proposed for resuscitation of systemic LA

overdose, and enthusiastically adopted worldwide, even though the mechanism of

action is incompletely understood. Intralipid is not an antidote against LA and

cannot replace meticulous conduct of regional anaesthesia. All LA are toxic,

dependent on dose. The question whether local anaesthetics protect against

perioperative tumour progression cannot be answered at this moment, and results

from clinical (retrospective) studies are equivocal. Future areas of interest will be

design of new subtype-specific sodium channel blockers. At the same time, older

LA such as 2-chloroprocaine are reintroduced into the clinical setting. Multimodal

perineural analgesia and liposomal bupivacaine may replace catheter techniques in

some indications.


19

Local anaesthetics (LA) are widely used for regional anaesthesia and pain

therapy. This review seeks to address 10 essential topics regarding LA use in daily

practice.

General physicochemical properties

Local anaesthetics are a heterogeneous group of compounds which block

voltage-gated sodium channels. Structurally, LA consist of a hydrophobic aromatic

group, an amide group, and the connecting intermediary chain. This means that they

possess both hydrophobic and –philic properties. According to the type of

intermediary chain, LA can be divided into ester-type and amide-type compounds.

According to duration of action, LA can be divided into short-acting (e.g.,

chloroprocaine), intermediate-acting (e.g., mepivacaine, lidocaine), and long-acting

(e.g., bupivacaine, ropivacaine) compounds. The metabolism of esters is by plasma

cholinesterases and tissue esterases, whereas amides are primarily metabolized

hepatically through the mixed-function oxidase system.1 Conversely, defects in the

metabolizing steps lead to higher systemic concentrations of LA. Concerning

allergic potential, currently used LA are widely considered to be among the safest

perioperative drugs.2

All currently used LA are chiral, which means they feature an asymmetric

carbon atom and two potential spatial molecular configurations, called enantiomers.

The only exception to this rule is lidocaine, which is achiral.3 The clinical relevance

of chirality is that in comparison to racemic mixtures containing both enantiomers,

pure enantiomers may offer pharmacologic advantages such as enhanced blockade

and decreased toxicity.4 However, the differences in clinical use when comparing

equipotent doses are modest, such that both conventional drugs as bupivacaine, as

well as the newer isoforms continue to be in widespread use.5

LA have distinct physicochemical properties which determine their mode of

action, most notably pKa value, lipophilicity, protein binding, and intrinsic

vasoactivity. The main characteristics of clinically used drugs are summarized in

Table 1. Importantly, the free systemic fraction of LA, not bound by plasma

proteins, is the principal determinant of systemic adverse effects. The rate of

Chapter 1.2

20

absorption into the systemic circulation depends upon the site of injection. For

example, equal plasma concentrations of lidocaine are attained when 300 mg are

given intercostally, or 500 mg epidurally, or 1000 mg subcutaneously.6 A

multifactorial approach to choosing safe doses on an individualized basis per patient

has been advocated, for which the reader is referred to the comprehensive review by

Rosenberg and colleagues.6

In plasma, LA are bound to albumine, an abundant protein with weak

affinity for LA, and, more importantly, alpha-1-acidic glycoprotein (AAG), less

abundant but potent in binding LA.7 Since AAG is an acute phase protein, synthesis

increases postoperatively and after trauma, decreasing free local anaesthetic, and

protecting against systemic toxicity. For example, Veering et al. demonstrated that

continuous postoperative infusion of epidural bupivacaine leads to increasing levels

of total local anaesthetic in the systemic circulation, but at the same time, increasing

AAG levels postoperatively bind bupivacaine, resulting in stable levels of systemic

free drug.8 Synthesis of AAG does not mature until one year of age, such that

neonates are theoretically at higher risk of elevated free LA plasma levels.7

Experimental evidence suggests that in pregnancy, protein binding of the more

lipophilic bupivacaine is decreased, similarly increasing risk of systemic toxicity.9

Conclusion: Local anaesthetics are a heterogeneous group of compounds

which block voltage-gated sodium channels. Each drug is characterized by

distinctive physicochemical properties. The free plasmatic share determines

systemic toxicity.

The primary target: voltage-gated sodium channels

Local anaesthetics (LA) are primarily characterized by their ability to block

voltage-gated sodium channels (VGSC), transmembrane proteins consisting of one

main alpha-subunit, linked to one or more beta-subunits.10

Crystallographic images

of sodium channels have become available, improving insight into function and

blockade.11

The alpha subunit of the VGSC constitutes the functional ion channel,

and harbours the binding site for LAs.12

This subunit consists of four domains

numbered DI-DIV, each consisting of 6 segments numbered S1-S6 (Figure 1).10 13

In


21

contrast, the beta subunits of the VGSC modulate kinetics and voltage dependence

of activation and inactivation.12

The S4 segments are considered the “voltage

sensor”, while specific amino acid sequences between segments S5 and S6 mediate

the channel’s specificity for sodium.14

The binding sites of LA are the S6 segments of domains I, III and IV.12

Figure 2 gives a schema of the VGSC in cross-section, with (from the outside) the

extracellular pore, the selectivity filter, the central cavity, and the innermost

activation gate. LA preferably bind to activated and resting channels, because in

these states, the activation gate is open, a property described as use-dependent

block.12

Under experimental conditions, binding of the LA to the receptor leads to

reversible and concentration-dependent reduction of peak sodium current 10

by

modulating the dynamic conformation of the voltage sensing segments S4 across

domains of VGSC,15

and by the creation of a positive charge within the channel’s

lumen, directly impeding sodium flux.16

Importantly, the family of VGSC consists of at least 10 different subtypes,

depending on the gene for the alpha subunit. Dysfunction or mutation has been

linked to several pathophysiological states, such as inherited erythromelalgia

(syndrome of pain and erythema),17

paroxysmal extreme pain disorder,18

congenital

insensitivity to pain,18

cardiac arrhythmias,19

and epilepsy.20

Lastly, drugs other than local anaesthetics can effectively block the

neuronal sodium channel. Interestingly, two members of the opiate family, pethidine

and buprenorphine, have clinically relevant local anaesthetic properties.21,22

Conclusion: Sodium channel block is caused by conformational change and

the creation of a positive charge in the channel lumen.

The three ways for local anaesthetics to block the primary target

To block the VGSC, classic LA in contemporary use need to attach to a

specific binding site on the inner surface of the channel, which they cannot access

through the sodium-specific channel itself. Following the “classic hydrophilic”

pathway, LA need to traverse the neuronal membrane as uncharged molecules first,

Chapter 1.2

22

and then conjugate with hydrogen ions and reach the binding site from the

cytoplasma (Figure 2).

Another pathway is observed with benzocaine, a permanently uncharged

LA, characterized by its low pKa value, and primarily used for topical anaesthesia.23

It reaches the sodium channel directly through the nerve membrane and lateral

fenestrations in the channel, a concept supported by recent crystallographic

investigations and described as the “hydrophobic pathway”.11

The “opposite compound” of benzocaine is the permanently charged

lidocaine derivative, QX-314. Because it is charged, QX-314 will only very slowly

cross nerve cell membranes. However, artificial activation of the transient receptor

potential vanilloid-1 (TRPV-1) channel allows for the influx of QX-314, and this

has been designated the “alternative hydrophilic pathway”.24

Conduction block by LA is first achieved in weakly or non-myelinated

fibers (e.g., fibers responsible for the sympathetic nervous system and nociception),

whereas myelinated fibers (e.g., motor fibers) are blocked later. However, this effect

cannot be explained solely on the basis of different myelin sheath diameters. For

example, Huang and co-workers demonstrated that C-type fibers were more resistant

to blockade than A-delta- and A-beta-type fibers, which have a larger myelin

sheath.25

Rather, it is increasingly appreciated that different neuron populations do

not only differ by myelin thickness and size, but also by different patterns of

electrophysiologic properties and ion channel composition.26

Conclusion: Different local anaesthetics can reach the LA binding site from

the cytoplasmic compartment (“classic hydrophilic pathway”), directly via the lipid

membrane (“hydrophobic pathway”), or can enter via large-pore channels

(“alternative hydrophilic pathway”).

The secondary targets: from ion channels to G-protein coupled receptors

Next to sodium channel blockade, local anesthetics interact with a wide

array of target structures, for example tetrodotoxin-resistant sodium channels,

potassium channels, calcium channels, n-methyl-d-aspartate (NMDA) receptors, and

G-protein coupled receptors.1 10

Not surprisingly, systemic local anaesthetics have


23

been in clinical use for decades. Specific areas of use include administration of

lidocaine as class Ib anti-arrhythmic to treat ventricular tachycardia / fibrillation /

extra systole, even if recent guidelines limit its use.27

Lidocaine has also been

proposed as a reserve treatment for refractory status epilepticus.28

Similarly,

intravenous lidocaine has long been in use for treatment of tinnitus without precise

knowledge of the mechanism of action.29

Interestingly, when therapeutic systemic

doses are exceeded, arrhythmia, seizure and tinnitus are at the same time hallmarks

of systemic toxicity.

Systemic LA have a positive stabilizing effect on some physiological

systems. Firstly, some studies have described an antinociceptive effect of LA,30

while others found no clinically relevant effect.31 32

The analgesic effect is

potentially caused by lidocaine metabolites modulating glycinergic pathways,33

while anti-hyperalgesic effects may, at least in part, be explained by antagonism at

the NMDA receptor.34

Secondly, lidocaine has for decades been used as a co-

anaesthetic, and recent evidence confirms an anaesthetic-sparing effect of

lidocaine.35

Thirdly, LA have a pronounced anti-inflammatory effect, as they

modulate virtually every step of the inflammatory cascade, including leukocyte

adhesion to the endothelium, shape change, transendothelial migration,

phagocytosis, priming and release of inflammatory mediators.36

One potential

mechanism underlying the anti-inflammatory effects of LA may be the modulation

of G-protein coupled receptor signalling, in particular interference with G-proteins

of the Gq/11 family, that predominantly mediate haemostatic and inflammatory

signalling, such as the LPA-, TXA2- or PAF-receptor.37

However, the complete

mechanisms of LA action on these receptors and certain G-protein families remain

to be finally determined. Lastly, LA reduce hypercoagulability without inducing a

clinically relevant bleeding risk.38

Blockade of potassium channels has been demonstrated for amide-type

local anesthetics, contributing to a more intense nerve fibre block,39

and explaining

symptoms of central nervous system (CNS) excitation such as tinnitus and seizures

through depolarization of thalamocortical neurons.40

Well-recognized syndromes

such as some forms of Long QT syndrome (LQTS) due to potassium channel

Chapter 1.2

24

mutation share important pathogenic features with systemic toxicity of local

anaesthetics.41

Another site of local anaesthetic blockade is the nicotinic

acetylcholine receptor, a nonselective cation channel. This mechanism of action is

thought to contribute to the enhancement of neuromuscular blockade by local

anesthetics,42

but the clinical importance seems minor.43

Alternative effects, however, depend on adequate systemic levels of LA.

Classic pharmacokinetic studies carried out on rodent sciatic nerves suggest that

only a minor fraction of LA actually participates in nerve block, the majority being

absorbed into the systemic circulation.44

The use of ultrasound-guided regional

anaesthesia has led to the administration of very small volumes.45

For example,

Renes and coworkers used approximately a tenth of the dose of LA for interscalene

block as compared to the original description by Winnie. 46 47

These small doses will

invariably result in decreased plasma levels of LA, potentially decreasing incidence

and severity of systemic toxicity,48

but at the same time the sharply decreased

plasma levels may result in the loss of the above mentioned beneficial effects which

are only beginning to be fully appreciated and understood. In the view of the

authors, the desire to benefit from systemic effects will need to be weighed against

the challenge to find the minimum effective dose for daily clinical practice.

Conclusion: Next to sodium channel blockade, LA exert beneficial effects

on pain, stabilize the inflammatory response and the haemostatic system.

Local anaesthetics and inflamed tissue

It has long been accepted that LAs fail in inflamed tissue. However, there is

literature that inflamed tissues may be anaesthetized sufficiently, given higher

concentrations of LA. For example, Rood and coworkers demonstrated that in dental

anaesthesia for inflamed teeth, lidocaine 2% frequently failed to provide a

satisfactory block, whereas the success rate of lidocaine 5% was excellent.49 50

It is

the authors’ opinion that the current viewpoint “inflammatory tissue renders LA

inactive” should be adapted to “LA may not function optimally in inflammatory

tissue”. Three theories have been put forward to explain decreased function of LA

under inflammatory conditions: an acidic shift in tissue pH, increased excitability of


25

nerves in inflamed tissues, and increased vascularity leading to enhanced absorption.

Harris described improved efficacy of LA under inflammatory conditions when

injected together with a vasoconstrictor 51

.

Conclusion: Inflamed tissue is harder, but not impossible, to anesthetize.

Increasing dose or adding a vasoconstrictor, can lead to successful blockade.

Systemic toxicity

Since the early 1980s, awareness regarding potentially fatal LA overdose

led to the introduction of several safety measures, such as incremental

administration of LA (including test doses), repeated aspiration tests, compulsory

monitoring of the patient, and recommendations regarding maximum dosage. Recent

large suggest a low incidence of relevant systemic toxicity. Barrington, in a case

series of >20.000 patients, reported an overall incidence of mild signs of local

anesthetic systemic toxicity of approximately 1 : 1.000, which was decreased to 1 :

1.600 when ultrasound guidance was used. In the entire case series, one LAST

progressed to cardiac arrest.52

Sites, in a case series of more than 12.000 patients did

not report a single case of cardiac arrest.53

In general, two forms of LAST can be differentiated: On one hand,

“instant” LAST results from intravenous or -arterial injection of LA, and occurs

immediately after injection. On the other hand, “slow” LAST results from excessive

plasma levels due to overdosing, excessive absorption, reduced metabolism, or

reduced plasma protein binding. Slow LAST may occur up to 30 minutes after

injection.54

The initial presentation of slow systemic toxicity will vary depending on

the plasma level of free LA.

The classic cascade of systemic toxicity encompasses central nervous

system (CNS) symptoms increasing in severity from excitation to seizure to coma.

CNS excitation first causes an initial tachycardia and hypertension, while

subsequently, cardiac side-effects predominate and lead to progressive circulatory

collapse (Figure 3). It should be kept in mind that LA have differential cardiotoxic

effects. Substances such as lidocaine and mepivacaine predominantly affect

Chapter 1.2

26

contractility, whereas ropivacaine, levobupivacaine and bupivacaine are both

negatively inotropic and highly arrhythmogenic.55

Conclusion: Systemic toxicity of LA is potentially fatal. When preventative

measures are taken, the incidence of cardiac arrest is low.

Intralipid: critical appraisal

Treatment of LA-induced systemic toxicity consists of supportive treatment

to interrupt seizures and support cardiocirculatory function, and most contemporary

guidelines advocate administration of Intralipid®,56,57

which was first shown in a

1998 to Intralipid® was first found to decrease the susceptibility of rodents to

bupivacaine-induced systemic toxicity.58

Treatment using Intralipid® is thought to induce three changes: first,

according to the lipid sink hypothesis, Intralipid®

can bind free LA. Secondly,

experimental findings suggest Intralipid®

to interact with the sodium channel, and

thirdly, the lipid emulsion may support mitochondrial metabolism.59

Interestingly,

the efficacy of Intralipid® to counteract bupivacaine toxicity is dependent on the

model used. While Intralipid® has generally shown good effects in rodent models,

studies in porcine models, widely used in resuscitation research, generally show no

beneficial effects, and the debate which model is more relevant continues.60

There

exists only one human trial on effects of Intralipid®. Litonius and coworkers infused

bupivacaine intravenously, followed by Intralipid®. The authors found reduced

context-sensitive half-life of bupivacaine, potentially due to increased tissue

distribution, but

failed to detect a relevant lipid sink effect.61

Therefore, the

evidence-base concerning Intralipid®

effects remains unclear, and extrapolation of

clinically relevant benefits is not straightforward.

Traditionally, bupivacaine has been considered more cardiotoxic based on

the smaller dose needed to elicit toxicity as compared to other LA. However, when

comparing bupivacaine with, e.g., ropivacaine, it is imperative to consider the 40-

50% potency difference. In animal models of toxicity, ropivacaine and bupivacaine

are near equipotent in eliciting CNS symptoms.62

Nevertheless, even if the toxic

threshold is similar for equipotent doses of LA, another consideration is that


27

bupivacaine will also remain at the sodium channel longer because of its slower

kinetics (slow-in, slow-out).63

This needs to be weighed against a possible larger

therapeutic effect of Intralipid® in bupivacaine-induced toxicity because in theory,

less lipophilic drugs will be affected less by the lipid sink mechanism.64

Recent pharmacokinetic studies have suggested that Intralipid® will

decrease the cardiac bupivacaine concentration has been estimated to drop by 11%

within 3 minutes of Intralipid administration, and bupivacaine content of the brain

by 18% within 15 minutes.65

Despite the theoretic nature of these findings, they are

notable because they underline that Intralipid®

reduces, but does not eliminate

bupivacaine. Intralipid®

should not be considered an antidote the same way that a

sufficient dose of protamine will reliably antagonize any amount of heparin. Rather,

limited evidence suggests that it will considerably reduce the bupivacaine

concentration in target organs, most likely improve metabolism, and potentially have

direct beneficial effects at the sodium channel. Intralipid® is a valuable contribution

to, but not a substitute for, careful and meticulous conduct of regional anaesthesia.

Conclusion: Intralipid®

has been proposed for resuscitation of systemic LA

overdose, and enthusiastically adopted worldwide, even though the mechanism of

action is incompletely understood. Intralipid®

is not an antidote against LA and

cannot replace meticulous conduct of regional anaesthesia.

Local anesthetic-induced neuro- and myotoxicity

All local anaesthetics are toxic on virtually any type of tissue. In the clinical

setting, neuro- and myotoxicity constitute the main focus of attention.

On neuronal cells, local anaesthetics exhibit dose-dependent toxicity.66

The

question whether some local anaesthetics are more toxic than others has not been

definitively answered and some experimental evidence suggests that equipotent

doses of local anaesthetics exhibit the same degree of toxicity,66 67

whereas other

investigations found that lidocaine was more toxic than, e.g., bupivacaine.68 69

In the

classical observational study by Auroy et al., spinal anesthesia performed using

lidocaine was associated with more neurological deficits than bupivacaine.70

While

Chapter 1.2

28

the short-term neurological dysfunction after peripheral nerve block is relatively

frequent, permanent loss of function is rarely observed.71

Transient neurological syndrome (TNS) typically presents after resolution

of a spinal block, and is typically characterized by radicular segmental pain without

motor deficit, and spontaneous recovery within 72 hours.72

Etiology is

multifactorial, and both positioning (lithotomy) and drug (especially lidocaine) are

important pathogenic factors.73

72

At the other end of the spectrum, cauda equina syndrome is a generalized

destruction of lumbar and sacral nerve fibers, resulting in pain and both sensory and

motor deficit, transcending segmental barriers. The incidence has been estimated

close to 1 : 10.000 70 74

and importantly, there are many causes other than LA-

induced neurotoxicity, including hematoma, abscess formation, tumours, epidural

lipomatosis, and ossifications. Classically, this syndrome was associated with the

pooling of hyperbaric lidocaine 5% in the dural sac when repetitively applied via a

spinal microcatheter,75

lending support to the concept of dose-dependent LA-

induced neurotoxicity.

Myotoxicity of local anaesthetics has been appreciated for a long time,76

but

the clinical relevance is unclear. Most likely, the regenerative potential of muscles

and the functional redundancy mask myotoxic effects after most instances of

peripheral nerve blockade. Therefore, potential transient myotoxic effects are

observed most frequently after regional blockade for eye surgery, where a delicate

balance between weak muscles is easily disturbed by myotoxicity.77

Conclusion: All LA are toxic, dependent on dose. Neurotoxicity is rare, but

disastrous for the patient when it happens. Myotoxicity is thought to occur

frequently, but is only clinically apparent after ocular anesthesia.

Local anaesthetics and tumour recurrence

The perioperative period of tumour surgery has long been recognized as a

vulnerable timeframe, during which tumour progression and metastasis is often

accelerated.78

In small retrospective studies, it was suggested that regional or local

anaesthesia may improve patient survival and disease-free interval after cancer


29

surgery.79 80

Several experimental studies support this assumption. For example,

Deegan and colleagues found that serum taken from patients undergoing tumour

surgery under regional anaesthesia halted growth in a tumour cell line in vitro.81

As

a consequence, several large-scale multicentre randomized controlled trials are

currently under way to assess beneficial effects of regional anaesthesia in tumour

surgery, but they are projected to last until the end of the decade because of long

follow-up times.82

In the meantime, a number of retrospective analyses has been

published, showing heterogeneous effects (Table 2).

The potential mechanisms of anti-metastatic effects can be divided into

direct and indirect effects of LA. Direct effects include the interference with tumour-

promoting pathways, 83

changes in the epigenetic signature of tumour cells,84

, or

direct toxic effects when used for local infiltration.80

Indirect effects result from

reduction of the perioperative stress response, and the preservation of the immune

response.85

Since many of these effects can be duplicated using intravenous LA,32

another avenue of research may be the employment of systemic LA in the

perioperative period of tumour surgery.

We forecast that even if large-scale outcome studies find positive effects,

these will most likely not be universal, but limited to specific types of cancer, and

possibly, cancer stage. It will be imperative to integrate knowledge obtained in these

trials into patient- and tumour-specific strategies to decrease tumour progression

during the perioperative period with all available techniques. This will necessitate an

overall patient care concept including, for well-described indications, regional

anaesthesia.

Conclusion: The question whether local anaesthetics protect against

perioperative tumour progression cannot be answered at this moment. Results from

clinical (retrospective) studies are equivocal.

The future of local anaesthetics

Currently, research efforts are being directed towards the generation of

isoform-specific sodium channel blockers, which will allow for the targeted

treatment of, e.g., neuropathic pain states.86

Still, the idea of targeting of local

Chapter 1.2

30

anaesthetics into nociceptors is of great potential interest.24

Even though the

combination of QX-314 and capsaicin does not lend itself to clinical application, the

concept is enticing. Substantial research efforts are undertaken to compare effects of

intravenous administration of local anaesthetics with regional blockade, leading to

new considerations concerning indications of regional anaesthesia.87

In this context,

it will be essential to define outcome-based patient- and procedure-specific

indications for preventive analgesia using either regional anaesthetic or multimodal

techniques,88

or, in selected patients and indications, both.89

At the same time, “old” local anaesthetics are being rediscovered for new

indications. For example, chloroprocaine, prilocaine and articaine have been

suggested as novel local anaesthetics for day-case surgery.90 91

The same way as multimodal systemic analgesia has been adopted for

perioperative pain treatment, multimodal perineural analgesia has been advocated.

This technique uses a combination of several drugs for peripheral nerve blocks to

avoid the need of catheter techniques.92

As an alternative to catheter techniques and adjuvants, the liposomal slow-

release formulation of bupivacaine, Exparel®, was recently granted FDA approval

for wound infiltration. When used for wound infiltration as part of a multimodal

treatment regimen, encouraging results were obtained,93

but application for nerve

block showed substantial inter-individual variation in block characteristics,94

such

that further studies are needed to ascertain fitness of Exparel® for regional

anaesthesia.

Conclusion: Future areas of interest will be design of new subtype-specific

sodium channel blockers. At the same time, older LA such as 2-chloroprocaine are

reintroduced into the clinical setting. Multimodal perineural analgesia and liposomal

bupivacaine may replace catheter techniques in some indications.

The mechanisms of action and access pathways of LA, and their local

pharmacokinetics are increasingly understood and appreciated. Only very small

amounts of LA actually take part in sodium channel block, while most is absorbed

into tissues or the systemic circulation. Systemic LA are responsible for a substantial


31

share of beneficial effects of regional anesthesia. The dose of LA administered

should be tailored to block site and the individual patient. LA, administered

systemically or locally, will remain of paramount importance in perioperative

medicine.

Acknowledgments

Presentation: This article is based upon the Refresher Course “Local anesthetics –

the essentials” held at the Euroanaesthesia congress, June 2014, Stockholm, Sweden.

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93 Cohen SM. Extended pain relief trial utilizing infiltration of Exparel((R)), a

long-acting multivesicular liposome formulation of bupivacaine: a Phase IV health

economic trial in adult patients undergoing open colectomy. J Pain Res 2012; 5:

567-72

94 Ilfeld BM, Malhotra N, Furnish TJ, Donohue MC, Madison SJ. Liposomal

bupivacaine as a single-injection peripheral nerve block: a dose-response study.

Anesth Analg 2013; 117: 1248-56

Figure legends

Figure 1 Spatial configuration of the alpha subunit of the voltage-gated sodium

channel. The most frequent mutations related to inherited erythromelalgia,17

paroxysmal extreme pain disorder,18

congenital insensitivity to pain18

are given.

Reproduced with permission from Elsevier Publishers: Brain Res Rev 60: 65-83.13

Figure 2 Crystallographic structure of the sodium channel

(A) Side view of the sodium channel, with (from top) extracellular pore, the

selectivity filter, the central cavity, and the innermost activation gate, and (B) Top

view of the same structure. Local anaesthetics exist in an equilibrium between the

uncharged form B and the charged form BH+, where B can cross the membrane but

BH+ is required to bind to the channel. H+ denotes hydrogen ions. Adapted by

permission from Macmillan Publishers Ltd: Nature 475: 353-359. Copyright 2011.11

Figure 3 Sequence of events during local anaesthetic induced systemic toxicity

Increasing systemic concentrations of free local anaesthetic lead to a cascade of

central nervous system and cardiac symptoms.

Chapter 1.2

40

Figure 1


41

Figure 2

Chapter 1.2

42

Figure 3


43

Section 2

Clinical applications

Spinal anaesthesia-induced hypotension

45

Chapter 2.1

Spinal anesthesia-induced hypotension

Based on

Lirk P, Haller I, Wong CA

Management of spinal anesthesia-induced hypotension: a European survey

Eur J Anaesthesiol 2013; 29: 452-3

Chapter 2.1

46

Introduction

Hypotension following administration of spinal anaesthesia for caesarean

delivery is common; the reported incidence varies between 50 and 100%.1 Several

strategies have been suggested to reduce the incidence, or mitigate the severity, of

hypotension, such as patient positioning, fluid administration, and use of

vasopressors to prevent or correct hypotension.2 The last decade has seen extensive

research efforts to devise the optimal regimen for prevention or treatment of spinal

anaesthesia-induced hypotension, including type of intravenous fluid (crystalloid or

colloid), timing of fluid administration (before or after initiation of spinal

anaesthesia), and choice of vasopressor (ephedrine or phenylephrine).3 However, the

degree to which these new findings have been incorporated into clinical practice is

unknown. A survey conducted among American obstetric anaesthesiologists in

20074 found considerable change in clinical practice compared to a UK survey

conducted in 1999.5 Specifically, the use of ephedrine had dropped from 95%

5 to

32%,4 likely reflecting increased awareness of the potential adverse effects of

ephedrine, i.e. tachyarrhythmia and foetal acidosis.6 7

There exist no recent data on

clinical practice among anaesthesiologists providing care for parturients in Europe.

The aim of the present survey was to determine the clinical practice of

spinal anaesthesia for caesarean delivery, focusing on prevention and treatment of

post-spinal anaesthesia hypotension among European anaesthesiologists in 2010.

Methods

We developed a web-based survey questionnaire using an online survey

engine (www.2ask.at). The initial pilot version was tested by several

anaesthesiologists not involved in the study. The survey questions were created by

two authors (P.L., I.H.) after reviewing literature about vasopressor and fluid

therapy use in obstetric anaesthesia. Following approval by the Scientific

Committees of the European Society of Anaesthesiologists (ESA) and the European

Society of Regional Anaesthesia and Pain Therapy (ESRA), an e-mail containing an

invitation to participate in the survey was distributed to members of the two

societies. A total number of 5540 emails were sent, of which 131 were returned


47

undelivered, resulting in 5409 contacted members. The invitation email was sent out

in May 2010, and the survey remained open for two months. To ensure

confidentiality, survey responses did not contain personal or institutional identifying

information. Moreover, the invitation emails were distributed by ESA and ESRA

headquarters anonymously. No follow-up of non-responders was done. All

responses were stored on a secure database and were exported to Microsoft Excel

2003 and SPSS 16.0 for analysis. Plausibility testing was performed and three

responses were discarded.

The survey collected demographic data and assessed practitioners’ routine

methods for preventing and treating hypotension during spinal anaesthesia,

focussing on intravenous fluid administration and vasopressor use. The

questionnaire employed multiple branching, allowing responders to loop through a

set of multiple-choice questions based on their initial responses to several key

questions. The complete survey questionnaire is found in the Appendix.

Results

A total of 5409 email invitations were delivered. We received 351

responses with fully completed questionnaires, which represented an overall

response rate of 6.5%. Sixty-seven incomplete questionnaires were excluded from

further analysis.

Demographics

Demographic characteristics of the responders are found in Table 1. The

responders were distributed among 36 European or Mediterranean countries, with 12

participants (3.4%) from the United States and Australasia. A third of the responders

were members of a professional obstetric anaesthesia society (Society for Obstetric

Anesthesia and Perinatology or Obstetric Anaesthetists Association). The most

common type of anaesthesia for cesaerean delivery was spinal anaesthesia.

Chapter 2.1

48

Performance of spinal anaesthesia

Details of monitoring and spinal anaesthesia technique are found in Table

2. Blood pressure, heart rate, electrocardiography and pulse oximetry were used by

almost all practitioners; other techniques such as bispectral index, fetal heart rate,

and urinary output were used by less than 1% of participants. The majority of

responders routinely administer oxygen. Spinal anaesthesia was initiated in the

sitting position by the majority of anaesthesiologists. The most common spinal

anaesthetic was bupivacaine. The use of adjuvants, particularly opioids, was

common.

Management of post-spinal anaesthesia hypotension

Details of preventative and treatment measures for spinal anaesthesia-

induced hypotension are found in Table 3. Responders estimated that the mean

incidence of post-spinal anaesthesia hypotension was 42% (range 1% to 100%).

Over 80% of responders routinely administer fluid, most commonly crystalloid,

between 500 and 1000 mL. Preload is administered more commonly than coload.

Eighty-three percent of responders agreed that patients generally fall into two

possible categories: hypotension associated with tachycardia, or hypotension

associated with bradycardia (Figure 1). Responders estimated tachycardia occurs

more often (63%) than bradycardia (36%). Slightly more than 60% of

anaesthesiologists routinely used a vasopressor to prevent hypotension. The most

common vasopressor was ephedrine; fewer used phenylephrine and a small minority

used another vasopressor. Almost half of the responders always used ephedrine as

the first choice vasopressor to treat hypotension, whereas only 14% always used

phenylephrine for this indication. Others chose the drug depending on the patient’s

heart rate.

Discussion

The main findings of this 2010 survey are that ephedrine was still routinely

used by more than 70% of survey responders practicing primarily in Europe, for the

treatment of hypotension associated with spinal anaesthesia in women undergoing


49

caesarean delivery; however, more than 40% also use phenylephrine. One quarter of

responders used either ephedrine or phenylephrine, depending on heart rate and / or

blood pressure.

Response rate and demographics

A limitation of our results is our low response rate. The low rate most likely

represents the anonymity with which participants were contacted, and the lack of

follow-up. Hazard Munro postulated that that the minimal number of survey

responses required for survey validity is equal to the number of questions times ten;8

therefore the current 28- question survey required at least 300 response, a number

which was exceeded in the present study. The preferred method to increase response

rate would be to decrease sample size while increasing efforts to contact non-

responders.9 Unfortunately, due to the distribution of the invitation email by the two

anaesthesiology societies, this technique was not possible in the present study. As in

all surveys, nonresponder bias may constitute a source of error. However, the

characteristics of the test persons in our study compare well with those of previous

surveys, with a slight majority working in community hospitals, and slightly less

than half practicing academic anaesthesiology.4 Also, our responders were

distributed over different European and Mediterranean countries, therefore meeting

our aim of surveying predominantly European anaesthesiologists. The share of

caesarean deliveries among overall deliveries was reported by responders to be

approximately 25%, corresponding well with previous investigations.10

Most

responders performed elective caesarean delivery under spinal anaesthesia or other

neuraxial anaesthesia modalities (epidural, combined spinal-epidural), although

16.2% of caesarean deliveries were estimated to occur under general anaesthesia.

These numbers correspond with recent European surveys on use of general

anaesthesia for caesarean deliveries. In Germany the rate of general anesthesia

decreased from 71% in 199811

to 27% in 2005, in Italy the rate was 34% in 2007,12

and in Israel the rate was 15% in 2010.13

Finally, the response rate may have been negatively affected by the specific

nature of the survey. Because almost 80% of responders perform obstetric

Chapter 2.1

50

anaesthesia routinely, we surmise that the survey was completed by a group of

anaesthesiologists with a dedicated and collective interest in obstetric

anaesthesiology.

Performance of spinal anaesthesia

The practice of administering oxygen to parturients in women with normal

oxygen saturation is controversial, although a majority of the survey’s responders do

so routinely. High inspired oxygen concentrations during elective caesarean delivery

under spinal anaesthesia have been controversially discussed to cause oxygen free

radical activity in both mother and foetus.14 15

Potential adverse effects of high

neonatal oxygen concentrations have been demonstrated in several neonatal diseases

such as bronchopulmonary dysplasia, and retinopathy of prematurity.14

Historically,

it has been thought that supplementary oxygen delivered to the mother might protect

the foetus against desaturation in the event of prolonged incision-to-delivery interval

during elective caesarean section, but this was disproved by Khaw and colleagues.16

Administration of 60% oxygen to parturients undergoing emergency caesarean

section under spinal anesthesia resulted in improved oxygenation indices in the

fetus, without increase in lipid peroxidation, and similar outcome.17

The clinical

relevance of these findings remains unclear, but the routine administration of oxygen

to every parturient irrespective of peripheral oxygen saturation is not supported by

literature. Some anaesthesiologists may be administering oxygen solely because

doing so allows simultaneous measurement of end-tidal carbon dioxide.

The majority of responders used bupivacaine as the local anaesthetic,

combined with fentanyl, sufentanil, or morphine. Very few used clonidine or

epinephrine, while 13% did not use any adjuvants. Interestingly, the majority of

responders base the anaesthetic dose on clinical experience or body-mass or height

nomograms, while approximately 20% use the same dose of local anaesthetics in all

patients. The latter practice is supported by recent investigations which found no

relationship between body mass index (BMI) and dose-response for spinal

anaesthesia for elective caesarean delivery.18 19

Hence, current literature does not

support altering the local anaesthetic dose based on body habitus.


51

Management of post-spinal anaesthesia hypotension

Many responders were not aware of guidelines for the prevention and

treatment of hypotension. The reported incidence of post-spinal hypotension varies

widely, with a range of 7% to 74% in prospective studies, depending on the

definition of hypotension and method of measurement.20

Wide heterogeneity exists

in the definition of hypotension, and consequently, the treatment goal. The most

common definitions of hypotension after spinal anaesthesia for caesarean delivery

are “below 80% of baseline” or a combined “below 80% of baseline or 100 mmHg

systolic”.20

In our survey, the target blood pressure goal among clinicians varied

widely. In a recent North American survey by Allen et al., the most common

threshold for treatment of hypotension was less than 20% of baseline pressure,

whereas 13% tried to maintain BP at baseline level.4 In survey of British obstetric

anaesthesiologists a systolic blood pressure of 100 mmHg was defined as threshold

for vasopressor administration by 44% of responders; 41% chose a threshold of 90

mmHg.5 Recent data suggest that maintaining blood pressure close to baseline

compared to 80% baseline is associated with higher umbilical artery pH values21

In

our survey, the overwhelming majority of responders tried to maintain blood

pressure at more than 80% of baseline level, which is in keeping with previous

investigations; less than 5% aimed to keep pressure at baseline.

Research has attempted to determine risk factors for post-spinal

hypotension. Retrospective analysis suggests chronic alcohol consumption, history

of hypertension, increased body mass index, sensory block height and urgency of

surgery as independent risk factors for post-spinal hypotension.22

Recently, body

mass index, sensory block level and older maternal age were identified as

independent risk factors for maternal hypotension, but inclusion of these factors into

a prospective risk assessment showed moderate sensitivity23

and despite preventive

measures, clinicians should continue to anticipate hypotension in every parturient in

which spinal anaesthesia is performed for caesarean delivery.

Chapter 2.1

52

Adverse effects of hypotension are both maternal (syncope, nausea and

vomiting) and foetal/neonatal (placental hypoperfusion, hypoxia, acidosis).2 In a

retrospective study of 919 mother-infant pairs, maternal hypotension was not found

to predict perinatal complications if promptly treated,24

but prolonged hypotension is

associated with foetal acidosis 25

and may cause maternal and foetal morbidity.26

The wide range of responses of our responders estimating the incidence of post-

spinal hypotension (1% to 100%), may reflect individual anaesthesiologists’

experiences with various preventive strategies, as well as prophylactic versus

treatment use of vasopressors. Maintenance of blood pressure was most often

achieved by a combination of fluid and vasopressor therapy. The crystalloid volume

range reported by responders was consistent with the volumes reported in most

clinical trials.27

Fluid loading appears insufficient on its own to prevent or treat hypotension

following spinal anesthesia in parturients. Most responders administered crystalloid

as a preload despite evidence suggesting that this practice is ineffective. 2 28-30

Measurements of cardiac output during caesarean delivery show that the beneficial

effects of a pre-load fluid bolus are not sufficient to maintain hemodynamic stability

after the initiation of spinal anaesthesia.31

It is likely that the increases in cardiac

output that result from fluid loading are insufficient to compensate for the decrease

in systemic vascular resistance caused by high-thoracic neuroblockade.31

Moreover,

rapid pre- or coload may lead to activation of atrial natriuretic peptide in response to

increased intravascular volume, thereby counteracting desired volume-expanding

effects in both healthy and pre-eclamptic parturients.32 33

Colloid is more reliable than crystalloid in preventing hypotension in a

systematic review,34

but a 2010 meta-analysis concluded that the incidence of

maternal hypotension remains high, no matter whether crystalloid or colloid is

administered as a preload or coload.27

Since colloids are associated with a small but

measurable risk of allergic reactions,34

choice of fluid must weigh the small risk of

allergic reactions (estimated at 0.03%35

) associated with colloid administration

against its value in preventing hypotension following spinal anaesthesia.34


53

In a recent trial, colloid pre-load was found to increase cardiac output

transiently for the first five minutes after spinal anesthesia, but there was no

difference in vasopressor requirements or blood pressure changes between colloid

preload and coload.36

The rationale behind fluid pre- and co-load is to counteract

decreased cardiac output due to decreased venous return following spinal anesthesia.

However, recent evidence emphasizes the importance of changes in systemic

vascular resistance in post-spinal anesthesia hypotension,28

with the possible

exception of patients who are hypotensive and bradycardic after spinal anesthesia.

This latter group of patients should preferentially be treated using fluid, ephedrine or

epinephrine, and possibly anticholinergic agents.28 37

The proportion of bradycardic

patients after spinal anesthesia was estimated by our responders, on average, to

comprise around a third of patients, whereas a much lower incidence was reported in

recent trials 37

and a recent review of hemodynamic changes following spinal

anaesthesia for caesarean section described the incidence as low.28

Despite preventive measures and fluid co-load, hypotension after spinal

anaesthesia is common.38

In a recent study, the combination of prophylactic

phenylephrine and rapid crystalloid cohydration was found to be effective in

preventing spinal-induced hypotension for caesarean delivery.26

Ngan Kee et al.

postulated that cohydration better coincides with peak sympathectomy effect of

spinal anaesthesia, and promotes faster circulation of vasopressor.26

This latest study

achieved a very low incidence of maternal hypotension, albeit at the cost of

overcorrecting blood pressure in a substantial share of patients.26

Perioperative measurements of cardiac output have shown that have shown

that the major changes induced by spinal anaesthesia are a decrease in systemic

vascular resistance accompanied by a compensatory increase in cardiac output.28

This pathogenic mechanism is directly counteracted by phenylephrine, which

increases systemic vascular resistance, and decreases cardiac output.37

Ephedrine, in

contrast, increases heart rate and (to a lesser extent) stroke volume and therefore

cardiac output,37

but is often not potent enough to correct changes in vascular

resistance.

Chapter 2.1

54

Whether ephedrine of phenylephrine is the vasopressor of choice for post-

spinal hypotension in elective caesarean delivery remains controversial. However,

recent editorialists suggest that based on current evidence, phenylephrine is the drug

of choice.39

Ephedrine has been associated with foetal acidemia, and both the spinal

anaesthesia - delivery interval and total ephedrine dose are positively correlated with

neonatal academia.40

In a secondary analysis of the EPIPAGE trial data, neonatal

mortality of preterm infants was higher after spinal than general or epidural

anaesthesia; it has been hypothesized that the widespread use of ephedrine may be

responsible for this finding.41

The mechanism of action of ephedrine’s potential

adverse effects has not been clearly elucidated. Ephedrine crosses the placenta to a

greater extent than phenylephrine, and it may exert beta-adrenergic mediated

stimulatory effects on foetal metabolism.42

Accumulating evidence the phenylephrine is superior to ephedrine appears

to have resulted in an increased use of phenylephrine for the prevention and

treatment of hypotension in the past 11 years. Results of surveys in North American

and the United Kingdom in 1999 and 2007 suggest that the use of phenylephrine in

the obstetric population is increasing. In the 1999 survey, ephedrine was used by

95% of responders.5 In contrast, a 2007 survey found 23% and 26% of responders

used phenylephrine for the prevention and treatment of hypotension, respectively. In

the same survey, 40% of anaesthesiologists used either agent based upon heart rate.4

In our survey, 26% of responders used either agent based upon heart rate. This is

also in concordance with recent literature, which recommends that phenylephrine

may be the better choice in situations when hypotension coincides with tachycardia,

whereas ephedrine may be better suited in situations when hypotension coincides

with bradycardia.37

However, in light of the strong evidence to support routine use

of phenylephrine, we note the relatively large number of responders who still always

use ephedrine as the first choice, while a much smaller number of responders

reported the exclusive use phenylephrine.

In conclusion, we describe results from a 2010 European survey

investigating contemporary clinical practice in the management of hypotension

following spinal anaesthesia for caesarean delivery. In contrast to previous surveys,


55

we found increased use of phenylephrine to treat hypotension, in keeping with a

growing evidence-base suggesting its side-effect profile is superior to that of

ephedrine in most cases. Recent literature supports the use of phenylephrine as the

first-line drug to treat most patients with hypotension. Nevertheless, many clinicians

appear to continue to use ephedrine routinely.

References

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theory of relativity. Minerva Anestesiol 2008; 74: 409-24

2 Cyna AM, Andrew M, Emmett RS, Middleton P, Simmons SW. Techniques for

preventing hypotension during spinal anaesthesia for caesarean section. Cochrane

Database Syst Rev 2006: CD002251

3 Dyer RA, Reed AR. Spinal hypotension during elective cesarean delivery: closer

to a solution. Anesth Analg 2010; 111: 1093-5

4 Allen TK, Muir HA, George RB, Habib AS. A survey of the management of

spinal-induced hypotension for scheduled cesarean delivery. Int J Obstet Anesth

2009; 18: 356-61

5 Burns SM, Cowan CM, Wilkes RG. Prevention and management of hypotension

during spinal anaesthesia for elective Caesarean section: a survey of practice.

Anaesthesia 2001; 56: 794-8

6 Cooper DW, Carpenter M, Mowbray P, Desira WR, Ryall DM, Kokri MS. Fetal

and maternal effects of phenylephrine and ephedrine during spinal anesthesia for

cesarean delivery. Anesthesiology 2002; 97: 1582-90

7 Saravanan S, Kocarev M, Wilson RC, Watkins E, Columb MO, Lyons G.

Equivalent dose of ephedrine and phenylephrine in the prevention of post-spinal

hypotension in Caesarean section. Br J Anaesth 2006; 96: 95-9

8 Hazard Munro E. Statistical methods for Health Care Research. Philadelphia:

Lippincott Williams and Wilkins, 2001

9 Burmeister LF. Principles of successful sample surveys. Anesthesiology 2003; 99:

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10 Stamer UM, Wiese R, Stuber F, Wulf H, Meuser T. Change in anaesthetic

practice for Caesarean section in Germany. Acta Anaesthesiol Scand 2005; 49: 170-

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11 Stamer UM, Messerschmidt A, Wulf H. Anaesthesia for caesarean section--a

German survey. Acta Anaesthesiol Scand 1998; 42: 678-84

12 Gori F, Pasqualucci A, Corradetti F, Milli M, Peduto VA. Maternal and neonatal

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13 Weiniger CF, Ivri S, Ioscovich A, Grimberg L, Evron S, Ginosar Y. Obstetric

anesthesia units in Israel: a national questionnaire-based survey. Int J Obstet Anesth

2010; 19: 410-6

14 Khaw KS, Wang CC, Ngan Kee WD, Pang CP, Rogers MS. Effects of high

inspired oxygen fraction during elective caesarean section under spinal anaesthesia

on maternal and fetal oxygenation and lipid peroxidation. Br J Anaesth 2002; 88:

18-23

15 Khaw KS, Ngan Kee WD, Chu CY, et al. Effects of different inspired oxygen

fractions on lipid peroxidation during general anaesthesia for elective Caesarean

section. Br J Anaesth 2010; 105: 355-60

16 Khaw KS, Ngan Kee WD, Lee A, et al. Supplementary oxygen for elective

Caesarean section under spinal anaesthesia: useful in prolonged uterine incision-to-

delivery interval? Br J Anaesth 2004; 92: 518-22

17 Khaw KS, Wang CC, Ngan Kee WD, et al. Supplementary oxygen for

emergency Caesarean section under regional anaesthesia. Br J Anaesth 2009; 102:

90-6

18 Carvalho B, Collins J, Drover DR, Atkinson Ralls L, Riley ET. ED50 and ED95

of Intrathecal Bupivacaine in Morbidly Obese Patients Undergoing Cesarean

Delivery. Anesthesiology 2011; 114: 529-35

19 Lee Y, Balki M, Parkes R, Carvalho JC. Dose requirement of intrathecal

bupivacaine for cesarean delivery is similar in obese and normal weight women. Rev

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20 Klohr S, Roth R, Hofmann T, Rossaint R, Heesen M. Definitions of hypotension

after spinal anaesthesia for caesarean section: literature search and application to

parturients. Acta Anaesthesiol Scand 2010; 54: 909-21

21 Ngan Kee WD, Khaw KS, Ng FF. Comparison of phenylephrine infusion

regimens for maintaining maternal blood pressure during spinal anaesthesia for

Caesarean section. Br J Anaesth 2004; 92: 469-74

22 Hartmann B, Junger A, Klasen J, et al. The incidence and risk factors for

hypotension after spinal anesthesia induction: an analysis with automated data

collection. Anesth Analg 2002; 94: 1521-9, table of contents

23 Brenck F, Hartmann B, Katzer C, et al. Hypotension after spinal anesthesia for

cesarean section: identification of risk factors using an anesthesia information

management system. J Clin Monit Comput 2009; 23: 85-92

24 Maayan-Metzger A, Schushan-Eisen I, Todris L, Etchin A, Kuint J. Maternal

hypotension during elective cesarean section and short-term neonatal outcome. Am J

Obstet Gynecol 2010; 202: 56 e1-5

25 Ngan Kee WD, Lee A. Multivariate analysis of factors associated with umbilical

arterial pH and standard base excess after Caesarean section under spinal

anaesthesia. Anaesthesia 2003; 58: 125-30

26 Ngan Kee WD, Khaw KS, Ng FF. Prevention of hypotension during spinal

anesthesia for cesarean delivery: an effective technique using combination

phenylephrine infusion and crystalloid cohydration. Anesthesiology 2005; 103: 744-

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27 Banerjee A, Stocche RM, Angle P, Halpern SH. Preload or coload for spinal

anesthesia for elective Cesarean delivery: a meta-analysis. Can J Anaesth 2010; 57:

24-31

28 Langesaeter E, Dyer RA. Maternal haemodynamic changes during spinal

anaesthesia for caesarean section. Curr Opin Anaesthesiol 2011

29 Husaini SW, Russell IF. Volume preload: lack of effect in the prevention of

spinal-induced hypotension at caesarean section. Int J Obstet Anesth 1998; 7: 76-81

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30 Jackson MR, Friedman SA, Carter AJ, et al. Hemostatic efficacy of a fibrin

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31 Tamilselvan P, Fernando R, Bray J, Sodhi M, Columb M. The effects of

crystalloid and colloid preload on cardiac output in the parturient undergoing

planned cesarean delivery under spinal anesthesia: a randomized trial. Anesth Analg

2009; 109: 1916-21

32 Pouta A, Karinen J, Vuolteenaho O, Laatikainen T. Pre-eclampsia: the effect of

intravenous fluid preload on atrial natriuretic peptide secretion during caesarean

section under spinal anaesthesia. Acta Anaesthesiol Scand 1996; 40: 1203-9

33 Pouta AM, Karinen J, Vuolteenaho OJ, Laatikainen TJ. Effect of intravenous

fluid preload on vasoactive peptide secretion during Caesarean section under spinal

anaesthesia. Anaesthesia 1996; 51: 128-32

34 Morgan PJ, Halpern SH, Tarshis J. The effects of an increase of central blood

volume before spinal anesthesia for cesarean delivery: a qualitative systematic

review. Anesth Analg 2001; 92: 997-1005

35 Karri K, Raghavan R, Shahid J. Severe Anaphylaxis to Volplex, a Colloid

Solution during Cesarean Section: A Case Report and Review. Obstet Gynecol Int

2009; 2009: 374791

36 Teoh WH, Sia AT. Colloid preload versus coload for spinal anesthesia for

cesarean delivery: the effects on maternal cardiac output. Anesth Analg 2009; 108:

1592-8

37 Dyer RA, Reed AR, van Dyk D, et al. Hemodynamic effects of ephedrine,

phenylephrine, and the coadministration of phenylephrine with oxytocin during

spinal anesthesia for elective cesarean delivery. Anesthesiology 2009; 111: 753-65

38 George RB, McKeen D, Columb MO, Habib AS. Up-down determination of the

90% effective dose of phenylephrine for the treatment of spinal anesthesia-induced

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154-8

39 Smiley RM. Burden of proof. Anesthesiology 2009; 111: 470-2


59

40 Cooper DW, Gibb SC, Meek T, et al. Effect of intravenous vasopressor on

spread of spinal anaesthesia and fetal acid-base equilibrium. Br J Anaesth 2007; 98:

649-56

41 Laudenbach V, Mercier FJ, Roze JC, et al. Anaesthesia mode for caesarean

section and mortality in very preterm infants: an epidemiologic study in the

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42 Ngan Kee WD, Khaw KS, Tan PE, Ng FF, Karmakar MK. Placental transfer and

fetal metabolic effects of phenylephrine and ephedrine during spinal anesthesia for

cesarean delivery. Anesthesiology 2009; 111: 506-12

Chapter 2.1

60

Table 1. Responder demographic characteristics.

Hospital setting

Community hospital 57%

University hospital 43%

Provider type

Trainee 14%

Consultant 66%

Director 18%

Obstetric anaesthesia practice

Full time 10%

Routinely 78%

Rarely 12%


61

Table 2. Characteristics of spinal anaesthesia.

Administration of oxygen yes/no 60%/40%

Local anesthetic (multiple answers possible)

Bupivacaine 89%

Levobupivacaine 7%

Ropivacaine 6%

Lidocaine/Mepivacaine 2%

Additives (multiple answers possible)

Fentanyl 48%

Sufentanil 29%

Morphine 21%

Clonidine 1%

Epinephrine < 1%

Dehydrobenzperidol < 1%

Pethidine, Diamorphine < 1%

Chapter 2.1

62

Table 3. Hemodynamic variables and treatment

Blood pressure goal

at/above baseline 4%

above 90% of baseline 40%

above 80% of baseline 54%

Fluid pre-load or co-load (multiple answers possible)

Fluid preload 85%

Crystalloid fluid preload 65%

Colloid fluid preload 27%

Fluid co-load 39%

Fluid volume 500-1000 ml 71%

Fluid volume 1000-1500 ml 16%

Preventive vasopressor (multiple answers possible)

Routinely used 63%

Ephedrine 72%

Phenylephrine 46%

Other 9%

Therapeutic vasopressor

Always ephedrine 48%

Always phenylephrine 14%

Phenylephrine 1st line, ephedrine 2

nd line 4%

Ephedrine 1st line, phenylephrine 2

nd line 9%

Choice based upon heart rate 26%

Others: metaraminol, theodrenaline, noradrenaline

Pulmonary effects of spinal anesthesia

63

Chapter 2.2


Based on

Lirk P, Kleber N, Mitterschiffthaler G, Keller C, Benzer A, Putz G

Pulmonary effects of bupivacaine, ropivacaine, and levobupivacaine in parturients

undergoing spinal anaesthesia for elective caesarean delivery: a randomised

controlled study

Int J Obstet Anaesthesiol 2010; 19: 287-92

Chapter 2.2

64

Introduction

Intrathecal anaesthesia has replaced general as the first-line method to

provide anaesthesia for elective caesarean delivery.1 The main reason for the

popularity of spinal anaesthesia is that general anaesthesia may be associated with

complications in airway management such as aspiration or failure to achieve tracheal

intubation.1 It is unclear whether spinal anaesthesia carries a risk of significant

respiratory deterioration due to motor block of respiratory muscles,2 3

since

caesarean delivery necessitates a spinal block extending as far cephalad as the fourth

thoracic segmental nerve.3 Motor blockade from the lumbar to the thoracic nerves

temporarily deactivates some muscles that contribute to respiration, including the

intercostal muscles4 and the abdominal wall muscles.

5

In clinical practice, bupivacaine is the most widely used local anaesthetic

for elective caesarean delivery.6 7

Numerous publications have reported that spinal

anaesthesia using bupivacaine significantly decreases dynamic pulmonary function

parameters in the parturient.2 3 5 8

It has been suggested that the newer local

anaesthetics ropivacaine and levobupivacaine do not cause the same degree of motor

block,9-11

but the pulmonary effects of these drugs when used for spinal anaesthesia

are unclear. For example, epidural and intrathecal levobupivacaine and ropivacaine

were shown in vivo to elicit less motor block than bupivacaine.12

Recently,

intrathecal bupivacaine was shown in parturients to have a higher potency for motor

block than levobupivacaine and ropivacaine.13

Therefore, the pulmonary effects of

ropivacaine and levobupivacaine may be less pronounced than those of bupivacaine.

The aim of the present study was to compare the performance ofbupivacaine 10 mg,

ropivacaine 20 mg, and levobupivacaine 10 mg for spinal anaesthesia in parturients

undergoing elective caesarean delivery. As relevant endpoints, we chose to

investigate dynamic maternal pulmonary function parameters and neonatal indices.

Our working hypothesis was that pulmonary function would be attenuated by all

three local anaesthetics.


65

Methods

In a single-blind study, 48 otherwise healthy parturients scheduled to

undergo elective caesarean section were enrolled and randomised to receive

bupivacaine, ropivacaine or levobupivacaine. Randomization of patients into the

three treatment groups was performed according to a preset randomization list

disclosed from sealed opaque envelopes before spinal anesthesia. Exclusion criteria

were pre-existing maternal pulmonary or cardiac disease, thoracic malformation,

non-singleton pregnancy, signs of fetal compromise and a body mass index (BMI)

>35 kg/m2, indicating severe obesity.

14 The study was approved by the Ethics

Committee of the Medical University, Innsbruck (Protocol No. UN2195_ZEK),

Austria, and registered with the European Union Clinical Trials Database (EudraCT

No. 2004-004649-17). Written informed consent was obtained from all patients.

Patients were blinded to the study drug, but the investigator and anaesthesiologist

administering spinal anaesthesia were not.

Premedication and spinal anaesthesia

All parturients were preloaded intravenously with 1000 mL of crystalloid

fluid (Ringer’s lactate) and received famotidine 20 mg before transfer to the

operating room. Non-invasive blood pressure, pulse oximetry and electrocardiogram

were monitored. Spinal anaesthesia was performed in the sitting position between

the second and fourth lumbar vertebrae using a 25-gauge Sprotte pencil-point needle

(Smith, Brisbane, Australia). Patients were randomised to receive 0.5% bupivacaine

10 mg (Curasan, Kleinostheim, Germany), 1% ropivacaine 20 mg (Astra Zeneca,

Södertälje, Sweden), or 0.5% levobupivacaine 10 mg (Abbott, Campoverde, Italy).

Fentanyl 15 µg (Torrex Pharma, Vienna, Austria) was added to the local anaesthetic

in all groups. Sensory block was tested every minute by pin prick in the mid-

clavicular line, testing for discrimination between sharp and blunt stimulus, until a

stable sensory block level was reached. The lowest mean arterial pressure and

vasopressor requirements following spinal anaesthesia were recorded.

Chapter 2.2

66

Measurement of pulmonary function

When the sensory block had stabilized, dynamic pulmonary function

indices were measured using an EasyOne spirometer (ndd, Zurich, Switzerland)

before spinal anaesthesia, and when a stable sensory level was achieved. The

spirometer used requires several measurements tested for consistency before the best

value is accepted as a result. Internal quality control software detects both

implausible spirometric results and intra-test variability. Accepted results require at

least two measurements of forced expiratory volume during the first second of

exhalation (FEV1) or forced vital capacity (FVC) to vary no more than 200 mL. A

single investigator (NK), experienced in performing spirometric measurements,

recorded all pulmonary function indices; quality control and tabulation of measured

data was performed blinded to group allocation. Pulmonary function tests took

approximately 2 minutes to perform.

The evening before surgery, the patients were instructed in the use of the

spirometer to simulate testing. Baseline measurements were obtained on the day of

surgery in the 15° left lateral tilt position. To explore the possibility that the 15° left

tilt and supine positions would yield different pulmonary function test results,

measurements were obtained in both positions in eight patients. As there were no

significant differences in pulmonary function tests, baseline results were pooled.

Pulmonary function following spinal anaesthesia were measured in the 15° left tilt

position. Caesarean delivery was conducted after measurements were completed.

The main outcome variables were FVC, FEV1, and peak expiratory flow

rate (PEFR). Secondary outcome variables were fetal blood gases and Apgar score.

Independent factors included maternal characteristics (age, BMI, smoking status,

gestational age) and perioperative data (site of puncture, time for the block to reach

T4, duration of surgery, time to delivery).


67

Statistical analyses

Power calculation based on previous investigations of spinal anaesthesia for

caesarean delivery5 assumed a mean change in FEV1 of 15-20%. Therefore, 16 test

parturients per group were predicted as being necessary to detect a statistically

significant alteration in function tests with a power of 85%. The primary purpose of

the study was to evaluate if any of the three local anaesthetic agents cause a 15-20%

decrease in FEV1 in parturients undergoing spinal anaesthesia; as such, the power

analysis was not designed to indicate differences between the three agents. Normal

distribution of data was checked by the Kolmogorov-Smirnov test. The time course

of the main variables was tested using analysis of variance (ANOVA) with

Bonferroni’s post hoc correction. Secondary variables were tested using the χ2 test

for qualitative, and ANOVA for quantitative data. P values <0.05 were considered

statistically significant. The statistical null hypothesis was that pulmonary function

would not be attenuated by any of the three local anaesthetics. Data were analysed

using SPSS version 12.0, Chicago, IL.

Chapter 2.2

68

Results

Demographics. The 48 parturients were between 22 and 43 years of age.

There were 16 patients in each group. There were no significant differences in mean

age, BMI, gestation age, smoking status or number of previous pregnancies or

cesarean sections between the three groups (Table 1).

Spinal anaesthesia and surgery. The documented site of puncture varied

between L2-3 (n=25) and L3-4 (n=23) but was not different between groups. The

upper level of block of pin-prick sensation and the time to reach T4 did not differ

between the three groups (Table 1). All caesarean deliveries were performed under

spinal anaesthesia, without the need for conversion to general anaesthesia. There

were no differences in duration of surgery, time to delivery after incision, lowest

recorded mean arterial pressure or numbers of patients requiring vasopressors

following spinal anaesthesia (Table 1).

Maternal pulmonary function tests. At baseline, the values for FVC, FEV1,

and PEFR were normally distributed, and mean values for the three study groups did

not differ significantly (Table 2). Forced vital capacity was decreased in the

bupivacaine and ropivacaine groups but not in the levobupivacaine group. Forced

expiratory volume during the first second was not decreased in any group. Peak

expiratory flow rate was decreased in the ropivacaine and levobupivacaine groups.

There were no significant differences in pulmonary function indices after spinal

anaesthesia between the groups. To examine whether the level of spinal block would

influence maternal lung function, all patients were pooled by block level, comparing

the subset of patients in whom the block spread to no higher than T4 vs. higher than

T4. The maternal lung function parameters after spinal anaesthesia were not

different between these two groups (Table 2).

Neonatal status: there were no significant differences in neonatal outcome

measures between the groups (Table 3).


69

Discussion

The effects of neuraxial bupivacaine on pulmonary function have

repeatedly been measued,2 3 8 15

but comparative data for ropivacaine and

levobupivacaine have not hitherto been available. We found a significant reduction

in selected lung function parameters after intrathecal bupivacaine 10 mg,

ropivacaine 20 mg and levobupivacaine 10 mg, all with fentanyl 15 µg, among

patients about to undergo elective caesarean delivery. These reductions were in the

region of 3-6% for FVC and 6-13%for PEFR. However, the clinical importance of

this finding remains unclear. No difference was found between bupivacaine,

ropivacaine and levobupivacaine in pulmonary function variables, Apgar scores or

umbilical artery pH.

The propensity of ropivacaine and levobupivacaine to cause motor block is

thought to be less than that of bupivacaine. One reason for this differential block

may be preferential blockade of sodium channels specific for nociceptive neurones

by ropivacaine.16

A recent investigation in parturients undergoing elective caesarean

section found that intrathecal bupivacaine, levobupivacaine, and ropivacaine had

high, intermediate, and low potency for motor block, respectively.13

In a similar

study, intrathecal ropivacaine was shown to cause less intense and shorter-lasting

motor block than bupivacaine.17

However, Camorcia et al. postulated that the

differences in motor block among local anaesthetics were less distinctive when used

for spinal than for epidural anesthesia.13

Caesarean delivery necessitates a block to pin-prick or cold extending as far

cephalad as the fourth thoracic segmental nerve. Since this blockade may affect

some of the intercostal muscles, a substantial share of respiratory capability may be

deactivated during spinal anaesthesia. Intercostal muscles play multifaceted roles

even during physiologic inspiration and expiration.4 Nevertheless, Egbert et al.

18 and

Askrog et al.19

failed to detect clinically significant alterations in the capacity to

initiate a forceful cough, presumably because of functional compensation by the

diaphragm.19

In addition to the diaphragm, and in contrast to respiration at rest,

additional muscles are required for forceful inspiration (i.e. sternocleidomastoid,

scalenus, external intercostals) and expiration (i.e. internal intercostals, abdominal

Chapter 2.2

70

wall). Intercostal muscles after spinal blockade seem to play a minimal role in

overall lung function. In our study, results for the subset of patients in whom the

block extended no higher than T4 were not significantly different from those for

patients in whom the block extended beyond T4. Similarly, Arai et al.6 recently

found that maternal pulmonary function was not significantly different when

comparing patients with block level of T1-T2 and T4. It remains unclear whether

compensation by the diaphragm is the only factor maintaining forceful expiration in

the presence of partially blocked intercostal muscles. Even patients with spinal cord

injury involving the mid to lower cervical cord may be able to augment expiration

using accessory muscles of respiration.20

The concentrations of drugs chosen for this clinical trial were extrapolated

from previous investigations, which found a relative anaesthetic potency between

bupivacaine and ropivacaine of 2:1 and between bupivacaine and levobupivacaine of

1:1.20 21

Therefore, we chose final doses for bupivacaine 10 mg, levobupivacaine 10

mg, and ropivacaine 20 mg. However, in studies published after our study was

initiated, different relative potencies were found. For example, Parpaglioni et al.22

found that the potency ratio between levobupivacaine and ropivacaine was 1:0.74,

and not 1:0.5 as assumed in our study. Moreover, Camorcia et al. reported the

relative ED50 for motor block after intrathecal administration for

ropivacaine:bupivacaine 0.59 and for levobupivacaine:bupivacaine 0.71,31

so

respiratory impairment with ropivacaine might have been less than with the other

two agents in our study if a more nearly equipotent dose had been used.

The addition of opioid (fentanyl 15 µg) to all local anaesthetic agents in our

study could have theoretically altered the block characteristics and motor function.

However, previous investigation in patients undergoing transurethral resection of the

prostate demonstrated that lung function was not altered when fentanyl was added to

bupivacaine for spinal anesthesia.23

Although fentanyl might reduce respiratory

drive, it would be unlikely to affect capacity.

The dynamic pulmonary function tests were affected to different degrees by

spinal anaesthesia. More specifically, FEV1 did not decrease significantly in any of

the study groups, which is consistent in other studies with bupivacaine.2,8

By


71

contrast, Kelly et al. found significant decreases in FEV1 after spinal anaesthesia,3

but their results could have been confounded by an open abdomen, which has been

previously shown to substantially influence pulmonary function.8

FVC was negatively influenced by spinal anaesthesia using bupivacaine

and ropivacaine, which is in accord with previous investigations.8,3

However,

Harrop-Griffiths et al.2 and Arai et al.

6 found no significant alterations in FVC after

bupivacaine spinal anaesthesia. Possible explanations for these differences include a

12% greater BMI in our patients than among those studied by Arai and colleagues;6

the higher BMI appears to predispose patients undergoing spinal anaesthesia to more

severe reductions in lung function.24 25

Finally, PEFR decreased significantly with ropivacaine and

levobupivacaine, but not with bupivacaine. While Conn et al.8 did not detect a

significant deterioration of PEFR after spinal anaesthesia with bupivacaine for

caesarean delivery, Harrop-Griffiths et al.2 and Kelly et al.

3 reported statistically

significant decreases. This disparity may be explained by different doses of

bupivacaine: Harrop-Griffiths and Kelly both gave a dose of 12.5 mg. In addition,

Kelly measured pulmonary function before spinal anaesthesia, and during surgery,

when the abdomen was open, which may further compromise ventilation.3

An important secondary endpoint was the analyses of Apgar scores and

umbilical blood gases. In accordance with previous literature,26

no significant

differences were found. To exclude the potential confounding of these results by

hypotension or vasopressor administration, the lowest measured mean arterial

pressure following spinal anaesthesia, and vasopressor requirements were recorded.

These values were not significantly different between the groups.

Some limitations of our study should be briefly addressed. Firstly,

spirometric testing depends to a large extent upon the compliance of the test person

and adherence to the experimental protocol may have been difficult during the very

stressful moments immediately before delivery. We sought to minimize this

confounding variable by preoperative instruction, including hands-on practice with

the spirometry device.2 3 8

Secondly, not all baseline measurements were taken in the

same position (supine vs. 15° left tilt). Even though we performed statistical testing

Chapter 2.2

72

to assure that these values were not different, we cannot exclude a small influence

upon our results. Thirdly, our power analysis was designed to test if the groups

achieved a certain decrease in pulmonary function tests, but not to evaluate

differences between the groups. Because clinically significant reductions in

pulmonary function were not observed in any of the groups, it seems highly unlikely

that significant differences would be found between the three groups. Finally,

although the present study was randomised and controlled, organisational problems

precluded the use of a double-blind technique. Nevertheless, we believe that results

from our study are valid and reproducible because the spirometric device required

several recordings tested for consistency before computing pulmonary function

indices.

The results of our study suggest that spinal anaesthesia with bupivacaine,

ropivacaine, or levobupivacaine, all with fentanyl 15 µg, resulted in comparable

maternal lung function (FVC, FEV1, PEFR), and neonatal outcomes. Based on our

findings, the three investigated local anaesthetics may be equally well suited for

spinal anaesthesia for elective caesarean delivery.

References

1 Dresner MR, Freeman JM. Anaesthesia for caesarean section. Best Pract Res Clin

Obstet Gynaecol 2001; 15: 127-43

2 Harrop-Griffiths AW, Ravalia A, Browne DA, Robinson PN. Regional

anaesthesia and cough effectiveness. A study in patients undergoing caesarean

section. Anaesthesia 1991; 46: 11-3

3 Kelly MC, Fitzpatrick KT, Hill DA. Respiratory effects of spinal anaesthesia for

caesarean section. Anaesthesia 1996; 51: 1120-2

4 De Troyer A, Kirkwood PA, Wilson TA. Respiratory action of the intercostal

muscles. Physiol Rev 2005; 85: 717-56

5 von Ungern-Sternberg BS, Regli A, Bucher E, Reber A, Schneider MC. Impact of

spinal anaesthesia and obesity on maternal respiratory function during elective

Caesarean section. Anaesthesia 2004; 59: 743-9


73

6 Arai YC, Ogata J, Fukunaga K, Shimazu A, Fujioka A, Uchida T. The effect of

intrathecal fentanyl added to hyperbaric bupivacaine on maternal respiratory

function during cesarean section. Acta Anaesthesiol Scand 2006; 50: 364-7

7 Stamer UM, Wiese R, Stuber F, Wulf H, Meuser T. Change in anaesthetic

practice for Caesarean section in Germany. Acta Anaesthesiol Scand 2005; 49: 170-

6

8 Conn DA, Moffat AC, McCallum GD, Thorburn J. Changes in pulmonary

function tests during spinal anaesthesia for caesarean section. Int J Obstet Anesth

1993; 2: 12-4

9 Stienstra R. The place of ropivacaine in anesthesia. Acta Anaesthesiol Belg 2003;

54: 141-8

10 Simpson D, Curran MP, Oldfield V, Keating GM. Ropivacaine: a review of its

use in regional anaesthesia and acute pain management. Drugs 2005; 65: 2675-717

11 Foster RH, Markham A. Levobupivacaine: a review of its pharmacology and use

as a local anaesthetic. Drugs 2000; 59: 551-79

12 Kanai Y, Tateyama S, Nakamura T, Kasaba T, Takasaki M. Effects of

levobupivacaine, bupivacaine, and ropivacaine on tail-flick response and motor

function in rats following epidural or intrathecal administration. Reg Anesth Pain

Med 1999; 24: 444-52

13 Camorcia M, Capogna G, Berritta C, Columb MO. The relative potencies for

motor block after intrathecal ropivacaine, levobupivacaine, and bupivacaine. Anesth

Analg 2007; 104: 904-7

14 Poobalan AS, Aucott LS, Gurung T, Smith WC, Bhattacharya S. Obesity as an

independent risk factor for elective and emergency caesarean delivery in nulliparous

women - systematic review and meta-analysis of cohort studies. Obes Rev 2008

15 Gamil M. Serial peak expiratory flow rates in mothers during caesarean section

under extradural anaesthesia. Br J Anaesth 1989; 62: 415-8

16 Oda A, Ohashi H, Komori S, Iida H, Dohi S. Characteristics of ropivacaine

block of Na+ channels in rat dorsal root ganglion neurons. Anesth Analg 2000; 91:

1213-20

Chapter 2.2

74

17 Whiteside JB, Burke D, Wildsmith JA. Comparison of ropivacaine 0.5% (in

glucose 5%) with bupivacaine 0.5% (in glucose 8%) for spinal anaesthesia for

elective surgery. Br J Anaesth 2003; 90: 304-8

18 Egbert LD, Tamersoy K, Deas TC. Pulmonary function during spinal anesthesia:

the mechanism of cough depression. Anesthesiology 1961; 22: 882-5

19 Askrog VF, Smith TC, Eckenhoff JE. Changes in pulmonary ventilation during

spinal anesthesia. Surg Gynecol Obstet 1964; 119: 563-7

20 McDonald SB, Liu SS, Kopacz DJ, Stephenson CA. Hyperbaric spinal

ropivacaine: a comparison to bupivacaine in volunteers. Anesthesiology 1999; 90:

971-7

21 Alley EA, Kopacz DJ, McDonald SB, Liu SS. Hyperbaric spinal

levobupivacaine: a comparison to racemic bupivacaine in volunteers. Anesth Analg

2002; 94: 188-93

22 Parpaglioni R, Frigo MG, Lemma A, Sebastiani M, Barbati G, Celleno D.

Minimum local anaesthetic dose (MLAD) of intrathecal levobupivacaine and

ropivacaine for Caesarean section. Anaesthesia 2006; 61: 110-5

23 Walsh KH, Murphy C, Iohom G, Cooney C, McAdoo J. Comparison of the

effects of two intrathecal anaesthetic techniques for transurethral prostatectomy on

haemodynamic and pulmonary function. Eur J Anaesthesiol 2003; 20: 560-4

24 von Ungern-Sternberg BS, Regli A, Schneider MC, Kunz F, Reber A. Effect of

obesity and site of surgery on perioperative lung volumes. Br J Anaesth 2004; 92:

202-7

25 Regli A, von Ungern-Sternberg BS, Reber A, Schneider MC. Impact of spinal

anaesthesia on peri-operative lung volumes in obese and morbidly obese female

patients. Anaesthesia 2006; 61: 215-21

26 Coppejans HC, Vercauteren MP. Low-dose combined spinal-epidural anesthesia

for cesarean delivery: a comparison of three plain local anesthetics. Acta

Anaesthesiol Belg 2006; 57: 39-43


75

Table 1. Patient demographics and outcome of spinal anaesthesia

Bupivacaine

(n=16)

Ropivacaine

(n=16)

Levobupi

(n=16)

Age (years) 31.9 ± 4.0 32.2 ± 5.1 33.9 ± 4.4

BMI (kg/m2) 27.0 ± 3.6 27.5 ± 3.7 28.1 ± 4.9

Smokers 1/16 5/16 2/16

Gestation (weeks) 38.6 ± 1.0 38.4 ± 1.0 38.2 ± 0.8

Parity 1 (0-4) 1 (0-4) 1 (0-4)

Previous caesarean sections 1 (0-3) 1 (0-2) 1 (0-2)

Maximum sensory block T4 [C5-T6] T4 [T1-T8] T4 [T1-T6]

Time to T4 (min) 6.3 ± 2.0 6.4 ± 1.7 6.7 ± 2.3

Time from incision to delivery

(min)

9.8 ± 3.7 7.3 ± 3.0 8.7 ± 4.0

Duration of surgery (min) 45.3 ± 12.5 46.5 ± 15.4 50.6 ± 12.8

Lowest mean arterial pressure

(mmHg)

68.5 ± 10.6 65.7 ± 9.7 65.8 ± 11.4

Number requiring vasopressor 13 14 12

Values mean ± SD, median [range] or number (range). No significant differences

between groups.

Chapter 2.2

76

Table 2. Maternal pulmonary function tests before and after spinal anaesthesia

FVC (L) FEV1 (L)

before after before after

Bupivacaine 3.6 ± 0.5 3.5 ± 0.4* 2.9 ± 0.5 2.8 ± 0.5

Ropivacaine 3.2 ± 0.4 3.1 ± 0.5* 2.6 ± 0.4 2.5 ± 0.5

Levobupivacaine 3.6 ± 0.5 3.4 ± 0.6 2.8 ± 0.4 2.7 ± 0.5

Block lower than T4 3.3 ± 0.6 2.6 ± 0.5

Block higher than T4 3.4 ± 0.4 2.8 ± 0.4

PEFR (L/s)

before after

Bupivacaine 6.2 ± 1.2 3.5 ± 0.4*

Ropivacaine 5.5 ± 1.5 3.1 ± 0.5*

Levobupivacaine 6.0 ± 1.1 3.4 ± 0.6

Block lower than T4 3.3 ± 0.6

Block higher than T4 3.4 ± 0.4

*significantly different from before: P <0.05 by t test, NS by ANOVA

** significantly different from before: P <0.01 by t test, NS by ANOVA


77

Table 3. Neonatal outcome

Bupivacaine Ropivacaine Levobupivacaine

Apgar scores 1/5/10

min

9/10/10 9/9/10 9/9/10

UA pH 7.3 ± 0.1 7.3 ± 0.0 7.3 ± 0.1

Data are median or mean ± SD

Failed epidural – causes and management

79

Chapter 2.3


Based on

Hermanides J, Hollmann MW, Stevens MF, Lirk P

Failed epidural: causes and management

Br J Anaesth 2012; 109: 144-54

Chapter 2.3

80

Introduction

In contrast to the subjective experience of many anaesthesiologists,

failure of epidural anaesthesia and analgesia is a frequent clinical problem

(feedback error). Current estimates concerning the incidence of failed epidurals

are hampered by lack of a uniform outcome parameter. The definitions given

cover a spectrum ranging from insufficient analgesia to catheter dislodgement to

any reason for early discontinuation of epidural analgesia (Table 1). In a

heterogeneous cohort of 2140 surgical patients, failure rates of 32% for the

thoracic, and 27% for the lumbar epidural were described.1 Of note, active

management of insufficient epidural anaesthesia, including a new block, results

in an almost complete success rate.2 In an imaging study investigating failed

epidurals, incorrect catheter localization accounted for half of the failures, while

the remaining patients experienced suboptimal analgesia through a correctly

positioned catheter.3 A flow chart adapted from Kinsella et al. illustrates in

exemplary fashion the problems encountered during epidural anaesthesia using

the example of a caesarean section, ultimately resulting in a success rate of just

76% (Figure 1).

The present review summarizes technical factors known to influence

block success, and gives an overview of the pharmacologic strategies available to

optimise epidural anaesthesia and analgesia. For each section, we performed a

comprehensive literature search for full published reports in MEDLINE covering

manuscripts until October 2011, with reference lists of retrieved articles searched

for additional trials or reports. We ranked meta-analyses and randomized

controlled trials highest, with other trials and reports resorted to in case no broad

evidence base could be discerned.


81

Technical factors influencing block success

Anatomical catheter location

Epidural catheters may primarily be placed erroneously, or dislodge

during the course of treatment. Collier described transforaminal migration of the

catheter tip and asymmetric epidural spread as the most important caveats during

epidural analgesia.4 Primary malposition of epidural catheters has been

described, among others, in the paravertebral space, in the pleural cavity, and

intravascularly. Even when the epidural space is correctly identified, the catheter

will not necessarily follow a straight line when being advanced, but may deviate

in different directions. The epidural catheter may exit the epidural space via the

intervertebral foramen at levels above or below the insertion site (Figure 2). In

obstetric patients, failure of epidural analgesia after initial success was observed

in 6.8% of a studied cohort.2 Furthermore, secondary migration of the catheter

after successful initial placement can occur. During normal patient movement,

epidural catheters may be displaced by centimetres.5 Hoshi et al investigated 60

patients undergoing lung surgery with a thoracic epidural, with chest radiographs

taken pre- and postoperatively. In 24% of the patients, the catheter had migrated

more than one vertebral level. In addition to gross body movements, changes in

epidural pressure and cerebrospinal fluid oscillations contribute to displacement

of epidural catheters.6 The epidural space is highly compartmentalized and

complex structure,7 which may influence catheter placement. Moreover, midline

fat pedicles may form a barrier to spread of local anaesthetics.7

Patient position

Patient positioning potentially affects needle placement by changing the

conformation of osseous and soft tissues. In addition to the obvious opening of

the posterior interlaminar space by spinal flexion, the position of spinal contents

is altered. The position of the spinal cord within the spinal canal is not precisely

Chapter 2.3

82

predictable using parameters such as sex, weight or height. However, the patient

assuming a flexed position with the head down will result in anterior motion of

the spinal cord at the thoracic level, while the spinal cord and cauda equina are

located more posteriorly at the lumbar level.8 The spinal cord is flexibly attached

within the dural sac, and changes position according to gravity when subjects are

positioned supine, or laterally.9

The sitting position has been described to result in shorter insertion

times and a trend toward higher accuracy at first attempt than the lateral position,

but at the cost of more vagal reflexes, and with comparable final success rates.10

When investigating combined spinal-epidural anaesthesia for caesarean section,

no differences were reported concerning insertion times,11

while another study

found more technical difficulties in the lateral compared to the sitting position.12

Lateral positioning increases the distance from skin to epidural space.13

Finally,

the sitting position leads to epidural venous plexus distension,14

which may

increase risk of vascular puncture, especially in parturients.15

Puncture site

Numerous studies have shown that anaesthetists tend to be inaccurate

when determining the precise dermatomal level for neuraxial puncture.e.g.,16

Of

note, most studies show that there is a clear tendency to puncture more cranially

than intended. Suggested approximate vertebral levels of puncture for various

types of surgery are given in Table 2.

Midline vs Paramedian

Few studies have examined the effect of median versus paramedian

needle placement upon block success. In cadavers, using epiduroscopy,

paramedian catheters were observed to cause less epidural tenting, and pass

cephalad more reliably than median catheters.17

In patients, Leeda et al. reported


83

faster catheter insertion times in the paramedian, and higher incidence of

paraesthesia in the median group.18

Adequate local infiltration is a prerequisite

for patient comfort during paramedian puncture.19 20

Finally, the paramedian

approach may be less dependent upon spine flexion.20

The risk of vascular

puncture during epidural catheter placement was not associated with lumbar

median or paramedian technique in parturients,19

while another study suggested

more paraesthesia and bloody puncture in non-pregnant adults when the median

approach was used.20

Localization of the epidural space

Inability to correctly insert an epidural catheter in the first place or the

number of attempts to catheter placement is not reported in most studies, while

differences are likely to exist between the thoracic vs. lumbar approach. For

example, Rigg and coworkers were completely unable to localize the thoracic

epidural space in 13/447 (2.9%) attempts.21

Placement of the epidural catheter in the correct position necessitates

tools to identify the epidural space. There is considerable variation in the

methods used to confirm epidural needle position.22

Loss of Resistance (LoR)

using saline has become the most widely used method, while LoR to air and the

hanging drop technique are less widely used.e.g.,22

A meta-analysis by Schier and

colleagues in 2009 included 5 RCTs comparing LoR to saline versus air: four in

the obstetric population and one in a general patient population, summarizing a

total of 4422 patients. No significant difference in any outcome was found,

except an absolute 1.5% reduction in postdural puncture headache when using

saline vs. air.23

In the meantime, a clinical trial comparing combined spinal-

epidural punctures using air vs. saline found no difference in success rate or

adverse events.24

A recent retrospective study of 929 obstetric epidurals found

that when using air for LoR, significantly more attempts were needed as

Chapter 2.3

84

compared to using saline, with comparable final success rates.25

Subgroup

analyses showed that the use of the “preferred technique” (defined as the

technique used by a practitioner >70% of the time) resulted in significantly fewer

attempts, a lower incidence of paresthesia, and fewer unintentional dural

punctures, irrespective of whether saline or air was used for LoR.25

The hanging drop technique depends on negative pressure within the

epidural space. Recent experimental evidence suggests that negative pressure is

suboptimal in reliably detecting the epidural space, and if at all, the hanging drop

technique is prudent only in the sitting position.26

Of note, identification of the

epidural space was reported 2 mm deeper for the hanging drop as compared to

LoR, possibly indicating increased risk of dural perforation.27

Finally, whatever technique is used, it is of importance to realise that the

ligamentum flavum is not continuous in all patients, and the presence of midline

gaps may make the loss of resistance to needle advancement and injection of

air/saline less perceptible when the median approach is used.28

A number of technical aids for epidural anaesthesia have been

described, none of them exhibiting sufficient accuracy and practicability to as yet

justify the increased effort and cost of their routine use in adults. Ultrasound may

serve as an educational tool and enhance the learning curve for epidural

anaesthesia,29

and pre-assessment of lumbar epidural space depth has been

shown to correlate well with actual puncture depth in obese parturients.30

In

children, ultrasound allows for identification of neuraxial structures, particularly

in neonates. Below an age of 3 months only the vertebral bodies are ossified,

enabling detailed visualization of spinal structures. At age 3 months or older, the

vertebral column further ossifies, leading to decreased visibility. At

approximately age 7 years, visibility of the neuraxial structures, especially the

thoracic segments, is significantly reduced and comparable with that of young

adults.31

Despite apparently obvious advantages of ultrasound-guided epidural


85

anaesthesia in children, only one randomized controlled trial has been conducted

to date. Willschke et al. compared ultrasound to LOR to identify the epidural

space. Use of ultrasound led to less bony contact, a shorter time to block success,

and decreased supplemental opioid requirements.32

Recently, visualization of

epidural spread of local anaesthetic has been used to predict optimal individual

epidural dose.e.g.,33

Catheter insertion and fixation

The catheter should be inserted at least 4 cm into the epidural space,5

and a recent study reported a higher success rate when slightly more than 5 cm

were achieved.34

Tunnelling the epidural catheter for 5 cm in a cohort of 82

patients was associated with less motion of the catheter, but the percentage of

catheters maintaining original position was not statistically different.35

In more

than 200 patients undergoing either thoracic or lumbar epidural anaesthesia,

tunnelling led to significantly decreased catheter migration, with a modest

clinical net result of 83% of functioning catheters after 3 days, as compared to

67% without tunnelling.36

Suturing of the epidural catheter was similarly

associated with less migration, but at the cost of increased inflammation at the

puncture site.37

Whereas erythema at the puncture site was not associated with

bacterial colonization in small-scale studies,38

one larger study described a

positive correlation.39

In a retrospective observational study involving more than

500 children, tunnelling a caudal epidural catheter reduced risk of bacterial

colonization to levels comparable to untunneled lumbar catheters.39

These results

may be related to the fact that tunnelling places the catheter entry point above the

diaper in babies and toddlers and may not be easily transferred to an adolescent

population undergoing lumbar or thoracic epidural anaesthesia. It seems prudent,

however, to consider tunnelling caudal epidural catheters in babies and toddlers.

In lumbar and epidural catheters, the advantages are less straightforward and the

Chapter 2.3

86

necessity to prevent dislodgement (often dependent on type of surgery) needs to

be weighed against the increased incidence of erythema at the puncture site,

potentially linked to increased risk of bacterial colonization. Catheter fixation

devices are available which may significantly reduce migration percentage and

reduce rates of analgesic failure.e.g.,40

Unfortunately there are no studies

comparing modern dressing devices with tunnelling techniques with respect to

migration, analgesic failure, or infection.

Test dose

The optimal way to pharmacologically determine position of the

epidural catheter has been debated. When administering a test dose, the two main

objectives are to detect intrathecal and intravascular catheter placement. The

optimal strategy to detect intrathecal catheter placement was long considered to

be lidocaine coupled with epinephrine. Specific regimens to detect intravascular

catheter position have been advocated for non-pregnant adult patients (fixed

epinephrine test dose), parturients (fentanyl test dose), and children (weight-

adjusted epinephrine test dose).41

It should be kept in mind that a non-significant

increase in heart rate (<15%) does not guarantee correct position. Furthermore,

patients sensitive to intravascular epinephrine (parturients, patients with cardiac

or vascular disease) may experience undesired effects in case the test dose is

“positive”. However, this risk is most likely outweighed by the deleterious

effects of local anaesthetic intoxication should intravascular placement not be

detected. Test doses consisting of lidocaine (to detect intrathecal placement) and

epinephrine (to detect intravascular placement) are recommended in patients

without contraindications to epinephrine.


87

Material

Problems with the material itself may be responsible for epidural failure.

The orifice of the catheter can be in the lateral or anterior epidural space, thereby

leaking the local anaesthetic preferably to one side and producing an unilateral

block.42

In general, multi-orifice catheters are considered superior to single-

orifice catheters.e.g.,43

Manufacturer’s errors may occur, such as faulty markings

on the epidural catheter. This can lead to erroneous depth of catheter

placement.44

Debridement in the catheter or disconnection may similarly cause

epidural failure.4 One important preventable cause for obstruction of the epidural

infusion system is air lock in the bacterial filter. Depending on the type, as little

as 0.3 to 0.7 ml of air is sufficient to cause obstruction.45

Knotting of the catheter inside or outside the body can cause

obstruction. Only 13% of lumbar catheters inserted in a group of 45 men were

advanced more than 4 cm without coiling, with coiling occurring at a mean

insertion depth of 2.8 cm.46

The frequency of knotting catheters is estimated to

be 1:20.00-30.000 epidurals.47

Based on 18 case reports, Brichant et al.

concluded that 87% of the knots occurred less than 3 cm from the tip of the

catheter and that 28% of the knots were associated with a loop in the catheter.47

Removal of a presumably knotted catheter can be attempted after sensitivity has

returned to monitor for neurological symptoms during catheter removal. When

radicular symptoms or pain occur during removal of a catheter, this should be

immediately stopped.4 It has been suggested that removal is easiest if position at

insertion and removal are similar.4 Surgical removal of a broken catheter is not

obligatory if the patient remains asymptomatic.47

Chapter 2.3

88

Pharmacologic optimisation of epidural anaesthesia

Local anaesthetic dose versus volume

The influence of dose, concentration and volume upon spread of

epidural anaesthesia and analgesia has been subject of considerable research, and

different constellations of volume and concentration have been assessed. In

general, the main determinant of epidural action is the local anaesthetic dose,

with volume playing a minor role. Thus, quality of epidural analgesia depends on

total local anaesthetic dose rather than volume or concentration, either in

conventional or patient-controlled epidural analgesia. Although there seems to be

a tendency towards more extended sensory block and lower blood pressures with

lower concentrations at higher volume 48 49

and one study even found a higher

rate of PONV,50

most studies did not detect such increased side effects 51-56

The situation may be different when local anaesthetic solutions are

applied as bolus. There is evidence supporting the role of volume in spread of

anaesthesia. For example, the number of dermatomes blocked during labour

analgesia was higher in a high-volume bupivacaine group than a low-volume

group when the same dose was administered.57

But again, the evidence is

equivocal. Sakura and coworkers found that the spread of lumbar epidural

anaesthesia for gynaecological surgery was similar whether 20 ml 1% lidocaine,

or 10 ml 2% lidocaine were used. However, the intensity of block was higher in

the lido 2% group.58

If the difference between injected volumes differs by more

than 200% for the same concentration, the block will spread further in the high-

volume group.59

For bolus application there is evidence that reducing dose

increases probability of differential blockade. In healthy volunteers, dose-

dependency of differential blockade was demonstrated at 0.075 and 0.125%

bupivacaine.60

Higher bupivacaine concentrations caused motor block. It should

further be kept in mind that differential blockade is a complex phenomenon, in


89

part caused by differential conduction block of spinal nerves and roots, and in

part by differential central somatosensory integration.61

Dose is the primary

determinant of epidural anaesthesia, with volume and concentration playing a

subordinate role during continuous or patient-controlled epidural anaesthesia

(PCEA) application. The effect of volume is more pronounced during bolus

application.

Motor block may be more extensive when performing lumbar epidural

anaesthesia because of the spatial proximity of motor fibers. This was recently

confirmed in audit form.34

In labour, low-dose epidural analgesia may be

associated with less operative vaginal deliveries.62

The use of smaller doses in

higher volumes has therefore been advocated for obstetric analgesia.63

Choice of local anaesthetic

The three main long-acting local anaesthetics for epidural anaesthesia

and analgesia are bupivacaine, levobupivacaine, and ropivacaine. The prospect

of improved differential blockade, and concerns regarding cardiac safety have

driven the increased use of the newer L-stereoisomers. The equipotency of these

three drugs has been the subject of many clinical studies. For example, equal

concentrations and dosing of bupivacaine and ropivacaine (0.125%, with

fentanyl 2mcg/ml) lead to equal efficacy concerning analgesia, but significantly

less motor block in the ropivacaine group.64

However, comparison of equal doses

of, e.g., bupivacaine and ropivacaine is difficult as the difference in potency is

approximately 40-50%.65

This has profound consequences because to interpret

differential toxicity, this difference in potency needs to be taken into account. If

one translates potency difference to the toxic threshold of local anaesthetic

causing convulsions in animal models,65

near equipotency of bupivacaine and

ropivacaine concerning toxicity becomes apparent. The likelihood of successful

resuscitation after local anaesthetic intoxication has been described as lower

Chapter 2.3

90

when bupivacaine is the causative agent based upon receptor binding.66

However, the therapy advocated to support resuscitation of patients intoxicated

with local anaesthetics, intralipid, may be more beneficial in bupivacaine- than in

ropivacaine-induced toxicity owing to the lipophilic properties of bupivacaine.67

There is no evidence to refute bupivacaine in favour of the newer stereoisomers

when used for epidural anaesthesia or analgesia in adults. Based upon

pharmacologic data, switching local anaesthetics is not likely to improve epidural

anaesthesia.

Addition of opiates

The addition of small doses of opiate allows for dose reduction of local

anaesthetics while improving quality of analgesia. The vast majority of studies

support the use of a combination of local anaesthetic and opioid over either drug

alone.68

In a 1998 meta-analysis, Curatolo et al. showed that epidural fentanyl

was a beneficial adjuvant to local anaesthetics administered for surgical

analgesia, improving pain therapy with a low incidence of nausea, and rare

occurrence of pruritus.69

The addition of opiates allows for smaller

concentrations of local anaesthetic, thereby possibly reducing motor block

postoperatively, or during labour. 70

In fact, it has been stated that the entire

concept of low-dose local anaesthetics for analgesia is feasible only when

opioids are used as adjunct therapeutics.71

Moreover, recent data suggest that

epidural opioids can enhance the quality of suppression of the surgical stress

response.72

Profound differences exist between hydrophilic opioids (such as

morphine) and lipophilic opioids (such as fentanyl and sufentanil). Microdialysis

studies demonstrate that epidural morphine has a longer residence time in the

epidural space, and results in higher CSF concentrations as compared to

sufentanil and fentanyl.73

This long residence time results in a spinal mechanism


91

of action, and consequently, clinical studies show a substantial dose reduction in

morphine when used epidurally instead of intravenously.74

Corresponding

evidence for lipophilic opioids such as fentanyl and sufentanil, however, is more

conflicting. While some studies show a clear benefit of adding epidural fentanyl

to bupivacaine,75

others suggest that effects of epidural fentanyl are primarily

mediated by supraspinal mechanisms after systemic absorption.76

In a recent

study undertaken in healthy volunteers, differences were observed between

continuous and bolus infusion. While continuous infusion resulted in non-

segmental analgesia (indicating supraspinal action), bolus injection resulted in

segmental analgesia (indicating significant spinal contribution).75

Therefore, the

elicitation of a spinal analgesic mechanism may depend on sufficient

concentrations of fentanyl in the epidural space to allow for diffusion into the

CSF, i.e., estimated above 10 mcg/ml, which exceeds current postoperative

analgesia regimens.77

Finally, some potential disadvantages of epidural opioid administration

should be discussed. First, the safety of opioids in obstetric analgesia has been

subject of discussion. Potential disadvantages of epidural opioid application

include possible interference with breast-feeding,78

but a recent randomized

controlled trial found no effect of epidural fentanyl on breastfeeding initiation

and duration.79

Second, biphasic respiratory depression may occur when

hydrophilic opioids are given epidurally. With hydrophilic opioids such as

morphine, the first peak corresponds to absorption from the epidural space into

the systemic circulation and occurs 30-90 minutes after injection, while a second

depression occurs 6-18 hours later as morphine spreads towards the brainstem. In

lipophilic opioids, there is only an early depression due to absorption and rostral

spread.80

Chapter 2.3

92

Addition of epinephrine

The addition of adrenaline to epidural solutions causes two desired

effects. First, vasoconstriction causes delayed absorption of local anaesthetic into

the systemic circulation, with higher effect-site and lower plasma concentrations.

Second, adrenaline has specific antinociceptive properties predominantly

mediated via alpha-2 adrenoreceptors. Effects of epinephrine with regards to

local anaesthetics and opiates are supra-additive. For example, the MLAC of

bupivacaine is reduced by 29% in labouring parturients.81

Adding epinephrine to

a low-dose thoracic epidural infusion of ropivacaine and fentanyl improved pain

relief and reduced nausea.82

Vasoconstriction plays a key role in the supra-additive effect of

adrenaline during epidural analgesia. Amide-type local anaesthetics are not

metabolized in the epidural space, liver, such that the main determinant for their

concentration there is absorption into the systemic circulation and subsequent

hepatic metabolism. This absorption is biphasic, with an initial fast peak

reflecting the fluid phase and later a slower second peak corresponding to

resorption from the lipid compartment at the site of injection.83

Addition of

epinephrine to local anaesthetic solutions slows down the first phase of systemic

absorption.84

The net clinical effect is a more profound block, or sufficient block

already at lower LA dose. The same mechanism seems to be valid for opioids.85

Further, epidural epinephrine has a specific alpha-2-mediated

antinociceptive effect causing decreased presynaptic transmitter release and

postsynaptic hyperpolarization within the substantia gelatinosa of the spinal cord

dorsal horn.86

Therefore, the full effect is only observed when the epidural

catheter is positioned within the vicinity of the spinal cord, i.e. above L1.

Lumbar catheters necessitate higher concentrations of local anaesthetic and

opioid, and here, adding epinephrine may increase the possibility of motor

block.86

Studies suggest a concentration of 1.5-2 µg/ml as effective.87


93

Finally, some potential risks of adding epinephrine should briefly be

discussed. Potential side effects have been described in obstetrics, the main

concern that adrenaline-containing solutions potentially cause longer labor, and

decreased uterine blood flow.88

At doses usually used clinically, spinal cord

ischemia seems to be no clinically significant problem.89

Bolus versus continuous dosing

The advent of PCEA has profoundly changed the administration of

postoperative pain treatment. In labour analgesia, a meta-analysis by van der

Vyver et al. demonstrated that patients undergoing obstetric PCEA needed less

co-analgesic interventions, while requiring less local anaesthetic, and potentially

having a decreased likelihood of motor block, albeit without a difference in

maternal satisfaction and no effect upon mode of delivery.90

Using pain scores

and cumulative local anaesthetic dose as outcome parameters, conflicting

evidence has been put forward to prove or refute background infusions. It is

important to keep in mind that PCEA requirement are determined by the site of

surgery, and surgery for malignant disease, as well as patient weight and age

seem to be the most important predictors.91

The addition of a continuous infusion

to PCEA during labour resulted in reduced total dose of local anaesthetic while

providing effective analgesia.92

A reduction in local anaesthetic dose was found

only in demand-only PCEA, but not in the group with background infusion by

Vallejo, who found similar outcomes in all groups.93

Recently, Lim and

colleagues showed that demand-only PCEA did result in less LA administered,

but also in more breakthrough pain, higher pain scores, and lower maternal

satisfaction during labour.94

More refined techniques such as programmed

intermittent epidural bolus (PIEB) combined with PCEA have shown potential

for even more accurate analgesia.95

Chapter 2.3

94

Conclusion

Failure of epidural anaesthesia and analgesia occurs in up to 30% in

clinical practice. Some technical factors can help to increase primary and

secondary success rate. Epidural catheters may be erroneously placed, or may

migrate secondarily after initial correct placement due to body movement and

oscillations in cerebrospinal fluid. Moreover, catheters may deviate from the

midline during insertion. The optimal depth of insertion in adults is

approximately 5 cm. The most widely used method with the least side effects for

localizing the epidural space is loss of resistance to saline. None of the additional

technical tools available has sufficient accuracy and predictability to justify

routine use, with the exception of a growing evidence-base for ultrasound in

obese patients and infants. The optimal test dose should combine lidocaine (to

detect intrathecal placement) and epinephrine (to detect intravascular placement)

in the absence of contraindictations to epinephrine. In the case of catheter

knotting, direct retrieval of the catheter should be attempted once anaesthesia has

worn off, while surgical intervention is rarely indicated. The choice of long-

acting local anaesthetic seems to be less important clinically. Dose is the primary

determinant of continuous epidural anaesthesia, with volume and concentration

playing a subordinate role. Addition of opiates may substantially increase the

effectiveness of epidural analgesia. Epinephrine strengthens analgesia by

delaying resorption of local anaesthetic from the epidural space, and by direct

antinociceptive action at the spinal cord. The method most supported by

literature for postoperative analgesia is patient controlled epidural analgesia with

background infusion.

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56 Snijdelaar DG, Hasenbos MA, van Egmond J, Wolff AP, Liem TH. High

thoracic epidural sufentanil with bupivacaine: continuous infusion of high

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58 Sakura S, Sumi M, Kushizaki H, Saito Y, Kosaka Y. Concentration of

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60 Brennum J, Nielsen PT, Horn A, Arendt-Nielsen L, Secher NH. Quantitative

sensory examination of epidural anaesthesia and analgesia in man; dose-response

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Quantitative sensory examination of epidural anaesthesia and analgesia in man:

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62 Effect of low-dose mobile versus traditional epidural techniques on mode of

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63 Olofsson C, Ekblom A, Ekman-Ordeberg G, Irestedt L. Obstetric outcome

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0.125% with sufentanil--a prospective randomized controlled study in 1000

parturients. Acta Anaesthesiol Scand 1998; 42: 284-92

64 Meister GC, D'Angelo R, Owen M, Nelson KE, Gaver R. A comparison of

epidural analgesia with 0.125% ropivacaine with fentanyl versus 0.125%

bupivacaine with fentanyl during labor. Anesth Analg 2000; 90: 632-7

65 Casati A, Putzu M. Bupivacaine, levobupivacaine and ropivacaine: are they

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66 Guinet P, Estebe JP, Ratajczak-Enselme M, et al. Electrocardiographic and

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68 Wheatley RG, Schug SA, Watson D. Safety and efficacy of postoperative

epidural analgesia. Br J Anaesth 2001; 87: 47-61

69 Curatolo M, Petersen-Felix S, Scaramozzino P, Zbinden AM. Epidural

fentanyl, adrenaline and clonidine as adjuvants to local anaesthetics for surgical

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70 Boulier V, Gomis P, Lautner C, Visseaux H, Palot M, Malinovsky JM.

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74 Negre I, Gueneron JP, Jamali SJ, Monin S, Ecoffey C. Preoperative analgesia

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76 Guinard JP, Mavrocordatos P, Chiolero R, Carpenter RL. A randomized

comparison of intravenous versus lumbar and thoracic epidural fentanyl for

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77 George MJ. The site of action of epidurally administered opioids and its

relevance to postoperative pain management. Anaesthesia 2006; 61: 659-64

78 Beilin Y, Bodian CA, Weiser J, et al. Effect of labor epidural analgesia with

and without fentanyl on infant breast-feeding: a prospective, randomized,

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82 Niemi G, Breivik H. Epinephrine markedly improves thoracic epidural

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after cesarean section: buprenorphine-0.015% bupivacaine with epinephrine

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86 Niemi G. Advantages and disadvantages of adrenaline in regional

anaesthesia. Best Pract Res Clin Anaesthesiol 2005; 19: 229-45

87 Niemi G, Breivik H. The minimally effective concentration of adrenaline in a

low-concentration thoracic epidural analgesic infusion of bupivacaine, fentanyl

Chapter 2.3

104

and adrenaline after major surgery. A randomized, double-blind, dose-finding

study. Acta Anaesthesiol Scand 2003; 47: 439-50

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or without epinephrine during epidural labor analgesia. Anesth Analg 2006; 103:


89 Neal JM. Effects of epinephrine in local anesthetics on the central and

peripheral nervous systems: Neurotoxicity and neural blood flow. Reg Anesth

Pain Med 2003; 28: 124-34

90 van der Vyver M, Halpern S, Joseph G. Patient-controlled epidural analgesia

versus continuous infusion for labour analgesia: a meta-analysis. Br J Anaesth

2002; 89: 459-65

91 Chang KY, Dai CY, Ger LP, et al. Determinants of patient-controlled

epidural analgesia requirements: a prospective analysis of 1753 patients. Clin J

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labour: the effects of adding a continuous epidural infusion. Anaesth Intensive

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continuous infusion versus patient-controlled epidural analgesia with background

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105

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parturients: a retrospective chart review. Int J Obstet Anesth 1998; 7: 220-5

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pain relief using thoracic epidural analgesia: outstanding success and

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Gastrointest Surg 2008; 12: 1207-20

99 Kinsella SM. A prospective audit of regional anaesthesia failure in 5080

Caesarean sections. Anaesthesia 2008; 63: 822-32

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bupivacaine plus sufentanil: high concentration/low volume versus low

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101 Sitsen E, van Poorten F, van Alphen W, Rose L, Dahan A, Stienstra R.

Postoperative epidural analgesia after total knee arthroplasty with sufentanil 1

microg/ml combined with ropivacaine 0.2%, ropivacaine 0.125%, or

levobupivacaine 0.125%: a randomized, double-blind comparison. Reg Anesth

Pain Med 2007; 32: 475-80

Chapter 2.3

106

Table 1 – Definitions and rates of failed epidural anaesthesia or analgesia

Type of surgery

Type of epidural

Definition Failure rate

Eappen et al. 96

IJOA 1998

Obstetric

Lumbar

Any reason for

intervention

550/4240

(13.1%)

Ready et al. 1

RAPM 1999

General surgery

Thoracic /

Lumbar

Technical defects

/ Insufficient

block

N=2140

thoracic (32%)

lumbar (27%)

McCleod et al. 97

Anaesthesia 2001

Major abdominal

surgery

Thoracic

Technical defects

/ Insufficient

block

83/640

(13.0%)

Rigg et al. 21

Lancet 2002

Gastrointestinal

surgery

Thoracic /

Lumbar

Failed insertion /

Removed early

203/431

(47.1%)

Neal et al. 89

RAPM 2003

Esophagectomy

Thoracic

Catheter

dislodgement

8/46

(14.2%)

Pan et al. 2

IJOA 2004

Obstetric

Lumbar

Inadequate

analgesia, spinal

tap

1099/7849

(14%)

Motamed et al. 3

A&A 2006

Abdominal

oncology

Thoracic

Failed block,

premature

removal of

catheter

31/125

(24.8%)

Pratt et al. 98

J GI Surg 2008

Whipple

Thoracic

Failed block,

premature

catheter removal

49/158

(31.0%)


107

Kinsella et al. 99

Aneasthesia,

2008

C-section

Lumbar

Inadequate

anesthesia

302/1286

(23.5%)

Königsrainer 34

Anaesthesia 2009

Major surgery

Thoracic /

Lumbar

Insufficient

analgesia, motor

block, catheter

dislodgement

124/300

(41.4%)

Table 2 - Landmarks for epidural anesthesia and analgesia

Desired dermatome level of neuraxial block

Type of

surgery

Upper dermatomal

block level

Anatomic

landmark

Optimal insertion

point

Esophagus,

lung

T1 Below clavicle T6-7

Upper

abdomen

T1 Below clavicle T9-10

Lower

abdomen

T6 Distal sternum T9-10

Cesarean

delivery

T4 Nipples L4-5

Lower

extremity

L1-2 Inguinal crease L4-5

109

Section 3

Regional anesthesia and risk of nerve damage:

influence of pre-existing neuropathy

Needle trauma and intraneural injection

111

Chapter 3.1

Effects of needle trauma and intraneural injection in

vivo

Based on

Kirchmair L, Loescher W, Voelckel W, Lirk P

Short-term neurophysiologic and morphologic effects of experimental needle

trauma and intraneural injection.

Br J Anaesth 2014; (in revision)

Chapter 3.1

112

Introduction

Peripheral nerve injury is a rare but severe complication after peripheral

regional anaesthesia.1 Several mechanisms are thought to contribute to transient

or even permanent neurologic deficit, such as local anaesthetic toxicity,

mechanical nerve damage, and ischemia.2 In the perioperative period, the

contribution of additional patient risk factors such as a pre-existing neuropathy is

controversially discussed.3 Substantial evidence supports the notion that injury to

a nerve because of needle trauma and subsequent intraneural injection is

harmful.2 On the other hand, some recent publications have suggested that

intraneural injection, if performed outside of individual nerve fascicles, is not

invariably deleterious 4 and may even be safe.

5 6

Previous investigations have been carried out, using

neurohistopathology and neurobehavioral testing as endpoints.7 8

No model has

been described which would allow for the detailed measurement of

electrophysiologic variables following nerve injury. We sought to describe a

large animal model of peripheral nerve blockade using the same equipment and

techniques as in clinical practice, to investigate functional effects of intraneural

needle placement and fluid injection.

The aim of this study was to test the hypothesis that intraneural fluid

injection, but not intraneural needle placement, would result in functional nerve

damage as evidenced by a decrease in compound motor action potential (cMAP)

amplitude.

Methods

Animals

The experimental study protocol was approved by the Animal Care and

Use Committee of the Austrian Federal Ministry of Science (GZ BMBWK-

66.011/0065-BrGT/2006). We tested 15 healthy research pigs, aged 12 to 16


113

weeks and weighing 22 ± 2 kgs. Animals were fasted overnight with free access

to water, and premedicated with azaperone 4 mg kg-1

i.m. and atropine 0.1 mg

kg-1

i.m.. After a bolus of ketamine 20 mg kg-1

i.m., peripheral venous access

was established. Anaesthesia was induced and maintained using propofol 2-3 mg

kg-1

, followed by 1.5 mg kg-1

h-1

. The trachea was intubated in the spontaneously

breathing supine animal. After intubation, animals were given piritramide 15 mg

i.v., repeated as necessary. Mechanical ventilation was initiated with an

inspiratory oxygen fraction of 35%, positive end-expiratory pressure of 5 cm

H2O, a respiratory rate of 20 breaths per minute, with tidal volume adjusted to

maintain normocapnia. In parallel, lactated ringer’s solution 6 ml kg-1

h-1

and

gelatine solution 3% 4 ml kg-1

h-1

were infused. Monitoring included a standard

two-lead electrocardiograph, pulse oximetry, and invasive blood pressure

monitoring. All animals were euthanized by means of propofol 3 mg kg-1

,

piritramid 30 mg, and potassium chloride 20 ml to induce cardiac arrest.

Experimental procedures

Animals were randomly assigned to one of four groups before baseline

measurements. In Group A (n=5), the sciatic nerve was pierced under ultrasound

guidance (needle trauma) and the needle was immediately retracted. In Group B

(n=6), 2.5 ml of saline were injected into the sciatic nerve, while in Group C

(n=6), 5 ml of saline were injected intraneurally. Using ultrasound, intraneural

injections were performed extrafascicularly. In control Group D (n=5), 5 ml of

saline were injected around the sciatic nerve to simulate a peripheral nerve block.

We obtained electrophysiological recordings at baseline, immediately following

nerve injury with or without fluid injection, and after 30, 60, 120, and 180

minutes. The diameter of the sciatic nerve was recorded parallel to the

electrophysiologic measurements to calculate its cross-sectional area.

Chapter 3.1

114

Sciatic nerve interventions

Sciatic nerve interventions were performed using both ultrasound

guidance and nerve stimulation. The sciatic nerve was visualized in a short-axis

view using a portable ultrasound device dedicated to veterinary use (SonoSite

Titan, SonoSite Inc., Bothell, WA) and its diameter (length, width) was recorded.

The cross-sectional area of the latter was assumed elliptic and therefore

calculated with the formula π*1/4*length (mm)*width (mm). A needle (UniPlex

NanoLine, 20 gauge, facet tip with 45° bevel, 150mm, Pajunk, Geisingen,

Germany) was introduced in plane with the ultrasound probe, and advanced to

the nerve. Correct needle position was confirmed by means of a standard nerve

stimulator (Stimuplex HNS 11, B Braun, Melsungen, Germany) set to deliver a

stimulus of 0.3 mA at a frequency of 1 Hz to elicit plantar flexion. Subsequently,

interventions were carried out as described above. After all interventions a

surgical suture was introduced through the needle to mark the site of intervention

for later examination after excision.

Fluid injection

To apply fluid with comparable hydrostatic pressure, we employed a

perfusion pump (Perfusor fm, B Braun, Melsungen, Germany) connected to a

pressure transducer via an arterial tubing line (Monitoring Set, Medex Medical

Inc., Haslingden, Rossendale, Great Britain). We continuously measured

pressures within the perfusion syringe, within the fluid line, and proximal to the

needle (given in mmHg), and determined average and peak pressures. Volume

application was performed using the bolus function of the perfusion pump preset

to deliver 10 ml min-1

(0,167 ml sec-1

), resulting in 15 and 30 seconds to inject

2.5 and 5 ml, respectively. Real-time visualization was used in order to prove

intraneural injections by nerve expansion. Baseline injection pressure (syringe


115

and tubing) without tissue resistance was obtained in a separate experiment

(n=10).

Electrophysiology

Compound motor evoked potentials (cMAP): The sciatic nerve was

stimulated at the gluteal level using a stimulating needle-electrode (Medtronic

A/S, Skovluunde, Denmark) which was placed under ultrasound guidance to

avoid needle-nerve contact. A surface electrode (TECA Disposable Surface

Electrodes, Oxford Instruments, Medical Systems Division, Pleasantville, NY,

USA) served as anode and was placed in the groin area opposite to the

stimulating electrode. The cMAPs were recorded by means of two surface

electrodes placed over the gastrocnemius muscle and its insertion point,

respectively. A standard ECG-electrode (Skintact, Innsbruck, Austria) was used

as reference electrode. To determine optimal intensity of stimulating current, we

used the “inching” protocol of the Nicolet Viking IV device (Nicolet Biomedical,

Madison, WI, USA) at the baseline of each experiment. In this protocol, the

sciatic nerve was stimulated with a square wave pulse of 0.2 ms duration, and

increasing stimulating current until the amplitude of cMAPs over the

gastrocnemius muscle did not increase further. This current was increased by a

additional 20-50% to assure supra-maximal stimulation intensities throughout the

experiment. Amplitude (negative peak given in mV, reflecting axonal damage)

and distal motor latency (given in ms, and reflecting myelin damage) of cMAPs

were recorded as the average of five single measurements.

Macroscopic examination

After animals had been euthanized, sciatic nerves and adjacent tissues

were excised, taking care not to distort anatomy. The sciatic nerves were exposed

Chapter 3.1

116

beyond the previously marked site of intervention and their macroscopic

appearance was documented by means of digital imaging.

Power Calculation and Statistics

The sample size used would permit a detection of a difference in

primary outcome (cMAP amplitude) characterized by difference in means of

40%, assuming a common standard deviation of 20%, a Power of 80%, and

assuming statistical significance at P < 0.05.

Analysis of Variance with post hoc correction (LSD) was used to

compare cMAPs (amplitudes, latencies) and sciatic nerve cross sectional areas.

Normal distribution of sample values was checked for using the Kolmogorov-

Smirnov Test. SPSS for Windows v11.0 (Chicago, IL) was used for all analyses,

and differences were considered highly significant at P < 0.001.

Results

Injection pressures

Baseline injection pressure (syringe and tubing) without tissue

resistance was 63.6 ± 3 mmHg. Maximal pressures were not different between

the four experimental groups (Table 1). In two instances, maximal pressures of

up to 320 mmHg were reached in the control group, and sonographic control of

needle position showed a fascial layer obstructing fluid outflow.

Sonography

The cross-sectional area of the sciatic nerve as determined by ultrasound

did not change after nerve trauma and in the control group. After injection of 2.5

ml or 5 ml of saline, significant changes in nerve diameter were discernible

(Figures 2a, 2b). One sciatic nerve injected with 5 ml saline exhibited rupture of

the epineurium observed by means of real-time ultrasound imaging,


117

accompanied by a sudden decrease of injection pressure. In some other nerves,

we observed circumstantial evidence of the same event, with a sudden drop in

nerve cross-sectional diameter after presumed rupture of the nerve epineurium.

Electrophysiology

We determined both amplitude and latency of cMAPs. Amplitudes and

latencies were normalized to baseline measurements before needle trauma or

injection. Absolute and normalized amplitudes as well as latencies were

distributed normally. Decreases in amplitude were significant after needle trauma

(Group A) and injection of 2.5 ml intraneurally (Group B), and highly significant

after injection of 5 ml (Group C) (Figure 1). Amplitude did not change in Group

D (controls). Normalized amplitudes are summarized in table 2. Latency of nerve

conduction did not change over time in any group.

Macroscopy

The harvested nerves showed apparent signs of injury (haematoma,

swelling) after needle trauma (group A) (Figure 3) and injection of 2.5 ml and 5

ml (groups B and C), respectively. In the control group (group D) the nerves

showed no signs of injury.

Discussion

The aim of this study was to test the hypothesis that intraneural fluid

injection, but not intraneural needle placement, would result in functional nerve

damage as evidenced by a decrease in cMAP amplitude. We report that cMAP

amplitude as a marker of axonal injury decreased significantly following both

intraneural needle placement and intraneural fluid injection.

Chapter 3.1

118

Injection pressure

Measurement of maximal injection pressures showed that values were

not different among the experimental groups and below the threshold of 15 psi (=

776 mmHg), suggesting extrafascicular injection.11

This is in line with recent

evidence in human cadavers showing high injection pressures during injection

into brachial plexus nerve roots exceeding by far any pressures encountered

during the present investigation.9 The fact that we observed pathological changes

even after extrafascicular injection cannot be extrapolated directly to the clinical

situation, but would lead us to question the purported safety of intraneural

extrafascicular injection.5

Sonography

In our study, ultrasound was used to perform intentional needle trauma

and fluid injections into the sciatic nerve. Real-time imaging was applied to

assure intraneural injection by monitoring nerve swelling as a reliable sign of

true intraneural injection, be it subepineural or subperineural.10

Our findings

confirm and widen previous reports by Chan et al.11

in as much as we visualized

the injection of fluid in real-time using ultrasound. We found no changes in

nerve diameter after needle trauma or extraneural injection whereas following

intraneural injection, the diameter of affected nerves was increased over several

hours. This provides circumstantial evidence of a fluid depot remaining within

the nerve, potentially leading to compression of nerve fascicles. We demonstrate

a fluid reservoir within nerves after injection, which persisted over the entire

experimental timescale, i.e. 3 hours (Figure 2b). Mechanical compression is a

well-established cause of neuronal damage and a valid method of temporary

neurolysis, e.g. in trigeminal neuralgia.12

In contrast, the extraneural fluid

reservoir injected in control animals was significantly decreased in volume after

three hours.


119

Whereas needle trauma typically led to formation of small haematoma

without further macroscopic evidence of nerve damage, we found no

macroscopic evidence of injury in our control group. In analogy, sonographic

appearance of nerves following needle trauma alone revealed no abnormal

findings. We note that in a recent study, paraesthesia during needle placement as

potential indicator of needle-nerve contact or even trauma was associated with

new-onset neurological symptoms.13

It should be noted that in the present study,

nerve diameter was not used as surrogate marker of nerve damage, rather it was

used to verify correct localization of injectate. We quantified nerve damage on

basis of electrophysiological parameters.

Electrophysiology

Using electrophysiological monitoring, we demonstrate a decrease in

amplitude of cMAP after needle trauma and intraneural injection of either 2.5 or

5 ml saline, reflecting neuronal damage by direct axonal injury, and, potentially,

mechanical compression of neuronal structures by fluid reservoirs. In detail,

cMAP amplitude decreased by about 30% of baseline values after needle trauma,

and by up to 70% after intraneural injection. Based upon the injection pressures

observed in our trial, and real-time imaging using ultrasound, we surmise that

our injections were intraneural, but extrafascicular. The present results lead us to

conclude that any impact on the integrity of a nerve leads to functional

impairment. The latter appears less pronounced after needle trauma alone as

compared to additional intraneural injection. However, the observed reductions

in cMAP amplitudes do not allow us to predict the degree of long term injury.

Intraneural injection in clinical practice

The question how often nerve injury occurs in clinical practice has been

controversially discussed, and based upon study design and definition of

Chapter 3.1

120

endpoint, the reported incidences range up to 80 %.14

While some investigators

have shown that even paraesthesia is a risk factor for neuropathy following

regional anaesthesia,13

some smaller studies found no neurological sequelae of

intraneural injection.4 14 15

However, some of the latter studies performed

injection within the connective tissue sheath surrounding tibial and common

peroneal nerve, with no evidence of structural harm to the constituent nerves,

such that these results may not fit the classical description of “intraneural”

injection. Our findings support the hypothesis that nerve damage by needle

trauma or intraneural fluid injection leads, at least temporarily, to impairment of

nerve function, even if we cannot exclude the possibility of long-term recovery

of nerve function in our model.

Methodology and Limitations

The main methodological difference between our study and others 11 16 17

was that we used online recorded electrophysiologic measurements as surrogate

markers of nerve damage. Ultrasound guidance was used throughout all

interventions to preserve the complex physiological environment of peripheral

nerves and monitor intraneural injections. This design is in contrast to „open“

animal models of nerve damage used in some other studies.8 18

The injection

speed chosen for the present investigations (10 ml min-1

) can be considered low

when compared with clinical practice. Therefore, if anything, our study design

underestimated the pressures generated during intraneural injection, and the

damage caused by pressure.12

Finally, some limitations of the present study should be briefly

discussed. First, since our experiment was conceptualized as a large animal

study, we were limited in the observation of long-term effects of nerve injury. It

would have been of interest to observe the time-course of nerve trauma over a

longer period, since a final determination of neurological damage is only


121

considered feasible after 10-14 days.19

An additional method offering valuable

information is neurohistopathology after nerve trauma. Other authors have

described inflammatory changes on sciatic nerves even after regular nerve

stimulation,20

noting lymphocytes and granulocytes six hours after nerve block.

Also, we note that our diagnosis of intraneural and extrafascicular injection was

based on surrogate parameters such as injection pressure and spread of injectate

within the nerve. In future studies, neurohistopathology should be integrated to

confirm structural damage and site of injection.

Ethical considerations

To minimize the number of test animals, our experiments were

conducted as addendum to a study investigating the inhalative application of N-

chlortaurin in the porcine model. We sought to minimize animal distress by

conducting all invasive procedures such as electrophysiology and nerve block

under general anaesthesia. Experimental procedures, data collection and

presentation were in accordance to the ARRIVE guidelines.21

Conclusion

Attesting to the fact that our model was not designed to detect long-term

recovery of nerve function, we report signs of acute functional neuronal damage

after needle trauma and intraneural extrafascicular injection.

References


after regional anesthesia: contemporary estimates of risk. Anesth Analg 2007;

104: 965-74

2 Hogan QH. Pathophysiology of peripheral nerve injury during regional

anesthesia. Reg Anesth Pain Med 2008; 33: 435-41

Chapter 3.1

122

3 Lirk P, Birmingham B, Hogan Q. Regional anesthesia in patients with

preexisting neuropathy. Int Anesthesiol Clin 2011; 49: 144-65

4 Bigeleisen PE. Nerve puncture and apparent intraneural injection during

ultrasound-guided axillary block does not invariably result in neurologic injury.


5 Bigeleisen PE, Chelly J. An unsubstantiated condemnation of intraneural

injection. Reg Anesth Pain Med 2011; 36: 95

6 Bigeleisen PE, Moayeri N, Groen GJ. Extraneural versus intraneural

stimulation thresholds during ultrasound-guided supraclavicular block.


7 Macfarlane AJ, Bhatia A, Brull R. Needle to nerve proximity: what do the

animal studies tell us? Reg Anesth Pain Med 2011; 36: 290-302

8 Lupu CM, Kiehl TR, Chan VW, El-Beheiry H, Madden M, Brull R. Nerve

expansion seen on ultrasound predicts histologic but not functional nerve injury

after intraneural injection in pigs. Reg Anesth Pain Med 2010; 35: 132-9

9 Orebaugh SL, Mukalel JJ, Krediet AC, et al. Brachial plexus root injection in

a human cadaver model: injectate distribution and effects on the neuraxis. Reg


10 Altermatt FR, Cummings TJ, Auten KM, Baldwin MF, Belknap SW,

Reynolds JD. Ultrasonographic appearance of intraneural injections in the

porcine model. Reg Anesth Pain Med 2010; 35: 203-6

11 Chan VW, Brull R, McCartney CJ, Xu D, Abbas S, Shannon P. An

ultrasonographic and histological study of intraneural injection and electrical

stimulation in pigs. Anesth Analg 2007; 104: 1281-4, tables of contents

12 Cheshire WP. Trigeminal neuralgia: for one nerve a multitude of treatments.

Expert Rev Neurother 2007; 7: 1565-79


123

13 Fredrickson MJ, Kilfoyle DH. Neurological complication analysis of 1000

ultrasound guided peripheral nerve blocks for elective orthopaedic surgery: a

prospective study. Anaesthesia 2009; 64: 836-44

14 Robards C, Hadzic A, Somasundaram L, et al. Intraneural injection with low-

current stimulation during popliteal sciatic nerve block. Anesth Analg 2009; 109:

673-7

15 Sala-Blanch X, Lopez AM, Pomes J, Valls-Sole J, Garcia AI, Hadzic A. No

clinical or electrophysiologic evidence of nerve injury after intraneural injection

during sciatic popliteal block. Anesthesiology 2011; 115: 589-95

16 Selander D, Sjostrand J. Longitudinal spread of intraneurally injected local

anesthetics. An experimental study of the initial neural distribution following

intraneural injections. Acta Anaesthesiol Scand 1978; 22: 622-34

17 Hadzic A, Dilberovic F, Shah S, et al. Combination of intraneural injection

and high injection pressure leads to fascicular injury and neurologic deficits in

dogs. Reg Anesth Pain Med 2004; 29: 417-23

18 Kapur E, Vuckovic I, Dilberovic F, et al. Neurologic and histologic outcome

after intraneural injections of lidocaine in canine sciatic nerves. Acta


19 Sawyer RJ, Richmond MN, Hickey JD, Jarrratt JA. Peripheral nerve injuries

associated with anaesthesia. Anaesthesia 2000; 55: 980-91

20 Voelckel WG, Klima G, Krismer AC, et al. Signs of inflammation after

sciatic nerve block in pigs. Anesth Analg 2005; 101: 1844-6

21 Galley HF. Mice, men, and medicine. Br J Anaesth 2010; 105: 396-400

Chapter 3.1

124

Table 1

Group N Injection

Pressure

Min Max

Group B 6 164.5 ± 77.2 100 300

Group C 6 112.8 ± 23.6 90 145

Group D 5 203.2 ± 103.6 100 320

Injection pressures for Group B (injection of 2.5 mL intraneurally), Group C

(injection of 5 mL intraneurally), and Group D (injection of 5 mL extraneurally),

using a predefined injection speed of 10 ml min-1

. No injection was performed in

Group A (needle trauma). Injection pressures given as mean ± standard deviation

(mmHg), supplemented by minimal and maximal values for each group.

Table 2

Group A B C D

(control)

baseline 1 1 1 1

intervention 0.9 (0.2) 0.7 (0.3) 0.6 (0.3) 1 (0.2)

30min 0.8 (0.1) 0.7 (0.4) 0.7 (0.3) 0.9 (0.2)

60min 0.7 (0.2) 0.6 (0.3) 0.5 (0.2) 1 (0.2)

120min 0.6 (0.2) 0.5 (0.3) 0.5 (0.1) 0.9 (0.1)

180min 0.6 (0.2) 0.4 (0.3) 0.3 (0.1) 0.1 (0.1)

Normalized amplitudes (mean, SD in parentheses) of cMAPs at baseline (bl),

immediately after intervention (i), and follow-up measurements at 30, 60, 120,

and 180 minutes. Group A (needle trauma), group B (injection of 2.5 ml

intraneurally), group C (injection of 5 ml intraneurally), group D (injection of 5

ml extraneurally).


125

Figure 1

Compound motor evoked potentials (cMAPs) after intraneural injection of 5 ml

saline. Recordings were obtained at baseline (top), immediately following

intraneural injection, and after 30, 60, 120, and 180 minutes. The corresponding

amplitudes are given in mV.

Figure 2a

Sciatic nerve in the gluteal region before intervention. +: sciatic nerve (short axis

view)

Figure 2b

Sciatic nerve in the gluteal region immediately after intraneural injection of 5 ml

saline. +: sciatic nerve (short axis view), x: fluid depot

Figure 3

Sciatic nerve after needle-trauma (excised). +: sciatic nerve. x: formation of

small haematoma at puncture site.

Chapter 3.1

126

Figure 1


127

Figure 2A

Figure 2B

Chapter 3.1

128

Figure 3

Regional anesthesia in neuropathic patients

129

Chapter 3.2

Regional anesthesia in patients with pre-existing

neuropathy: current knowledge

Based on

Lirk P, Birmingham B, Hogan QH

Regional anesthesia in patients with pre-existing neuropathy

Int Anesthesiol Clin 2011; 49: 144-65

Chapter 3.2

130

Introduction

Regional anesthetic (RA) techniques are increasingly advocated in the

perioperative management of ambulatory surgery.1 Promising evidence shows

that RA (i) leads to a reduction in the amount of opioid consumption, thus

decreasing post-operative nausea / vomiting (PONV), and (ii) improves pain

relief. Considering that pain and PONV are the top two reasons for prolonged

hospitalization 2 or re-hospitalization

3 after ambulatory surgery, the use of

regional techniques is likely to continue to rise for the foreseeable future.

Moreover, recent evidence has widened the spectrum of use of RA blocks into

the post-discharge period.4 As the indications for regional anesthesia and

analgesia procedures in ambulatory surgery increase,5 and the contemporary

boundaries of patient selection are pushed further outward,6 an increasing

number of ambulatory surgery patients will present for RA with substantial co-

morbidities, including neurologic disease.

Neuropathies are a heterogeneous group of neurological conditions

secondary to genetic, inflammatory, metabolic, or mechanical injury of nervous

tissue. Their relevance in the perioperative management for ambulatory surgery

lies, to a large extent, in the deliberation of whether to include a regional

technique in the anesthesia plan. This becomes especially important in the case

of patients suffering from long-standing diabetes mellitus (DM), whose

occurrence in daily practice is projected to rise steadily, and who are often

affected by diabetic peripheral neuropathy (DPN).7 Intuitively, it would seem

logical that preexisting metabolic or structural neural defects increase the risk of

nerve damage after RA. For decades, anesthesiologists have cautioned against

RA in patients with preexisting neural disease.8 The widely accepted notion by

Selander et al. that every nerve block entails a certain degree of (reversible)

neuronal injury would lend support to this theory.9 However, current literature

does not give definitive answers.


131

The aim of this review is to review the pathogenesis and incidence of

nerve injury, to discuss implications of preexisting neurological disease upon the

occurrence of nerve injury in the context of RA, and to describe strategies that

may minimize the risk of iatrogenic aggravation of a preexisting neuropathy.

Nerve Injury during RA

Nerves are exposed to multiple risks during RA from the converging

effects of local anesthetic (LA) pharmacology, technical aspects of nerve block,

patient factors, and surgical factors (e.g., tourniquet). Only a minority of nerve

injuries are caused by direct solitary mechanical trauma sufficient to damage a

nerve permanently (e.g. intraneural injection).

Often, the toxicity of LAs and adjuvants is directly implicated in nerve

injury, and a large body of literature supports the notion that LAs are directly

neurotoxic.10

In cell cultures, dose- and time-dependent elicitation of cell death

from LAs is observed,11 12

while in animal models, signs of nerve damage are

evident even at clinically relevant doses.13

In patients, application of LA during

nerve block results in very high concentrations of these drugs in target tissues,14

resulting in disruption of calcium signaling,15

triggering a cascade of events

leading to programmed cell death.12 16

A plethora of contributing events has been

investigated, some providing a potential link to preexisting neuropathy. For

example, lidocaine selectively activates the pro-apoptotic p38 mitogen activated

protein kinase in primary sensory neurons, causing neurotoxic effects.12

The

same pro-apoptotic pathway is activated in neurons during DPN, while its

inhibition diminishes the functional impact of neuropathy.17

It is thus

conceivable that lidocaine is more toxic in nerves that are already prone to

apoptosis owing to activation of the same deleterious pathway by both

neuropathy and LA. This concept would, on a molecular level, be in analogy

with the “double-crush hypothesis”, which states that preexisting injury of a

Chapter 3.2

132

neuron leads to a supra-additive effect of a second injury harming the same

nerve.18

LAs can also damage nerves by interrupting blood flow, owing to

vasoconstrictive properties of anesthetics such as lidocaine and bupivacaine.14

Direct mechanical damage by laceration of the perineurium leads to a disruption

of the blood-nerve barrier and nerve edema.14

However, it is thought that

permanent nerve damage is unlikely unless needle penetration is followed by

intraneural injection of LA. Structural examinations show that injection of saline

produces no long-lasting pathologic changes.19

Recently, it has been suggested

that intraneural injection may not be necessarily harmful,20

and it would seem

that nerve anatomy is supportive of this notion. Peripheral nerves are composed

of nerve fibers as well as abundant amounts of connective tissue, especially at

regions where nerves are exposed to mechanical and shear stress, such as across

joints, so, a needle may pierce a nerve without damaging neural structures.14 21

Nevertheless, it must be recognized that for commonly used LAs, toxicity is

most likely influenced by the time and concentration of exposure of the

peripheral nerve to the LA.14

Intraneural injection prolongs the exposure and

increases the concentration of LA, and may reduce intraneural blood flow.

Because of the potential risk of injuring nerve fibers upon intraneural needle

placement and subsequent injection of LA, we believe the safest option is to

avoid intraneural needle placement during anesthetic injection procedures. This

concern particularly applies to neuropathic nerves.

Nerve injury can also be a consequence of general anesthesia (GA),22 23

sedation,24

or surgery per se.25

For example, after shoulder surgery, interscalene

block is a less frequent cause of postoperative neuropathy than surgical factors.25

The term “coincident injury” describes an injury occurring during nerve block

which is unrelated or only indirectly related to RA.26

One example is neuropathy

caused by tourniquet inflation during total knee arthroplasty.27

RA is perhaps


133

more conspicuous than surgical events as a risk factor for nerve injury in the

perception of patients and surgical colleagues, such that any injury evident after

surgery during which RA was used is often, perhaps incorrectly, first attributed

to the nerve block technique. This has the potential to mask the true cause of

neurological injury and to delay appropriate therapy.26

At times, surgical risk

factors may overlap with anesthesia-related factors to elicit nerve irritation or

injury. For example, the association of toxicity (spinal lidocaine) 28

and surgical

positioning (lithotomy) 29

in the pathogenesis of transient neurological syndrome

has been demonstrated.

Incidence of Nerve Injury after RA

The incidence of nerve injury after RA has been reported with widely

diverging results. RA procedures feature prominently in closed claims databases,

with most complications, and all fatalities, occurring after neuraxial blocks, and a

smaller number of injuries being reported after peripheral nerve blocks.30

Data of

outcome studies shows that the risk for neuropathy following epidural and spinal

anesthesia is 3.78 : 10,000 and 2.19 : 10,000, respectively. After peripheral nerve

block, the incidence of transient neuropathy is less than 3 : 100, whereas

permanent nerve damage is very rare.31

Recently, Watts et al. investigated

outcomes after 1065 peripheral nerve blocks, and found one transient and one

permanent case of nerve injury, amounting to an overall incidence of block-

related nerve injuries of 0.22%.32

The Australasian Regional Anesthesia

Collaboration investigated 7156 blocks in 6069 patients, and found thirty

patients with potential nerve damage (0.5%), but at analysis, only 3 had

neuropathy attributable to RA, giving an incidence of 0.04%.33

Another

investigation reported two cases of neuropathy following continuous popliteal

sciatic nerve block in 400 patients, resulting in a risk of 0.5%.34

Chapter 3.2

134

Evidence for elevated risk in patients with preexisting neurological disease

For several decades, there has been a growing consensus that

preexisting nerve injury increases the risk of neuropathy in patients undergoing

surgery. Originating from the first recommendations to avoid neuraxial

anesthesia in patients with preexisting neuropathy,35

it was later hypothesized

that preexisting neuropathies may predispose peripheral and central nerves to

subsequent injury during RA.18

Today, a more nuanced view has been adopted.

While in some preexisting conditions, we are now more ready to administer RA

and analgesia after a careful weighing of risks and benefits (e.g., obstetric

epidural analgesia in patients suffering from multiple sclerosis), other

neuropathies such as diabetic neuropathy are increasingly recognized as serious

risk factors for postoperative neuropathy that may warrant a change of current

practice.

In many patients, preexisting neurologic disease may represent an

important risk factor, most notably in diabetic peripheral neuropathy, multiple

sclerosis, Guillain-Barre syndrome and Post-polio syndrome.

Specific disease patterns

Diabetic peripheral neuropathy

Diabetes mellitus is a disease increasing in prevalence in most high-

income countries,7 leading in turn to an increasing prevalence of DPN. It is

estimated that about 10% of diabetic patients present with DPN at the time of

diagnosis, while this number rises to more than 50% of diabetic patients 5 years

later,36

probably even faster in poorly controlled DM type I.37

Diabetic

neuropathy is the most common neuropathy worldwide, and can be subdivided

into distal symmetric, autonomic, and focal/multifocal forms.38

Most patients

suffer from a chronic distal symmetric nerve injury involving both sensory and

motor fibers. Symptoms include hypesthesia and dysesthesia. Due to the


135

development of symptoms in long fibers of the lower extremity, and the gradual

spread to shorter axons, this variant has been designated length-dependent

diabetic polyneuropathy.38

Whereas the traditional distal symmetric neuropathy

is predominantly driven by metabolic factors, focal neuropathies seem to be

caused by a combination of ischemic and inflammatory processes.38

Progression

of DPN is difficult to predict in the individual patient, but poor glycemic control

seems to be the main risk factor.39

Recently, genetic polymorphisms in the aldose

reductase gene AKR1B1 have been reported to influence the severity and

progression of the disease.40

Current evidence suggests that once structural

damage has occurred, damage to end organs is irreversible.38 39

This is relevant to

daily anaesthetic practice because on the one hand, cardiovascular and renal

complications of this disease lead to many surgical procedures for which RA

may be highly suitable. On the other hand, limited epidemiological 41

and

experimental 42

evidence suggests an increased risk of local anaesthetic-induced

neurotoxicity in this growing patient population. It should be kept in mind,

however, that up to 30% of neuropathies in diabetics may be unrelated to

diabetes.43

Concern regarding the performance of RA in diabetic patients has been

promoted by epidemiological, experimental, and anecdotal observations.

Retrospective data compiled by Hebl et al. indicate that preexisting DPN may be

a risk factor for nerve injury after neuraxial anesthesia.41

Even though direct

extrapolation of the results from this study is difficult owing to the small number

of complications observed (n=2), it is worth noting that these iatrogenic injuries

occurred in diabetic patients using full doses of 0.5 or 0.75% of neuraxial

bupivacaine.41

The risk of nerve damage was described as 10-fold higher in DPN

41 than the estimated 0.04% reported for the general patient population.

31

Experimental evidence supports increased toxicity of LA in diabetic test

animals.42 44

In a landmark study, Kalichman and Calcutt found that 2% lidocaine

Chapter 3.2

136

used for sciatic nerve block in rats produced moderate nerve injury in healthy

animals, but induced severe degeneration in diabetic animals.42

Recently, Kroin

et al. showed that lidocaine 1% was safe in a comparable setting for peripheral

nerve blockade, whereas there was slight evidence of nerve injury when

lidocaine was used with additives (clonidine, epinephrine), or ropivacaine 0.5%

was used.44

Anecdotal evidence lists several patients who experienced worsening

of neurologic function after peripheral 45 46

or neuraxial 47-49

anesthesia. The

extent of preexisting DPN in these patients ranged from subclinical 45

to severe,49

and included the combination of both diabetic and alcoholic neuropathies.48

Other concerns in diabetic patients pertain to pharmacology of LA on

diabetic nerves, and the extent to which diabetic nerves are amenable to

peripheral nerve stimulation. While Kalichman and Calcutt did not find

prolonged block in test animals,42

the recent study by Kroin et al. showed

prolongation of block duration in diabetic animals as compared to healthy

controls.44

It seems, therefore, that diabetic nerves are more sensitive to LA. In

fact, regional blockade may be paradoxically efficient in diabetic nerves 50

owing

to three reasons. First, nerves may be more sensitive to the LA itself,42 44 50

second, the sensory area of a nerve may be partly anesthetized by neuropathy

itself,50

and third, microangiopathy may contribute to delayed absorption of LA.

The possibility of altered responsiveness of diabetic nerves to electric stimulation

was first raised in case reports demonstrating difficult stimulation of peripheral

nerves in diabetic patients.51-53

It would seem logical that a nerve that does not

transmit usual impulses properly might be relatively resistant to nerve

stimulation, itself a variable undertaking.54

We have reported that the risk of

intraneural needle placement is increased in diabetic dogs compared to controls

when using a nerve stimulator to guide needle placement.55

A more recent report

combining ultrasound and nerve stimulation showed that for successful

supraclavicular block, higher thresholds are necessary in diabetic versus non-


137

diabetic patients.56

DPN may thus complicate nerve stimulation, possibly

resulting in increased incidence of unsuccessful nerve stimulation, or even

intraneural needle placement, which may predispose to nerve injury.20

It may be

appropriate in patients with severe DPN to adapt the stimulating patterns to

longer pulses, as has been suggested in diabetic / ischemic neuropathy (i.e., from

0.1 msec to 1 msec),57

or employ ultrasound imaging to improve the accuracy of

needle placement.

Whether for central or peripheral blockade, the degree of risk from the

use of RA in diabetic patients cannot be exactly defined on the basis of current

knowledge. Nonetheless, nerves in DPN are probably more sensitive to LA

effects, including toxicity,50

we recommend reduction of the LA concentration

for neural blockade in such patients. Retro-fitting of data from animal

experiments using US FDA guidelines allows for estimation of safest doses in

patients on the basis of current knowledge (Table 1). Dose reduction may be

aided by the employment of ultrasound-guided RA in peripheral nerve blocks.58

The addition of adjuvants without a corresponding decrease in LA concentration

is, in general, not recommended.44 59

Diabetic nerves may already be at risk of

neuronal ischemia and infarction owing to changes in endoneurial small

vessels.59

Since lidocaine is vasoconstrictive itself,60

we recommend avoiding

epinephrine during nerve blocks in DPN patients.

Multiple Sclerosis

Multiple sclerosis (MS) is a chronic demyelinating neuroinflammatory

disease. Its etiology is still debated, but a crucial issue is the interplay between

polygenetic risk factors (HLA haplotypes) and environmental factors (viral

infections, vitamin D deficiency, smoking).61

Manifestations typically appear in

early adulthood, and as in many autoimmune diseases, women are predominantly

affected. MS is the most common debilitating disease in young adults.62

Chapter 3.2

138

Diagnosis is primarily based upon the triad of neurologic symptoms, positive

findings on magnetic resonance imaging, and the presence of oligoclonal bands

on cerebrospinal fluid (CSF) examination. The evidence concerning RA risks in

MS patients is limited. Data on peripheral nerve blocks are scarce, and most data

on neuraxial techniques are from the obstetric population.

Traditionally, the use of peripheral blocks in patients with MS has been

regarded as safe, since blocks are performed far from the main pathogenic

demyelination and scarring processes occurring within the central nervous

system (e.g., 63

). However, there is evidence for involvement of the peripheral

nervous system in MS, with peripheral nerves showing demyelination.64

Anecdotal 65

(albeit equivocal 66 67

) reports of brachial plexopathy after

peripheral nerve block have heightened awareness of the fact that a substantial

share of MS patients may suffer from subclinical peripheral neuropathy, the

incidence of which is reported inconsistently,68

and the clinical relevance of

which remains unclear.69

Based upon circumstantial evidence, the notion that

peripheral nerve blocks are most probably safe in MS patients still holds true,

and peripheral RA continues to be a valid alternative to GA or central blocks.

The possibility that RA may worsen neurological status is a particular

concern in spinal anesthesia. Despite the theoretical consideration that LA may

have increased neurotoxic effects on demyelinated and thus pre-damaged

neurons, there are no clinical studies to support this notion.70

Anecdotal evidence

reports de novo exacerbation of previously undiagnosed MS through spinal

anesthesia,71

but this could also be explained by transient and reversible

worsening of negative symptoms through exposure to LA (see below).72

Initial

reports demonstrating a higher risk of MS exacerbation after spinal anesthesia 73

have not been replicated in recent literature. A recent retrospective study found

no evidence of relapse after epidural or spinal anesthesia in series of 18 and 17

patients, respectively.74


139

Research pertaining to neuraxial techniques in MS has been performed

mostly in obstetric patients. Even though this patient population is not directly

comparable to day-surgery patients, insights may be derived from these

investigations. First, large-scale controlled studies have not linked any form of

obstetric RA to worsening of symptoms of multiple sclerosis.70

Epidural

anesthesia and analgesia, according to large-scale studies, appear safe.75 76

Confavreux et al. reported that epidural labor analgesia was not a risk factor for

disease relapse in parturients with multiple sclerosis in a trial involving 241

patients, of whom 42 had received epidural analgesia,75

confirming earlier results

by Nelson and colleagues.77

This is reflected by the results of a recent survey

among obstetric anesthesiologists in which the risk of epidural anesthesia /

analgesia was judged to be low based on clinical experience.78

Furthermore,

transient worsening of symptoms or de novo relapse after childbirth may be

mistakenly attributed to RA. After delivery, intrinsic nerve injury,79

fatigue,80

or

pyrexia may exacerbate MS symptoms.78

Moreover, in patients with a relapsing

form of MS, although pregnancy is associated with a decreased risk of flare-ups,

the postpartum period is characterized by an increased risk of relapse. Pregnancy

induces a transition from cellular immunity towards humoral immune responses,

driven by the secretion of IL-10 from the feto-placental unit, thereby promoting

immunological tolerance of the fetus. This cellular immunosuppression rebounds

after delivery, causing increased relapse rate.75

The limited evidence that exists is not sufficient to provide a definitive

answer to the question of whether neuraxial blocks are safe in MS patients.

Should symptoms arise in the perioperative period, there is an abundance of

patient- and procedure-related risk factors potentially contributing to worsening

of neurologic function. Moreover, the case of obstetrics illustrates that the

neurological condition of the patient is not the only factor determining whether

or not to perform RA. In obstetric patient, the risks of neuraxial anesthesia must

Chapter 3.2

140

be weighed against the risks of GA in a parturient, which may explain why 90%

of responders in a recent survey among obstetric anesthesiologists stated that

after weighing risks and benefits, they would still perform spinal anesthesia for

emergency caesarean section in MS patients.78

Recommendations have been made to limit neuraxial doses to the

lowest possible level,81

although this may increase the risk of insufficient

analgesia. One argument in favor of limiting doses comes from a case-control

study of 20 MS parturients, in which bupivacaine administered in doses above

0.25% was associated with what seemed to be a higher incidence of relapse.82

In

general, definitive studies on pharmacological properties of LA in MS are

lacking.72

Whether LA act longer on nerves from MS patients has been debated,

with case reports describing normal 83

versus prolonged 84

block duration, and

there is as yet no answer as to whether dynamics of nerve block are altered in

MS. Although not tested in clinical or experimental MS settings, non-neurotoxic

adjuvants such as clonidine and buprenorphine may hold promise for the

future.85

Finally, LA may directly interact with MS lesions. In patients, lidocaine

can reversibly worsen symptoms of MS,86

presumably by blocking sodium

channels in demyelinated areas just enough to produce symptoms, while healthy

areas are not affected. These changes were noted at doses also observed

systemically after epidural anesthesia in other investigations (low micromolar

range).87

Paradoxically, lidocaine can worsen negative symptoms (i.e. paresis

and hypesthesia resulting from demyelination), but can be used to effectively

treat positive symptoms (i.e. spasticity, dysesthesia resulting from ectopic

impulses) of MS, although the therapeutic range is very narrow.88

Interestingly,

endogenous extracellular pentapeptides are observed in the CSF of patients with

Guillain-Barre Syndrome and MS, and these pentapeptides share many

electrophysiological properties with LA.89


141

In summary, peripheral nerve blocks have not been proved to be

harmful to MS patients, and MS should not be considered to be an absolute

contraindication for neuraxial blockade. Since LA toxicity may be more

pronounced in demyelinated fibers, epidural anesthesia appears safer than spinal.

Overall, it seems justifiable to perform peripheral nerve blocks and epidural

anesthesia in patients with MS, with due attention to restricting LA

concentration. Use of spinal anesthesia in MS patients in the presence of safe and

feasible alternatives should be avoided. MS patients need to be informed about

potential symptom aggravation irrespective of whether RA is employed.

Post-polio syndrome

Post-polio syndrome (PPS) is the most prevalent motor neuron disease

in North America. A recent review contradicted the notion that PPS affects only

the aged. Indeed, many patients with PPS are of working age, and through new

infections in the developing world, PPS will remain an anesthetic challenge for

decades to come.90

PPS is characterized by central fatigue, pain and muscle

weakness, frequently associated with sleep-disordered breathing

(hypoventilation).90

Although the exact pathogenic mechanism remains obscure,

current avenues of investigation implicate overuse of the enlarged motor units

formed during recovery from polio, accelerated aging of motor units, persistent

viral infection, and chronic inflammation.90

Patients with PPS are not well suited to undergo day-case surgery

because of postoperative monitoring necessities,91

but the sheer number of

patients suffering from this disease will necessitate discussion on the

perioperative use of RA, especially since these patients frequently have to

undergo orthopaedic procedures.92

Risks of RA must be balanced against the

dangers of GA, such as controversies regarding use of depolarizing 93

and

nondepolarizing muscle relaxants,94

sensitivity to sedative or analgesic

Chapter 3.2

142

medication, and risk of hypoventilation and aspiration.91

Concerns with regard to

regional anesthetic techniques include temporary deactivation of muscles that

contribute to respiration during neuraxial anesthesia,95

difficulty in puncture

during neuraxial blockade in patients with secondary abnormal spinal anatomy,96

97 and the potential of iatrogenic worsening of symptoms.

91 Two case reports

have described spinal anesthesia using normal doses of tetracaine 98

and

bupivacaine 96

without post-procedural worsening of symptoms. Similarly, no

complications were reported in a small PPS group of three patients undergoing

seven surgeries.92

In the largest series of patients with PPS, 79 patients

undergoing neuraxial anesthesia or analgesia showed no worsening of

neurological symptoms.74

The data provided in current literature allows no clear recommendation

to choose or avoid neuraxial techniques or peripheral nerve blocks in PPS.

However, there is no evidence that the performance of spinal or epidural

anesthesia increases the risk of disease progression, and it seems unlikely that

peripheral nerve blocks are associated with an increased risk of morbidity.

Considering the unusual sensitivity towards sedative and analgesic agents, is

recommended to avoid the use of opioid or sedative adjuvants to both central and

peripheral blocks.

Guillain Barre Syndrome

Guillain-Barre syndrome (GBS) is a heterogeneous cluster of conditions

characterized by acute neuromuscular paralysis, probably due to post-infectious

inflammation.99

In many patients, antibodies against neuronal membrane

gangliosides are detectable.100

The disease develops weeks after the initial

trigger. Paralysis is variable in extent and involvement of sensory, cranial, or

autonomic nerves. The initial symptoms peak between 2 and 4 weeks after onset

is followed by a much slower recovery. Diagnosis is not always straightforward,


143

and is based upon clinical signs, electrophysiological and CSF diagnostics. The

incidence is about 2 cases per 100,000 annually, increasing linearly with age, and

predominantly affecting men.101

GBS is not typically associated with

autoimmune or systemic diseases.99

Any individual’s prognosis is difficult to

predict, and many patients continue to have moderate to severe impairment of

neurologic function years after the initial onset of disease. Risk factors for

prolonged neurologic deficit include older age at onset (>50 years), severe initial

disease, and antecedent infection with campylobacter jejuni or

cytomegalovirus.100

The disease is usually differentiated into acute inflammatory

demyelinating polyneuropathy, which features demyelinative conduction block,

and acute motor axonal neuropathy, in which modifications at neuronal sodium

channels are thought to play a pathogenic role. In the latter form, antibodies that

act as functional blockers of sodium channels and channel phosphorylation have

been implicated. 100 102

As in MS, endogenous extracellular pentapeptides are

observed in CSF (see above).89

Reports of RA in GBS are few. The performance of peripheral nerve

blocks has not been described in enough detail to allow definitive

recommendations. However, in the setting of acute neuronal inflammation,

peripheral blockade at these very nerves should probably be avoided. Most

experience with neuraxial techniques has been gathered in obstetric patients.

General comments are that the use of neuraxial anesthesia may be associated

with exaggerated hypotension and bradycardia,103

but many patients with GBS

respond normally to neuraxial anesthesia.104

Pharmacologically, both normal 105

and higher-than-normal 106

spread of LA administered using an epidural

technique have been reported, but most case reports describe usual block

properties.

Anecdotal evidence suggests that epidural anesthesia may activate the

onset or recrudescence of GBS 107 108

hours 109

or weeks 110

after surgery under

Chapter 3.2

144

epidural anesthesia. The hypothetical link between RA and GBS is interaction of

LA with peripheral myelin, or nerve trauma due to epidural block, with

subsequent inflammation as the precipitating factor.110

However, definitive

attribution of GBS to RA beyond a temporal association is difficult. Occurrence

or recurrence of GBS has been described for surgery without RA,111-113

and often

the time-lag between initiating trigger and onset of GBS symptoms is unclear.108

Case reports have described successful use of epidural anesthesia at

high doses, and even spinal anesthesia, in parturients with residual symptoms of

GBS.105 114 115

Recently the use of combined spinal and epidural anesthesia in a

parturient with GBS was reported without unusual hemodynamic effects or

exaggerated effects of LA.104

Weighing of risks and benefits of RA in patients with GBS needs to take

into account the alternative risks of GA in this patient population, and the status

of the disease. It has been suggested that acute neuronal inflammation should be

a relative contraindication for RA 116

and the possibility of a postoperative

diagnostic dilemma from the combined effects of RA and GBS argue for caution.

In patients with residual symptoms of GBS, the safe use of neuraxial blockade

has been described using both high and low volumes and doses of LA. The few

case reports describing a temporal relationship between RA and the development

of GBS do not suffice to classify RA as a risk factor for GBS.

Strategies to minimize risk in peripheral nerve blocks

Preoperative Assessment

Preoperative assessment should always include a review of medical

conditions that would predispose a patient to peripheral neuropathy. Subclinical

peripheral neuropathy and increased vulnerability to LA toxicity may already be

present in these patients. Next, documentation of existing neurologic deficits,

both by history and physical examination, should be performed. Any decision to


145

proceed with RA must include a risk-benefit analysis that considers the possible

increased risk of peripheral nerve blockade in patients with a peripheral

neuropathy compared with the risks of GA in the same patient. Importantly, the

risks and benefits of RA should be discussed with the patient and documented in

the medical record. Recent surveys of regional anesthesiologists both in

academic and community settings have suggested that this practice is not

consistently performed.117

Technique of Regional Anesthetic

Choice of nerve localization technique and its relation, if any, to the risk

of peripheral neuropathy has long been a topic of controversy. The goal of nerve

and needle localization has traditionally been to deposit the LA around the

epineurium without penetration or laceration, using nerve stimulation. One

limitation of stimulator techniques is the lack of a predictable relationship

between the threshold for nerve stimulation and needle-to-nerve distance.

Significant variability exists between patients and for different nerve localization

sites. This relationship may even further be complicated in patients with

underlying neuropathies, such as DPN.55

Ultrasound guidance may improve

needle placement accuracy and reduce LA dose,58

which are both desirable in

neuropathic patients. While this seems plausible and of particular interest in

patients with neuropathy, superior safety by using ultrasound imaging has yet to

be confirmed.118

Choice of Local Anesthetic

A second area of controversy is type and concentration of LAs. All LAs

are neurotoxic at high concentrations,119

and potentially toxic even at clinical

concentrations.85

Neurotoxic effects are comparable between LAs at equipotent

doses. Diabetic nerves in animal studies have demonstrated an increased

Chapter 3.2

146

sensitivity to LAs. Diabetic nerves require a lower concentration of LA for

successful nerve block and have a longer duration of block.44

Concentration of

LA and duration of nerve block correlate with histological fiber damage.44

It is

noteworthy that in Kalichman’s study of sciatic nerves of diabetic rats,

significantly increased nerve fiber damage was noted at 4% lidocaine versus 2%

lidocaine.42

In the study by Kroin et al, 1% lidocaine caused no significant nerve

fiber injury in diabetic rats.44

Thus, use of the lowest effective concentration and

amount of LA may reduce the risk of nerve fiber damage. This raises the

question of what concentration of LA is necessary for adequate surgical

anesthesia for, e.g., DPN patients. A recent study by Kocum and colleagues

showed that 0.25 % bupivacaine may be employed to produce effective femoral

and sciatic blocks in patients with diabetic foot syndrome.120

Further

investigations are needed to determine the optimal concentration of LA that

achieves successful surgical anesthesia while minimizing risk.

Role of Adjuvants

Epinephrine reduces blood flow in the peripheral nerve.121

Although the

temporary reduction in blood flow is well tolerated by most peripheral nerves,

the nerves of patients with compromised vascular integrity due to diabetes,

arteriosclerosis, or other injury may not tolerate severe reductions in blood flow.

Furthermore, by reducing neural blood flow, epinephrine decreases the washout

of LA and prolongs the duration of LA exposure. These properties prolong the

LA block for intermediate acting LA and may potentiate toxicity. In diabetic

nerves, epinephrine 5 mcg/ml prolongs the block by lidocaine 1%, causing

histologic evidence of axonal degeneration, which is absent with lidocaine or

epinephrine alone.44

In another study, epinephrine 5 mcg/ml caused significant

reduction in blood flow of healthy nerves, whereas epinephrine 2.5 mcg/ml did

not.122

On this basis, some have advocated using reduced doses of epinephrine in


147

patients with peripheral neuropathy to reduce the risk of nerve damage.118 123

We

advocate a more conservative stance of avoiding epinephrine in DPN patients

altogether. The the absence of this marker for intravascular injection, we also

recommend slow, incremental administration of the LA dose, and use of

lidocaine in preference to agents capable of producing malignant ventricular

dysrhythmias such as bupivacaine and ropivacaine.124 125

Similarly, clonidine has

been advocated as a useful adjuvant for peripheral nerve blockade.123

In doses as

low as 0.1 mcg/kg, clonidine has been shown to significantly increase the

duration of anesthesia for intermediate acting LA.126

The effect of clonidine is

peripherally mediated and is likely the result of activity dependent

hyperpolarization of the peripheral nerve fibers.127

In diabetic sciatic nerves, the

addition of clonidine 7.5 mcg/ml to 1% lidocaine significantly prolonged block

duration compared with lidocaine 1% plain but also produced histologic

evidence of axonal degeneration.44

Of note, this represents a higher dose of

clonidine than would be used clinically. Further investigations using lower

concentrations of clonidine may reveal a dose where anesthesia may be

augmented without augmenting axonal toxicity.128

Clonidine and buprenorphine

85 may prove to be useful adjuvants or replacements

129 for LA.

Summary

Based on limited data, mostly from animal studies, the risk of axonal

injury to peripheral nerves compromised by peripheral neuropathy may be

reduced by limiting the concentration and duration of LA exposure. Strategies to

accomplish this may include reducing the concentration of the selected LA,

reducing or avoiding added epinephrine, and utilizing a nerve localization

technique that minimizes the incidence of intraneural injections. Further

investigations should clarify the safe and effective concentrations of LA and

adjuvants in patients with peripheral neuropathy, and whether the potential for

Chapter 3.2

148

combined adjuvants reduces or eliminates the need for LA. Similarly, the safety

of different nerve localization techniques and nerve block selection should be

explored to optimize safety in this vulnerable, and growing patient population.

Strategies to minimize risk in neuraxial blocks

Evaluation of Risk

Several authors have speculated that patients with peripheral neuropathy

may be more vulnerable to symptomatic complications after neuraxial block.

First, due to the “double-crush” phenomenon, otherwise asymptomatic lesions

along a conduction pathway may produce symptoms following even a mild

degree of anesthetic-induced neurotoxicity.41

Second, “peripheral neuropathies”

such as diabetic polyneuropathy, may also produce changes in the spinal cord.130

Choice of Local Anesthetic

Similarly to effects on the peripheral nervous system, all LA can cause

dose dependent neurotoxicity.9 Some studies have raised concern that lidocaine

in clinically used concentrations may have a propensity for increased

neurotoxicity compared with similarly potent doses of bupivacaine.10 131

The

presence of peripheral neuropathy has not been associated with transient

neurologic symptoms in ambulatory patients.132

Limiting the dose and

concentration of LA in neuraxial blocks has been advocated to minimize the risk

of neurotoxicity.133

In ambulatory patients, the use of the lowest effective dose of

LA may also enable rapid return of sensory and motor function.134-136

Use of Adjuvants

Hydrophobic opioids such as fentanyl have been added to both low dose

bupivacaine and lidocaine spinal anesthetics to augment sensory blockade.137 138

A relative paucity of animal data exists regarding the toxicity of spinal fentanyl.


149

In clinical practice, few concerns about neurotoxicity have been raised regarding

low dose fentanyl or sufentanil used for ambulatory anesthesia. The limited

animal studies which have been done have not revealed significant histologic or

physiologic evidence of neurotoxicity.10 139

Therefore the addition of lipophilic

opioids to low doses of LA appears to be a useful adjunct of to decrease the total

dose of LA needed for successful surgical anesthesia in ambulatory patients, but

with the concomitant undesirable side-effects such as pruritus, nausea, and

somnolence in the elderly.

Epinephrine is rarely used in ambulatory patients as the resulting

delayed resolution of neuraxial anesthesia is usually not desirable for ambulatory

patients undergoing relatively short procedures. Epinephrine in most clinically

used doses does not significantly affect spinal cord blood flow.121

However,

epinephrine does worsen spinal cord injury when given with 5 % lidocaine or 1

to 2 % tetracaine in rats. It is likely that this effect is due to reduced clearance

and therefore prolongs exposure to the LA than to any direct effect of the

epinephrine. Also, when used with chloroprocaine, epinephrine can cause flu-

like symptoms.140

Because of its undesirable prolongation of anesthesia and

possible enhancement of toxicity, neuraxial epinephrine is best avoided in

patients with peripheral neuropathy presenting for ambulatory anesthesia.

Summary

Neuraxial anesthesia can be provided to patients with preexisting

neuropathy on the basis of thorough risk-benefit-analysis. Strategies to minimize

the risk of nerve toxicity include the use of the lowest effective dose of LA,

avoidance of epinephrine, and possible use of adjuncts such as lipophilic opioids.

Careful selection of agents can provide optimal surgical anesthesia and time to

discharge readiness as well as maximizing safety.

Chapter 3.2

150

Conclusion

RA is a reasonably safe and at the same time highly effective method of

providing pain relief to day-surgery patients. There is no universal answer to the

question of whether a regional anesthetic technique is the best choice for patients

with preexisting neuropathy. In most patients, the decision is complex and

requires a careful and individual weighing of risks and benefits of both general

anesthesia and regional anesthesia. In patients with preexisting neurological

disease, function can deteriorate perioperatively irrespective of whether RA is

employed. In diseases such as DPN and MS, reducing local anesthetic

concentration is appropriate. Focused informed consent regarding RA versus GA

with these patients should be followed by close observation for potential

sequelae.

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patient. Anesth Analg 2004; 98: 825-7, table of contents

108 Gautier PE, Pierre PA, Van Obbergh LJ, Van Steenberge A. Guillain-Barre

syndrome after obstetrical epidural analgesia. Reg Anesth 1989; 14: 251-2

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pancreatic cancer after an epidural-general anesthetic. Anesth Analg 2005; 100:

1197-9

110 Steiner I, Argov Z, Cahan C, Abramsky O. Guillain-Barre syndrome after

epidural anesthesia: direct nerve root damage may trigger disease. Neurology

1985; 35: 1473-5

111 Kuok CH, Tsai PS, Hsu YW, Ko YP, Huang CJ, Chen CC. Postoperative

delayed respiratory failure caused by Guillain-Barre syndrome--a case report.

Acta Anaesthesiol Taiwan 2007; 45: 43-6

112 Hogan JC, Briggs TP, Oldershaw PJ. Guillain-Barre syndrome following

cardiopulmonary bypass. Int J Cardiol 1992; 35: 427-8

113 Olivier N, Laribi H, Pages M, Camu W, Juntas Morales R. [Recurrent

Guillain-Barre syndrome after surgery]. Rev Neurol (Paris) 2010; 166: 644-7


161

114 Brooks H, Christian AS, May AE. Pregnancy, anaesthesia and Guillain

Barre syndrome. Anaesthesia 2000; 55: 894-8

115 Kocabas S, Karaman S, Firat V, Bademkiran F. Anesthetic management of

Guillain-Barre syndrome in pregnancy. J Clin Anesth 2007; 19: 299-302

116 Kattula A, Angelini G, Arndt G. Regional anesthesia in the presence of

neurologic disease. In: Finucane BT, ed. Complications of Regional Anesthesia.

New York: Springer Science, 1999

117 Brull R, McCartney CJ, Chan VW, et al. Disclosure of risks associated with

regional anesthesia: a survey of academic regional anesthesiologists. Reg Anesth

Pain Med 2007; 32: 7-11

118 Neal JM, Bernards CM, Hadzic A, et al. ASRA Practice Advisory on

Neurologic Complications in Regional Anesthesia and Pain Medicine. Reg


119 Bainton CR, Strichartz GR. Concentration dependence of lidocaine-induced

irreversible conduction loss in frog nerve. Anesthesiology 1994; 81: 657-67

120 Kocum A, Turkoz A, Bozdogan N, Caliskan E, Eker EH, Arslan G. Femoral

and sciatic nerve block with 0.25% bupivacaine for surgical management of

diabetic foot syndrome: an anesthetic technique for high-risk patients with

diabetic nephropathy. J Clin Anesth 2010; 22: 363-6

121 Neal JM. Effects of epinephrine in local anesthetics on the central and

peripheral nervous systems: Neurotoxicity and neural blood flow. Reg Anesth

Pain Med 2003; 28: 124-34

122 Partridge BL. The effects of local anesthetics and epinephrine on rat sciatic

nerve blood flow. Anesthesiology 1991; 75: 243-50

123 Neal JM, Hebl JR, Gerancher JC, Hogan QH. Brachial plexus anesthesia:

essentials of our current understanding. Reg Anesth Pain Med 2002; 27: 402-28

Chapter 3.2

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124 Rutten AJ, Nancarrow C, Mather LE, Ilsley AH, Runciman WB, Upton RN.

Hemodynamic and central nervous system effects of intravenous bolus doses of

lidocaine, bupivacaine, and ropivacaine in sheep. Anesth Analg 1989; 69: 291-9

125 Nancarrow C, Rutten AJ, Runciman WB, et al. Myocardial and cerebral

drug concentrations and the mechanisms of death after fatal intravenous doses of

lidocaine, bupivacaine, and ropivacaine in the sheep. Anesth Analg 1989; 69:

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126 Singelyn FJ, Gouverneur JM, Robert A. A minimum dose of clonidine

added to mepivacaine prolongs the duration of anesthesia and analgesia after

axillary brachial plexus block. Anesth Analg 1996; 83: 1046-50

127 Kroin JS, Buvanendran A, Beck DR, Topic JE, Watts DE, Tuman KJ.

Clonidine prolongation of lidocaine analgesia after sciatic nerve block in rats Is

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adrenoreceptors. Anesthesiology 2004; 101: 488-94

128 Williams BA. Toward a potential paradigm shift for the clinical care of

diabetic patients requiring perineural analgesia: strategies for using the diabetic

rodent model. Reg Anesth Pain Med 2010; 35: 329-32

129 Whiting DJ, Williams BA, Orebaugh SL, Toshok RR. Case report:

postoperative analgesia and preserved motor function with clonidine and

buprenorphine via a sciatic perineural catheter. J Clin Anesth 2009; 21: 297-9

130 Eaton SE, Harris ND, Rajbhandari SM, et al. Spinal-cord involvement in

diabetic peripheral neuropathy. Lancet 2001; 358: 35-6

131 Lambert LA, Lambert DH, Strichartz GR. Irreversible conduction block in

isolated nerve by high concentrations of local anesthetics. Anesthesiology 1994;

80: 1082-93

132 Moen V, Dahlgren N, Irestedt L. Severe neurological complications after

central neuraxial blockades in Sweden 1990-1999. Anesthesiology 2004; 101:

950-9


163

133 Drasner K. Local anesthetic neurotoxicity: clinical injury and strategies that

may minimize risk. Reg Anesth Pain Med 2002; 27: 576-80

134 Van Zundert AA, Grouls RJ, Korsten HH, Lambert DH. Spinal anesthesia.

Volume or concentration--what matters? Reg Anesth 1996; 21: 112-8

135 Liu S, Pollock JE, Mulroy MF, Allen HW, Neal JM, Carpenter RL.

Comparison of 5% with dextrose, 1.5% with dextrose, and 1.5% dextrose-free

lidocaine solutions for spinal anesthesia in human volunteers. Anesth Analg

1995; 81: 697-702

136 Korhonen AM. Use of spinal anaesthesia in day surgery. Curr Opin

Anaesthesiol 2006; 19: 612-6

137 Ben-David B, Solomon E, Levin H, Admoni H, Goldik Z. Intrathecal

fentanyl with small-dose dilute bupivacaine: better anesthesia without prolonging

recovery. Anesth Analg 1997; 85: 560-5

138 Liu S, Chiu AA, Carpenter RL, et al. Fentanyl prolongs lidocaine spinal

anesthesia without prolonging recovery. Anesth Analg 1995; 80: 730-4

139 Fukusima S, Takenami T, Nara Y. Neurotoxicity of intrathecally

administered fentanyl and bupivacaine in the rat spinal model. American Society

of Anesthesiologists Annual Meeting. San Diego, 2010

140 Smith KN, Kopacz DJ, McDonald SB. Spinal 2-chloroprocaine: a dose-

ranging study and the effect of added epinephrine. Anesth Analg 2004; 98: 81-8

Chapter 3.2

164

Table 1

Proposal for concentration and maximum single dose of local anesthetics in

patients with DPN based upon equipotency.

Drug Concentration DPN-adjusted

Concentration

Lidocaine 2 % 1 %

Mepivacaine 1.5 % 0.75 %

Ropivacaine 0.75 % 0.375 %

Bupivacaine 0.5 % 0.25 %

Equipotency values based on Hadzic A (ed.) Textbook of Regional Anesthesia

and Acute Pain Management, 2006, The McGraw-Hill Companies. We are

indebted to Brian Williams MD, MBA, for his kind permission to use his

calculations in this manuscript.

Regional anesthesia in subclinical neuropathy

165

Chapter 3.3

Effects of subclinical diabetic neuropathy on sciatic nerve

block

Based on

Lirk P, Flatz M, Haller I, Hausott B, Blumenthal S, Stevens MF Suzuki S,

Klimaschewski L, Gerner P

Subclinical Diabetic Neuropathy Increases In Vivo Lidocaine Block

Duration But Not In Vitro Neurotoxicity.

Reg Anesth Pain Med 2012; 37: 601-606.

Chapter 3.3

166

Introduction

Local anesthetics are widely used to block nerve conduction for surgical

anesthesia, or to manage acute and chronic pain. Unfortunately, local tissue damage,

in particular neurotoxicity induced by local anesthetics, remains a substantial

concern.1 This concern may apply even more to nerves with pre-existing pathology.

2

Neuropathy due to diabetes mellitus (DM) may be a relevant risk factor for regional

anesthetic procedures.3 4

It is the most common neuropathy worldwide, affecting a

large proportion of diabetes patients.5 The relevance of diabetic neuropathy for daily

anesthetic practice lies in the fact that because of their frequent comorbidities,

diabetic patients are predestined to undergo many types of surgery under regional

anesthesia. Results from some experimental trials suggest that diabetic neuropathic

nerves are more susceptible to local anesthetic-induced neurotoxicity than healthy

control nerves during peripheral nerve blockade,6 7

while another recent trial found

no difference in toxicity when effects of spinal anesthesia were examined.8

Epidemiological data suggest that nerve damage following regional anesthesia may

be up to ten times more frequent in diabetic patients as compared to the general

patient population.2 9

The effect of isolated hyperglycemia on local anesthetic-

induced neurotoxicity has not been investigated.

According to the “double-crush-hypothesis”, pre-existing nerve damage

may further predispose nerves to suffer from local anesthetic-induced

neurotoxicity.10

One pathogenic pathway activated in both diabetic neuropathy and

local anesthetic-induced neuropathy is activation of the p38 Mitogen-Activated

Protein Kinase (MAPK). 3 Neurotoxicity of the prototype local anesthetic, lidocaine,

is mediated by specific activation of the p38 MAPK, while its inhibition

significantly attenuates toxicity.11

Similarly, expression of the p38 MAPK has been

correlated with the development of diabetic sensorimotor neuropathy in a

streptozotocin-induced rat diabetic model, while its inhibition resulted in restoration

of normal nerve conduction.12

Further, it has been controversially discussed whether diabetic peripheral

neuropathy affects block duration.3 Three studies investigated this in a

streptozotocin-induced rat model, with one study demonstrating normal,6 and two


167

studies showing prolonged block duration.7

8 In diabetic patients, spinal anesthesia

has been reported to last longer than in healthy control patients.13

The present study therefore aimed to test in a rat model of Type II diabetes,

the hypothesis that pre-existing subclinical neuropathy, but not isolated

hyperglycemia, would predispose nerves to injury by local anesthetics. Our

secondary goals were to determine length of nerve block, and examine potential

neuroprotective effects of an inhibitor of the p38 MAPK, SB 203580.

Methods

Experimental procedures were approved by the Animal Care and Use

Committee of the Austrian Federal Ministry of Science, Education and Culture

(BMWF-66.011/0097-C/GT/2007) and the Harvard Medical Area Standing

Committee on Animals (Boston, MA).

Drugs

For in vitro experiments, the pH of the lidocaine stock solution 1 M was

4.65 (in dimethylsulfoxide). The pH of the final solution added to medium was 7.38

for lidocaine 40 mM (Sigma Aldrich, Vienna, Austria), which corresponds to a

concentration of approximately 1%. The concentration of dimethylsulfoxide (Sigma,

Vienna) in treated cultures was not significantly higher than in control cultures

incubated with vehicle only. The inhibitor of the p38 MAPK, SB203580, was co-

incubated with lidocaine in selected cultures at a final concentration of 10 µM. D-

Glucose (Sigma, Vienna) was prepared in a 1 M stock solution in distilled water. For

experiments, we diluted glucose in Roswell Park Memorial Institute (RPMI)-

medium supplemented with B27- and antibiotic additives to achieve a final

concentration in medium of 5 mM (control) or 25 mM (hyperglycaemic).14

Incubation of neuronal cell cultures with lidocaine, with or without addition of

SB203580 (Sigma, Vienna), was performed for 24 hours. This timeframe was used

in previous investigations and, combined with the lidocaine concentration of 40

mM, resulted in approximately 50% of cell death.11 15 16

Chapter 3.3

168

Animals

We employed adult male Zucker Diabetic Fatty (ZDF) rats, a combined

congenital / dietary inbred model of type II diabetes mellitus obtained from Charles

River Laboratories (Sulzfeld, Germany or Wilmington, MA). The main pathogenic

mechanism in these animals is a homozygous missense mutation in the leptin

receptor (ZDFfa/fa

genotype). ZDF rats develop metabolic syndrome, including

hyperglycemia.17

Two types of ZDF rats were used: the genotype homozygous for

obesity and metabolic syndrome (ZDFfa/fa

, “diabetic”), and heterozygous

normoglycemic animals (ZDFfa/-

, “control”). The progression from pre-diabetic to

diabetic state has been described in detail.18

Diabetic rats suffer from obesity, insulin

resistance, and overt type II diabetes mellitus starting at 7 to 10 weeks of age. At age

12-14 weeks, ZDF rats develop slowing of conduction velocity indicative of diabetic

neuropathy.19

Animals were used at 6 weeks of age for cell culture experiments into

acute and chronic hyperglycemia, and at 12 weeks of age for in vitro and in vivo

experiments into subclinical diabetic neuropathy. Until experiments, diabetic rats

received water and irradiated Purina diet #5008 (Charles River Laboratories) ad

libitum. This diet has been shown to most reliably promote the progression of

metabolic syndrome in ZDF rats and has been used in previous investigations on

diabetic neuropathy in this model, e.g.,20

Primary sensory neuron culture

Dorsal root ganglion (DRG) cultures were obtained as described

previously.21

Neurons were acutely harvested from (ZDFfa/fa

) or ZDF control rats,

which were euthanized by carbon dioxide narcosis according to institutional

protocol (Animal Care and Use Committee of the Austrian Federal Ministry of

Education, Science and Culture, Vienna, Austria). DRGs were de-sheathed and

incubated in collagenase 5000 U/ml for 90 min at 37°C, followed by 15 min in

0.25% trypsin/EDTA. After dissociation in Roswell Park Memorial Institute (RPMI)

medium containing 10% horse/5% fetal bovine serum, neurons were plated in RPMI

medium supplemented with B27 additives (1:100) and antibiotics (penicillin, 1000

units/ml; streptomycin, 1000 µg/ml; and amphotericin B, 25 µg/ml in 0.85% saline),

all purchased from Invitrogen (Carlsbad, CA). Neurons were allowed to adhere to


169

the glass floor of dishes coated with poly-D-lysine/laminin for 24 h. Poly-D-lysine

was applied at a concentration of 0.1 mg/ml in distilled H20 and laminin at 7 µg/ml

in RPMI solution. Cell cultures were kept at 37°C in a humidified atmosphere

containing 5% CO2.

In vitro model of diabetic metabolism

After harvesting ganglions from healthy ZDFfa/-

control rats, cell cultures

had 24 hours time to stabilize until continued treatment with glucose. Subsequently,

we maintained control (5 mM) and high (25 mM) glucose concentrations for 24 or

72 hours to simulate acute or hyperglycemia, or diabetic metabolism as described

before.14

Inhibition of p38 MAP kinase

To determine whether blocking of p38 MAPK would increase neuronal cell

survival in neurons treated with lidocaine, we co-incubated some cultures with an

inhibitor of the p38 MAPK, SB203580, at a concentration of 10µm.11

Quantification of neurotoxicity in vitro

We quantified neurotoxicity using the cytotoxicity detection kit (Roche

Diagnostics, Graz, Austria). This assay detects lactate dehydrogenase (LDH)

released from injured cells. We used sterile 96-well tissue culture plates, and

incubated cells as described above with experimental drugs. Cell injury results in

release of LDH, which is subsequently present in the culture supernatant.

Supernatant is extracted and mixed with reagent containing a tetrazolium salt, which

is cleaved by LDH to result in the formation of formazan after incubation at 30

minutes at room temperature under light protection. Detection of water-soluble

formazan is done by determining absorption at 492 nm, interpolating values with

low and high controls included in the kit. Plausibility control was undertaken to

remove extreme outliers. Due to measurement variability, some LDH values resulted

in survival values of just above 100%.

Chapter 3.3

170

Peripheral nerve blockade

In vivo experiments were conducted according to a protocol approved by

the Harvard Medical Area Standing Committee on Animals (Boston, MA).

Neurobehavioral investigation was performed as described before by an investigator

blinded to experimental group allocation.22 23

24

We performed in vivo experiments

on ZDFfa/fa

(“diabetic”) and ZDFfa/-

(“control”) rats. All rats were weighed before

experiments. We tested nociceptive and sensory function with mechanical pinch

with serrated forceps. Tests were performed bilaterally at all measurement time

points. In male diabetic ZDF animals, a specialized diet such as applied in our

animals results in hyperlipidemia and hyperglycemia by 8 weeks of age, and

diabetes by 12 weeks of age (Charles River Laboratories). In functional tests, motor

nerve conduction velocity as a sign of subclinical diabetic neuropathy is decreased at

12-14 weeks of age, indicating first underlying pathological processes.19

Test animals were anesthetized with Sevoflurane (Abbott, Abbott Park, IL)

during all procedures. Sciatic nerves were surgically exposed by lateral incision of

the thighs and division of the superficial fascia and muscle as described before 21

. A

volume of 0.2-mL test drug was injected directly beneath the clear fascia

surrounding the nerve but outside the perineurium, proximal to the sciatic

bifurcation. We chose lidocaine 2% (corresponding to approximately 80 mM) as

described previously.21

The wound was sutured with 3-0 vicryl suture.

Motor function was assayed before injection, and after 15, 30, 45, 60, 75,

90, 120, and 150 minutes by holding the rat upright with the control hind limb

extended so that the distal metatarsus and toes of the target leg supported the

animal’s weight; the extensor postural thrust was recorded as the force (in grams)

applied by each of the two hind limbs to a digital platform balance (Ohaus Lopro;

Fisher Scientific, Florham Park, NJ). The reduction in this force, representing

reduced extensor muscle contraction caused by motor block, was calculated as a

percentage of the control force (preinjection control value 90–130 g). The percent

reduction in force was assigned a “range” score: 0 = no block (or baseline); 1 =

minimal block, force between the preinjection control value of 100% and 50%; 2 =

moderate block, force between 50% of the preinjection control value and 12 g


171

(approximately 12 g represented the approximate weight of the flaccid limb); 3 =

complete block, force 12 g or less.

Nociception was evaluated by the nocifensive withdrawal reflex and

vocalization to pinch of a skin fold over the lateral metatarsus (cutaneous pain) with

a serrated forceps; the force and duration of this pinch was held as constant as

possible. The extent of the nocifensive withdrawal reflex and vocalization were

combined on a scale of 0–3 for each examination. Grading was as follows: 3 =

complete block, no nocifensive reaction or vocalization; 2 = moderate block,

vocalization accompanied by slow withdrawal or flexion of the leg; 1 = minimal

block, brisk flexion of the leg, with some sideways movement of the body or other

escape response and loud vocalization; 0 = baseline with quick nocifensive

responses listed above. The contralateral limb was used as control in all

experiments. We defined the time to complete absence of block (motor block and

nociceptive block score = 0) as block time.

Statistics

We used ANOVA and if significant unpaired t-test to compare data

between two groups. Statistical significance was assumed at P < 0.05. Bonferroni-

Holm was used to correct for multiple group comparisons. SPSS 16.0 was used for

statistical analyses.

Results

In vitro, acute or chronic hyperglycemia do not increase lidocaine neurotoxicity

The application of control or high concentrations of glucose for 24 hours

prior to lidocaine incubation did not alter neurotoxicity. In comparison to control

cultures and high glucose alone, incubation with lidocaine alone led to a reduction in

survival of about 50% in both control and high-glucose cultures (Table 1). Co-

application of SB203580 with lidocaine did not prevent lidocaine-induced

neurotoxicity (52 ± 15%, n = 16).

In both cultures incubated with control medium or high glucose levels for

72 hours, lidocaine incubation led to a reduction in cell survival of approximately

Chapter 3.3

172

50% (Table 1). Co-application of SB203580 with lidocaine resulted in a cell number

of 50 ± 25% (n = 16, non-significant).

In vitro, lidocaine is more neurotoxic in diabetic than in control cells, and co-

incubation with SB203580 reduced neurotoxicity

In neuron cultures from diabetic animals harvested at 12 weeks of age,

control cultures had a viability of 117 ± 7 % (n = 18). Cultures from control animals

incubated with lidocaine had a cell count of 64 ± 9 % (n = 33, P < 0.001 as

compared to controls), whereas neurons from diabetic animals had a cell count of 57

± 19 % (n = 64, P < 0.001 as compared to controls, P < 0.05 as compared to non-

diabetic animals after multiple-group correction, Figure 1).

Neurons from diabetic animals incubated with lidocaine had a survival rate

of 57 ± 19 % (n = 64), and addition of SB203580 to lidocaine in neurons from

diabetic animals resulted in a survival of 71 ± 12 % (n = 66, P < 0.001 to controls, P

< 0.001 as compared to lidocaine in diabetic animals). Some non-diabetic cultures

were incubated with lidocaine and SB203580, resulting in a survival of 66 ± 9 % (n

= 33, non-significant as compared to lidocaine in diabetic animals).

In vivo, subclinical diabetic neuropathy prolongs lidocaine duration of action

At baseline, behavioural tests were not significantly different between

diabetic and non-diabetic rats. In detail, motor testing revealed a force of 111 ± 12 g

in control and 114 ± 12 g in diabetic animals. Nociceptive testing was 0 in all

control, and 0 in all diabetic animals. In diabetic animals, motor block lasted

significantly longer than in control animals (137 ± 16 min versus 86 ± 17 min, P <

0.001, Figure 2A). Response to deep pinch was abolished longer in diabetic than in

non-diabetic rats (128 ± 22 min versus 77 ± 10 min, P < 0.001, Figure 2B).

Response to superficial pinch was similarly abolished longer in diabetic than in non-

diabetic rats (128 ± 22 min versus 81 ± 8 min, P < 0.001, Figure 2C). All animals

regained the values of the baseline behavioural test within a day. Return to baseline

values for neurobehavioral tests was observed in all animals.


173

Discussion

The main findings of our study are that (A) in vitro, lidocaine neurotoxicity

is not altered by acute (24h) or chronic (72h) hyperglycemia; (B) in vitro, despite

reaching statistical significance, subclinical diabetic neuropathy does not appear to

substantially increase lidocaine neurotoxicity; (C) in vitro, inhibition of p38 MAPK,

at least partly, reversed lidocaine neurotoxicity in diabetic neurons; and (D) in vivo,

subclinical diabetic neuropathy increases block duration without apparent adverse

behavioral effects after the blocks resolve. However, we did not histologically

examine these lidocaine-exposed sciatic nerves to address potential disruption of

fiber integrity after behavioral tests had returned to baseline. In nerve cell cultures

from non-diabetic animals, lidocaine resulted in a reduction in cell survival of about

50%, confirming results from previous investigations and corroborating the

experimental in vitro paradigm.11 21

In vitro, acute or chronic hyperglycemia do not increase lidocaine neurotoxicity

We first sought to determine whether acute or prolonged hyperglycemia per

se would be sufficient to increase lidocaine neurotoxicity in vitro. Hyperglycemia

alone did not enhance lidocaine-induced neurotoxicity. Even though a period of 72

hours has previously been described as sufficient to elicit first general changes

indicative of diabetic metabolism in PC12 cell line cultures,14

this approach may not

result in long-standing alterations characteristic of severe diabetic neuropathy which

may predispose nerves to injury following exposure to local anesthetics. Using doses

of glucose which reflect previous investigations seeking to simulate diabetic

conditions in vitro,14

we were unable to detect a negative impact of acute or chronic

hyperglycemia on local anesthetic neurotoxicity.

In vitro, lidocaine neurotoxicity is comparable between diabetic and control cells

Secondly, neurons explanted from animals with longer-lasting diabetes (12

weeks) were compared to neurons harvested from non-diabetic animals with regards

to sensitivity towards lidocaine neurotoxicity. The slight survival advantage of non-

diabetic neurons observed was small, and, despite reaching statistical significance,

appears not to reflect clinically relevant neurotoxicity. No previous investigations

Chapter 3.3

174

have investigated the potential of neurotoxicity in neurons explanted from a

genetically modified rodent model. The timing of neuron harvesting was chosen to

precede development of manifest diabetic neuropathy. It is generally thought that

diabetic nerves are more susceptible to exogeneous trauma / ischemia / toxins.6 25 26

However, the results obtained by us are not as pronounced as those reported

previously in vivo by others.6 7

Two reasons may account for this discrepancy.

Firstly, at the time of harvesting, animals had long-standing hyperglycemia, but

presumably only mild subclinical neuropathy, while animals in other trials 6 7

were

pronounced neuropathic. Zucker rats reliably develop progressive neuropathy,19

so

by choosing a later timepoint for experiments, we may have found a more

pronounced neurotoxic effect. Also, a recent study in Type I diabetic rats subjected

to repeated intrathecal injections failed to demonstrate significant neurotoxicity.8

Secondly, the model employed by us is a model of DM type II, reflecting metabolic

syndrome rather than the streptozotocin (STZ) model used in previous

investigations.6 7

It has been stated that there is no single best animal model for

research into regional anesthesia in diabetic neuropathy.27

We believe the model

chosen by us relates reasonably well to the clinical situation, in which the majority

of neuropathic patients presenting for surgery are type II diabetics.

In vitro, co-incubation of lidocaine with SB203580 reduces neurotoxicity in diabetic

neurons

Physiologically, the p38 MAPK is a member of the family of mitogen-

activated protein kinases, key regulators of eukaryotic cell regulation.28

p38 MAPK

is activated following a broad variety of stressors such as environmental factors

(cytotoxic substances, radiation, osmotic stress, heat shock), inflammatory cytokines

(e.g. Tumor Necrosis Factor α), and growth factors.28

P38 MAPK potently activates

transcription factors and other protein kinases.28

Therapeutic inhibition of p38

MAPK activity has been shown to be of potential benefit in, among others,

experimental nerve trauma,29

excitotoxicity,30

and growth factor withdrawal.31

In the setting of diabetic neuropathy, activation of p38 MAPK (leading to

neuronal degeneration) coincides with the development of functional deficits in a

STZ-based rat model, while pharmacologic inhibition of MAPK greatly attenuates


175

functional changes.12

Similarly, p38 MAPK activation occurs when primary sensory

neurons are exposed to toxic doses of local anaesthetics,11 21 32

and inhibition of p38

MAPK leads to decreased neurotoxicity in vitro and in vivo.21

We therefore

hypothesized that activation of the same neurodegenerative p38 MAPK pathway by

both diabetic neuropathy and local anaesthetic aggravates neuronal damage. This

should be most prominent when lidocaine is applied, because the latter selectively

activates p38 MAPK in neurons.11

We found only minimally increased neurotoxicity

when lidocaine was applied to nerves harvested from diabetic animals as compared

to control animals. Inhibition of the p38 MAPK, at least in part, abolished this effect

in vitro. Inhibition of p38 MAPK reduced the lidocaine-induced neurotoxicity in

diabetic neurons, confirming our experimental hypothesis. Effects on healthy

neurons were not statistically different. The protective effect of p38 MAPK in

healthy neurons was smaller compared to previous investigations.15 16 21

Several

factors may account for the unexpected finding that lidocaine neurotoxicity in

control cells was unaffected by p38 MAPK inhibition. The model used in this study

differs from that used in previous studies as it represents a different genotype of rats

(ZDF versus Sprague Dawley rats or PC12 cell line cultures).

In vivo, subclinical diabetic neuropathy prolongs lidocaine duration of action

We next investigated influence of diabetic subclinical neuropathy upon

lidocaine block duration. At baseline, we found no difference in sensitivity,

supporting our model of subclinical diabetic neuropathy. When lidocaine was

applied to diabetic nerves, it led to a longer-lasting sensory and motor block

compared to wild-type controls. In a previous study using the STZ diabetes model,

Kalichman and Calcutt showed that lidocaine exhibited normal duration of block in

diabetic nerves. 6 Our results are, however, in concordance with a recent

investigation by Kroin et al.,7 who demonstrated increased sciatic nerve block

duration in STZ diabetic rats, using a slightly longer pretreatment time.7 In the same

STZ experimental model in rats, spinal anesthesia was reported to result in a longer

block duration in diabetic animals without a concomitant increase in neurotoxicity.8

In diabetic patients, spinal anesthesia was reported to last longer than in non-diabetic

patients.13

Chapter 3.3

176

The reason for increased block duration may be multifactorial, involving

changes in nerve metabolism and physiology, and nerve fiber damage.7 Previous

investigations had used open 6 or percutaneous

7 nerve block. We cannot exclude

that the open approach resulted in inflammation around the nerve fascia, but in a

recent experimental study, Kroin et al. found no difference in block duration

whether nerve block was performed open or closed.7

Limitations

Some limitations of the present study should be briefly addressed. First, our

in vitro data cannot be directly correlated with clinical practice. As for peripheral

nerve blockade (rat sciatic nerve block model), the drugs are applied to the axon

rather than the DRG, making dissociated neuronal cultures an imperfect

approximation. Moreover, glial cells, usually found in standard DRG cultures, could

influence LA-induced neurotoxicity. We used lidocaine as the prototype amide-type

local anesthetic. For peripheral nerve block in clinical practice, other drugs such as

mepivacaine (short-acting) or long-acting local anesthetics are more frequently used.

We suggest the ZDF model to be used to investigate other local anesthetics in

regular clinical use, and to include a group of animals with even longer-standing

diabetes to evaluate fully developed diabetic neuropathy, as well.

The ZDF rat has been described as a model of type II diabetes mellitus, but

features other conditions as well, most notably obesity. In the ZDF model, male rats

develop manifest hyperglycemia at 7 weeks of age. At the age of 12-14 weeks (the

timepoint used for our experiments), ZDF rats feature an overt neuropathic deficit

discernible by electrophysiologic investigation.19

We attest to the fact that we did

not confirm these investigations in our test animals, such that presence of subclinical

nerve damage was inferred from the literature showing a predictable clinical course

of diabetes and complications,17

and a definable window when this damage

appears.19

The ZDF model chosen to investigate the effects of long-standing

hyperglycemia on regional anesthesia has been widely used in metabolic research.

Nevertheless, a recent editorial highlighted the fact that all current diabetes models

feature confounding factors.27

In the case of ZDF, it has been argued that it is more a

model of metabolic syndrome than exclusively for diabetes, while the same applies


177

for STZ-induced diabetes Type I, which is confounded by hepatic and renal damage,

and substantial behavioural changes.27 33

Our model is most likely more

representative of clinical reality for anesthesiologists, in which patients with

neuropathy due to Type II diabetes represent the most controversial patient

collective.

Finally, we concluded from behavioural data that no overt neurotoxicity

was observed in our model, without performing neurohistopathological

investigations once tests had returned to baseline. Future investigations should

include histological examination to corroborate our findings. We did not carry out

neurobehavioural testing after tests had returned to baseline.

Conclusion

In conclusion, we demonstrate that in vitro, acute or chronic hyperglycemia

per se does not increase lidocaine-induced neurotoxicicity. We observed a small,

albeit statistically significant, increase in neurotoxicity when lidocaine was applied

to diabetic as compared to non-diabetic neurons, but the difference in our model

appears small. The inhibitor of the p38 MAPK, SB203580, can at least partially

reverse lidocaine-induced neurotoxicity in diabetic nerves. In vivo, block duration is

substantially prolonged by subclinical diabetic neuropathy.

References

1 Auroy Y, Benhamou D, Bouaziz H, et al. [Peripheral nerve block: yesterday's

facts and tomorrow's challenges]. Ann Fr Anesth Reanim 2006; 25: 82-3

2 Hebl JR, Kopp SL, Schroeder DR, Horlocker TT. Neurologic complications after

neuraxial anesthesia or analgesia in patients with preexisting peripheral


9

3 Lirk P, Birmingham B, Hogan Q. Regional anesthesia in patients with preexisting

neuropathy. Int Anesthesiol Clin 2011; 49: 144-65

4 Blumenthal S, Borgeat A, Maurer K, et al. Preexisting subclinical neuropathy as a

risk factor for nerve injury after continuous ropivacaine administration through a

femoral nerve catheter. Anesthesiology 2006; 105: 1053-6

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5 Said G. Diabetic neuropathy--a review. Nat Clin Pract Neurol 2007; 3: 331-40




block and nerve fiber damage in diabetic rats. Reg Anesth Pain Med 2010; 35: 343-

50

8 Kroin JS, Buvanendran A, Tuman KJ, Kerns JM. Safety of Local Anesthetics

Administered Intrathecally in Diabetic Rats. Pain Med 2012; 13: 802-7



965-74

10 Hebl JR, Horlocker TT, Pritchard DJ. Diffuse brachial plexopathy after

interscalene blockade in a patient receiving cisplatin chemotherapy: the

pharmacologic double crush syndrome. Anesth Analg 2001; 92: 249-51





12 Price SA, Agthong S, Middlemas AB, Tomlinson DR. Mitogen-activated protein

kinase p38 mediates reduced nerve conduction velocity in experimental diabetic

neuropathy: interactions with aldose reductase. Diabetes 2004; 53: 1851-6

13 Echevarria M, Hachero A, Martinez A, et al. Spinal anaesthesia with 0.5%

isobaric bupivacaine in patients with diabetes mellitus: the influence of CSF

composition on sensory and motor block. Eur J Anaesthesiol 2008; 25: 1014-9

14 Lelkes E, Unsworth BR, Lelkes PI. Reactive oxygen species, apoptosis and

altered NGF-induced signaling in PC12 pheochromocytoma cells cultured in

elevated glucose: an in vitro cellular model for diabetic neuropathy. Neurotox Res

2001; 3: 189-203





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not by inhibiting caspase activity. Anesth Analg 2007; 105: 1657-64, table of

contents

17 Chen D, Wang MW. Development and application of rodent models for type 2

diabetes. Diabetes Obes Metab 2005; 7: 307-17

18 Etgen GJ, Oldham BA. Profiling of Zucker diabetic fatty rats in their progression

to the overt diabetic state. Metabolism 2000; 49: 684-8

19 Oltman CL, Coppey LJ, Gellett JS, Davidson EP, Lund DD, Yorek MA.

Progression of vascular and neural dysfunction in sciatic nerves of Zucker diabetic

fatty and Zucker rats. Am J Physiol Endocrinol Metab 2005; 289: E113-22

20 Brussee V, Guo G, Dong Y, et al. Distal degenerative sensory neuropathy in a

long-term type 2 diabetes rat model. Diabetes 2008; 57: 1664-73




22 Gerner P, Haderer AE, Mujtaba M, et al. Assessment of differential blockade by

amitriptyline and its N-methyl derivative in different species by different routes.


23 Thalhammer JG, Vladimirova M, Bershadsky B, Strichartz GR. Neurologic

evaluation of the rat during sciatic nerve block with lidocaine. Anesthesiology 1995;

82: 1013-25

24 Gerner P, Binshtok AM, Wang CF, et al. Capsaicin combined with local

anesthetics preferentially prolongs sensory/nociceptive block in rat sciatic nerve.


25 Zochodne DW, Cheng C, Sun H. Diabetes increases sciatic nerve susceptibility

to endothelin-induced ischemia. Diabetes 1996; 45: 627-32

26 Zochodne DW, Cheng C. Diabetic peripheral nerves are susceptible to

multifocal ischemic damage from endothelin. Brain Res 1999; 838: 11-7

27 Williams BA, Murinson BB, Grable BR, Orebaugh SL. Future considerations for

pharmacologic adjuvants in single-injection peripheral nerve blocks for patients with


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28 Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal

transduction pathways activated by stress and inflammation. Physiol Rev 2001; 81:

807-69

29 Obata K, Yamanaka H, Kobayashi K, et al. Role of mitogen-activated protein

kinase activation in injured and intact primary afferent neurons for mechanical and

heat hypersensitivity after spinal nerve ligation. J Neurosci 2004; 24: 10211-22

30 Park JY, Kim EJ, Kwon KJ, et al. Neuroprotection by fructose-1,6-bisphosphate

involves ROS alterations via p38 MAPK/ERK. Brain Res 2004; 1026: 295-301

31 Horstmann S, Kahle PJ, Borasio GD. Inhibitors of p38 mitogen-activated protein

kinase promote neuronal survival in vitro. J Neurosci Res 1998; 52: 483-90

32 Haller I, Lirk P, Keller C, Wang GK, Gerner P, Klimaschewski L. Differential

neurotoxicity of tricyclic antidepressants and novel derivatives in vitro in a dorsal

root ganglion cell culture model. Eur J Anaesthesiol 2007; 24: 702-8

33 Tomlinson KC, Gardiner SM, Hebden RA, Bennett T. Functional consequences

of streptozotocin-induced diabetes mellitus, with particular reference to the

cardiovascular system. Pharmacol Rev 1992; 44: 103-50


181

Table 1

Survival in response to lidocaine after acute or chronic hyperglycemia

Treatment group Survival n Significance

Acute hyperglycemia (24 hours)

1. Control 105 ± 4 9 S vs. 2 and 4

NS vs. 3

2. Lidocaine 45 ±15 17 S vs. 1 and 3

NS vs. 4

3. High glucose 107 ± 6 17 S vs. 2 and 4

NS vs. 1

4. High glucose plus

lidocaine

55 ±13 17 S vs. 1 and 3

NS vs. 2

Chronic hyperglycemia (72 hours)

1. Control 103 ± 4 9 S vs. 2 and 4

NS vs. 3

2. Lidocaine (as above) 45 ± 15 17 S vs. 1 and 3

NS vs. 4

3. High glucose 102 ±7 17 S vs. 2 and 4

NS vs. 1

4. High glucose plus

lidocaine

53 ± 27 16 S vs. 1 and 3

NS vs. 2

Legend: S significant NS non-significant.

Chapter 3.3

182

Figure legends

Figure 1. Comparison of viability of dorsal root ganglia primary cultures of diabetic

(ZDFfa/fa

) and control (ZDFfa/-

) animals. Cells were exposed for 24 h to control

medium, lidocaine or lidocaine and MAP kinase inhibitor SB203580. Data are

displayed as mean and standard deviation. Comparisons were made by means of

student-t-test with Bonferroni-Holm correction. * delineates p < 0.0001; ‡ = p <

0.01; # = p < 0.05.

Figure 2. The time course of motor (A) and deep sensory (B) block in control and

diabetic animals is displayed during 150 min after injection of lidocaine. Predefined

four-point scales were chosen with 0 denoting no block and 3 complete block. Data

presented as mean ± standard error, testing was done by means of ANOVA with

posthoc Benforoni test. * denotes p < 0.05.

Note: For all nerve conduction tested a significantly prolonged block was seen in

diabetic animals.


183

Figure 1

Chapter 3.3

184

Figure 2

Regional anesthesia in early and late neuropathy

185

Chapter 3.4

Effects of early and diabetic neuropathy on sciatic nerve

block duration and toxicity

Based on

Lirk P, Verhamme C, Boeckh R., Stevens MF, ten Hoope W, Gerner P,

Blumenthal S, de Girolami U, van Schaik IN, Hollmann MW, Picardi S

Effects of early and late diabetic neuropathy on

sciatic nerve block duration and neurotoxicity.

Br J Anaesth 2014 (in revision).

Chapter 3.4

186

Introduction

Diabetic peripheral neuropathy (DPN) is a frequent complication of both

Type I and Type II diabetes mellitus (DM), and the most prevalent neuropathy in the

Western world.1 Diabetics undergo surgery more often than non-diabetic patients,

2

and several surgical procedures for typical complications of long-standing DM, e.g.

creation of arteriovenous fistula in patients with end-stage renal disease, might be

preferably performed under regional anaesthesia.3

However, diabetic neuropathic nerves may be more sensitive to local

anesthetics and their toxicity, and this hypothesis is supported by two lines of

evidence. Firstly, regional anaesthesia in diabetic neuropathic patients may be

associated with increased risk of neurological injury.4 Limited epidemiological

evidence suggests higher risk of neurotoxicity in diabetic neuropathic patients,5,6

even if experimental evidence has been equivocal.7 Secondly, DPN may influence

nerve block duration.4 Clinical

8 9 and experimental

10 11 evidence suggests that block

duration may be prolonged in diabetic neuropathic nerves. However, most studies

were carried out in models of streptozotocin-induced Type I DM, which does not

reflect clinical reality, in which the huge majority of patients suffer from Type II

DM.5

Our aim was to determine the impact of regional anaesthesia in DPN in an

animal model for Type II DM. We therefore sought to devise a comprehensive

model using behavioural, electrophysiological, and histopathological investigations

to determine neurotoxicity of a lidocaine 2% peripheral nerve block and duration of

this nerve block in Zucker Diabetic Fatty rats with early diabetic neuropathy,

advanced diabetic neuropathy, and advanced diabetic neuropathy under partial

glycemic control. Our working hypothesis was that in a rodent model of Type II

DM, the presence of advanced (18 weeks) but not early (10 weeks) neuropathy

would lead to increased neurotoxicity and block duration following sciatic nerve

block with lidocaine as compared to age-matched healthy control animals. The main

endpoint was neurohistopathology one week after nerve block.


187

Materials and Methods

The present study protocol was approved by the Institutional Animal Care

and Use Committee of the Academic Medical Center, University of Amsterdam,

protocol number LEICA102868-1. Methods and results are reported according to

ARRIVE guidelines.12

Animals

Experiments were carried out on the left sciatic nerve of Zucker diabetic

fatty (ZDF) rats, obtained from Charles River Laboratories (L’Arbresle, France).

This inbred model of Type II DM combines a genetic predisposition (homozygous

leptin receptor mutation fa/fa, “diabetic”, or heterozygous mutation fa/+, “control”)

with a dietetic component (Purina #5008 diet, Charles River, L’Arbresle, France).11

13 Animals were obtained at 9 weeks of age and were given one week for

acclimatization. For all electrophysiological measurements, sciatic nerve block, and

placement of insulin release implants, animals were anaesthetized using isoflurane

(Baxter, Utrecht, The Netherlands) with an inspiratory concentration between 2 – 3

Vol%, since this regimen least affects electrophysiological measurements in rodent

models.14

Adequacy of anaesthesia was ascertained using sensory testing. We only

carried out procedures when the animal did not react to a forceful pinch to the

forefoot using a forceps. All procedures were performed percutaneously to minimize

animal distress, and analgesic rescue was buprenorphine 0.05 mg/kg body weight.

Detailed welfare assessment concerning appearance and behaviour was carried out

at least weekly by an animal care technician unrelated to the experiment. After the

last measurements, while still under isoflurane anaesthesia, animals were euthanized

using CO2 narcosis.

Experimental groups

The timeline of experimental procedures is given in Figure 1. In all

experimental groups, baseline measurements of electrophysiological parameters (see

below) were taken at 10 weeks of age.

Group “early control (EC)” were 6 ZDF fa/+ animals, and group “early

diabetic (ED)” were 10 ZDF fa/fa animals undergoing left sciatic nerve block

Chapter 3.4

188

immediately after baseline testing at 10 weeks. One week later, behavioral and

electrophysiologic measurements were repeated, and the left sciatic nerve was

excised for neurohistopathologic evaluation.

The group “late control (LC)” consisted of 10 fa/+ animals kept until 18

weeks of age. The group of diabetic animals for the late experiments were

randomized into one of two groups according to a pre-existing randomization list:

group “late diabetic without insulin (LD)” were 10 ZDF fa/fa diabetic animals kept

until 18 weeks of age. Group “late diabetic with insulin (LDI)” were 10 ZDF fa/fa

animals, which received 1½ subcutaneous insulin implants (LinPlant, LHR-10BV,

LinShin, Toronto, Canada) using a custom-made trocar G12-SS, LinShin) at 10

weeks of age. The latter were dosed according to weight at 10 weeks, approximately

300g, and released approximately 3U insulin per day for a period of 60 days,

covering our experimental period.15

Groups LC, LD, and LDI underwent a second,

“late baseline” electrophysiologic testing at 18 weeks of age, followed by sciatic

nerve block. One week later, behavior and electrophysiology tests were repeated,

and tissue excision for neurohistopathology was performed.

Serum glucose levels were measured in all experimental groups in blood

drawn from the left tail vein, using a commercially available glucose meter (Blue,

FIA Biomed, Emsdetten, Germany), regularly calibrated using the LI and LII

calibration solutions (FIA Biomed). In groups EC and ED, this was done at 10 and

11 weeks, and in groups LC, LD, and LDI, this was done at 10, 14, 16, and 18

weeks of age.

Electrophysiology

With temperature maintained well above 34°C using a warming blanket

(HK25, Beurer, Ulm, Germany), we studied the sciatic and caudal nerve with

monopolar needle electrodes as described previously using a Nicolet Viking IVP

electromyography system (Nicolet, Madison, WI).16

In brief, for motor conduction

studies of the sciatic nerve the recording cathode was placed in the intrinsic muscles

between the hallux and the second digit, and the recording anode was placed

subcutaneously on the lateral surface of the fifth digit. Stimulating electrodes were

inserted 3 mm apart at the medial ankle, and just cranial to the sciatic notch. A


189

grounding electrode was attached between the stimulating and the recording

electrodes. Supramaximal square-wave pulses of 0.1 ms duration were delivered.

Supramaximal stimulation was achieved by increasing the intensity by 25-30%

above the maximal stimulation. Compound muscle action potential (CMAP)

amplitudes (peak to peak) were recorded. Motor nerve conduction velocity (MNCV)

was calculated over the segment between the sciatic notch and the ankle, and

minimal F-wave latency was measured from seven F-wave recordings.

For studies of the mixed sensory and motor caudal nerve,17

the recording

cathode and anode were inserted at the base of the tail just 5 mm apart. The

stimulating cathode was placed laterally in the tail at exactly 3 cm from the base of

the tail; the stimulating anode was inserted 5 mm distal to the stimulating cathode.

The earth electrode was attached halfway between the stimulating and recording

electrodes. Supramaximal square-wave pulses of 0.5 ms duration were delivered.

Compound nerve action potential (CNAP) amplitudes (negative peak) were

recorded. The nerve conduction velocity (NCV) of the tail nerve was calculated

form the latency of the stimulus artefact to the onset of the negative peak of the

action potential elcited and the distance between the stimulating and the recording

cathodes, which was a standard distance of 3 cm. All measurements were carried out

by one investigator (P.L.), and electrophysiological measurements underwent

blinded assessment and validation by an experienced neurophysiologist (C.V.).

Sciatic nerve block

Nerve block was performed percutaneously combining the technique

described by Thalhammer et al.18

modified by nerve stimulation as described by

Kroin et al.10

In brief, a 25G needle was introduced just caudal to the sciatic notch

directed cephalad, and connected with a clip to the Viking electromyography system

programmed to deliver a pulse of 0.1 ms duration, and 0.6 mA current, triggered

manually. Ipsilateral hind-leg kick in the absence of local stimulation was taken as

sign of proximity of needle to nerve, and injection of 0.2 mL of lidocaine 2% was

performed. We defined a successful nerve block on the basis of three signs:

1) before injection, successful nerve stimulation at 0.6 mA current,

Chapter 3.4

190

2) gradual disappearance of the CMAP in electrophysiological recordings

after injection of lidocaine, and

3) subsequent behavioural testing showing absence of the toe-spreading

reflex.

The latter reflex, used to test sciatic nerve fibres, was tested as described

before by Kroin et al.10

Animals were gently lifted, resulting in a physiologic

vestibular reflex where toes are extended and spread. We noted the presence or

absence of these findings to characterize block duration every 15 minutes until the

block subsided. This gross behavioural testing was repeated before the animals were

subjected to anaesthesia one week after nerve block to detect any permanent nerve

injury.

Neurohistopathology

The main outcome parameter was the nerve injury score of the left sciatic

nerve one week after nerve block. To this end, after the last electrophysiological

measurements and animal euthanasia, left sciatic nerves were excised from animals.

The segment proximal and distal from the site of injection was harvested, and fixed

with a 2% buffered formalin solution, embedded in paraffin, cut at six microns in

longitudinal and transverse sections and stained with hematoxylin-eosin (H.E.) and

Masson trichrome. Samples were examined under the light microscope for evidence

of inflammation in the epineurial, perineurial and endoneurial compartment,

vascular injury and nerve fibre injury. Nerve fibre injury was assessed employing a

simple, semi-quantitative four point score where 0 represents a normal nerve and 4

represents extreme injury with inflammation and destruction of all components of

the nerve including axons and myelin, extending throughout the nerve bundle.19

The

pathologist (U. de G.) was blinded to experimental group allocation.

Statistical analysis

Semiquantitative neurohistopathological data such as the primary outcome

were compared by Friedman test followed - if significant - by Mann-Whitney U test.

Power analysis revealed that a group size of 10 animals would have 80% power to

reject the null hypothesis that the neurohistopathology score in the one group is


191

significantly different from another group of nerves using Mann-Whitney U test

with a 0.05 two-sided significance level. Body weight, blood glucose level and

neurophysiologic data were compared by analysis of variance (ANOVA) between

groups followed by posthoc Bonferroni test for multiple comparisons. Variations of

neurophysiological data over time were compared by paired samples T test. P < 0.05

was considered significant. Statistical analysis was performed with IBM SPSS®

Statistics Version 20 (IBM, San Fransciso, CA). Power analysis was done with the

aid of nQuery Advisor® 7.0 (Statistical Solutions Ltd., Cork, Ireland).

Results

All animals survived to the end of the experiment, no animal needed

analgesic rescue, and no animal fulfilled predefined criteria for termination of

experiments (humane endpoints). Welfare assessment showed no abnormalities

concerning appearance or behaviour at any time point. All animals showed clinical

recovery from sciatic nerve block.

Early diabetes

At baseline (10 weeks), mean glucose value was 7.1 ± 1.0 in EC and 13.1 ±

4.9 mmol/l in ED animals (P < 0.01). Mean body weight was 288.8 ± 8.5

respectively 344.1 ± 16.7 g (P < 0.001). In EC versus ED nerves, sciatic nerve

MNCV was 39.3 ± 4.2 versus 35.7 ± 2.5 m/s at baseline (P = 0.02), minimal F-wave

latency was 7.3 ± 0.5 ms versus 7.9 ± 0.6 (P < 0.005).

Sciatic nerve block duration was 45 ± 13 min in the EC, and 67.5 ± 27 min

in the ED group (P = 0.08).

The differences between electrophysiological parameters at baseline and 1

week after sciatic nerve block were calculated. Mean MNCV was decreased 1 week

after block compared to baseline across EC and ED animals (P < 0.01). There was

no difference between EC and ED animals in their change over time. We found no

significant differences in electrophysiological CMAP parameters for controlled and

EC animals at baseline and after nerve block.

In histopathological investigations most specimens in the EC and ED group

showed mild chronic inflammation in the epineurium and in the adipose tissue. In

Chapter 3.4

192

the EC group, 1/6 animals had a minimally elevated nerve injury score of “1+” (out

of 4), compared to 3/10 animals in the ED group with an elevated nerve injury score

of “1+” (n=1) and “2+” (n=2, n.s.).

Late diabetes

Weight and glucose levels of test animals are given in Table 1. Diabetic

animals were randomized at 10 weeks of age to receive insulin treatment (LDI) or

no treatment (LD). At 14 weeks of age, the mean glucose values were significantly

lower in LDI than in LD animals. However, thereafter the difference became not

significant (Table 1). Neurophysiologic data of conduction velocity and minimal F-

wave latency are given in Figure 2. Conduction velocity increased over time in the

LC group whereas it remained unaltered in LD and LDI animals. The conduction

velocity at 18 weeks in the LD animals was significantly lower than in the LC group

and remained significantly different one week after sciatic nerve block. After nerve

block, conduction velocity tended to decrease in all groups, but this decrease did

neither reach statistical significance in any of the separate groups nor when data of

all groups were pooled (p = 0.15, Figure 2A). We found no significant differences in

electrophysiological CMAP parameters for LC and LD animals before and after

nerve block. Caudal NCV was slowed significantly when comparing animals in

groups LC (65.3 ± 12 m/s) and LD (57.1 ± 8 m/s, P < 0.05).

At 18 weeks, minimal F-wave latencies were significantly prolonged in the

LD as compared to the LC group. At one week after nerve block the latency

increased significantly, if data of all three groups (LC, LD, LDI) were pooled

(Figure 2B). Decreases in minimal F-wave latency between the three experimental

groups were not significant.

Block duration was shortest in the LC group, longest in the LD group, and

intermediate in the LDI group (Figure 2C). The LDI animals had a longer block than

the LC animals, but there was no significant difference in block duration between

LD and LDI animals. LD animals had a block duration significantly longer (94.5 ±

33.2 min) than ED animals (67.5 ± 27 min, P < 0.05).

In histopathological investigations, we noted only minor changes. LC

animals showed minimal, variable, multi-focal chronic inflammatory infiltrates in


193

epineural connective tissue, generally not extending into the endoneurial or

perineurial compartments. One animal out of the LDI group and one animal out of

the LD group showed focal oedema or focal area of acute myelin injury and axonal

damage, associated with scattered inflammatory cells. The neurohistopathological

changes were not significant between groups (Figure 3).

Discussion

Zucker Diabetic Fatty rats at 10 weeks of age did not show prolongation of

duration of sciatic block with lidocaine 2% and did not show a discernible impact on

nerve damage one week after sciatic nerve block. In rats 18 weeks of age, with more

longstanding diabetes and clear signs of neuropathy, sciatic nerve block duration

was substantially prolonged, but no gross behavioural signs, or increased

neurohistopathological signs of nerve injury were found one week after sciatic nerve

block. Electrophysiologic changes suggestive of subtle nerve dysfunction after nerve

block were present in all experimental groups, but this was not related to the

duration or severity of the neuropathy.

The ZDF model used in the present study represents the important patient

collective of Type II DM more accurately than the previously used STZ-induced

Type I DM model.7 Notably, pathogenesis differs considerably between Type I and

Type II diabetes in experimental models,20

and in humans,1 such that implications

for regional anaesthesia should preferably be undertaken in a model most closely

resembling the clinical situation.7 However, all previous investigations had been

conducted in models of Type I DM in vivo,10 21-23

or in Type II DM in vitro.11

Our

investigation is the first to investigate the effects of a DPN secondary to Type II DM

on toxicology and function of sciatic nerve blockade.

Early diabetes

In our model, ED rats at 10 weeks of age had mildly decreased nerve

conduction velocities, indicating a mild diabetic neuropathy. The neuropathy in

these rats develops over time and our measurements of neuropathy correspond well

with previous literature.24

Duration of nerve block was not significantly prolonged

(P = 0.08) in ED as compared to EC animals. We found no significant

Chapter 3.4

194

neurohistopathologic or gross behavioural signs of nerve damage one week after

sciatic nerve block. Our results concerning neurotoxicity correspond to previous in

vitro investigations in the ZDF model at 12 weeks of age,11

and with similar findings

in an in vivo model of Type I DM.23

Late diabetes

LD animals at 18 weeks of age had decreased nerve conduction velocities

as compared to LC animals, indicating the development of a more severe diabetic

neuropathy over time. This corresponds to previous literature.25

We found that

while there was a slight increase in MNCV in LC over time, the MNCV of LD did

not show such an increase. This is in concordance with previous work by Oltman

and colleagues, where a slight increase in MNCV in lean (healthy) animals over

time until the 20th

week can be observed.24

Also, we corroborated our MNCV

findings by simultaneous measurements of the caudal NCV, confirming diabetic

neuropathy also in this predominantly sensory nerve.

We found no histopathological signs of increased neurotoxicity in LD

animals as compared to age-matched LC animals, when lidocaine 2% was used to

elicit sciatic nerve block. Our results mirror previous investigations in type I DM, in

which lidocaine at clinical concentrations had limited neurotoxic effects.10 21

There

have been no in vivo local anaesthetic neurotoxicity investigations in type II diabetic

models at all, but recent in vitro data show only modest neurotoxicity of lidocaine

2%.11

Our data confirm and widen these in vitro results using a multifaceted testing

setup in vivo. Further, epidemiologic clinical outcome data suggest that even when

long-lasting local anaesthetics are used, nerve injury following neuraxial blockade in

patients with pre-existing neuropathy is rare. Specifically, in one retrospective study,

the incidence of apparent neuropathic complications after neuraxial anaesthesia was

2 in 325 patients.6 Across the past 20 years, 6 case reports describing association of

nerve damage with diabetic neuropathy following regional anaesthesia have been

published.5

We note highly significant prolongation of minimal F-wave latency as a

subtle marker of nerve dysfunction one week after sciatic nerve block. This has not

been previously described, but there was no difference between LD and LC animals,


195

such that this most likely represents a minor and unspecific sequel of nerve block.

The clinical importance of this remains unclear, and is most probably very limited.

There has been discussion whether regional anaesthesia in diabetic patients may

induce clinically unapparent damage which may promote progression of diabetic

neuropathy.7 However, the changes observed here are very small, and need to be

investigated in detail before any clinical relevance can be ascribed.

In LD animals, block duration was significantly prolonged, while LDI

animals had an intermediate increase in block duration. This finding was expected

on basis of previous findings. Our results differ in magnitude from our previous

manuscript, in which several tests were used to quantify motor, deep sensory, and

superficial sensory block.11

In comparison, our rather crude “on/off” testing for a

vestibular reflex in this study more closely reflects the data obtained by Kroin, who

used the same method.10

The main difference with the latter study is that we used 0.2

mL of 2% lidocaine as in our previous study,11

whereas Kroin used 0.1 mL of 1%

lidocaine.10

Several studies assessed block duration in a streptozotocin-induced rat

model of type I DM, and while one study found no difference,21

three studies

showed prolonged block duration in diabetic rats.10 22 23

This latter finding was

confirmed in the ZDF rat model of type II DM by us.11

Recently, two clinical studies

demonstrated increased sciatic nerve block duration in diabetic patients.8 9

Therefore, the evidence strongly indicates that diabetic neuropathy will prolong the

duration of peripheral nerve block. This prolongation may be caused by

pharmacokinetic 26

or pharmacodynamic (e.g., modulation of sodium channels by

neuropathy 27

) mechanisms, but the respective contributions remain unclear.

Potential clinical implications are to consider the diabetic nerve “more sensitive” to

the effects of local anaesthetics, and it has been proposed to reduce dose of local

anaesthetics when performing nerve blocks for perioperative analgesia.4 Also, it had

been suggested to reduce or omit epinephrine from peripheral nerve blocks in

neuropathic patients,4 and the results obtained in experimental and clinical settings

would indicate that nerve block duration in diabetic neuropathy will be prolonged

anyway, even without the need to add adjuvant epinephrine.

Chapter 3.4

196

Limitations

In the LDI rats the effects of insulin were not sufficient to cause glucose

levels to be similar to those in control animals, even though the same dose achieved

good glucose control in type I 23

and type II 28

models of DM. This may be because

insulin was dosed according to body weight at baseline (10 weeks), and diabetic

animals were substantially heavier at the end of the experimental period, leading to

relative under-dosing towards the end of the experimental period, which is supported

by the increasing blood glucose levels over time in these animals. Therefore, our

insulin regimen was more representative of loose glycaemic control than of strict

glycaemic control.

In diabetic as well as control animals, small inflammatory changes were

noted on neurohistopathological investigation in all groups, which may be explained

by repeated stimulation. Sciatic nerve inflammation after repetitive stimulation, as

occurred in our study due to neurophysiological measurements, has been described

in vivo before.29

The timepoint of excision was chosen on basis of earlier

experiments by our group, but differ with the timepoint chosen by Kroin et al.10

(2

days post block). Seen that the mild changes after block were seen both in the study

by Kroin and our study, these results can be interpreted to support and strengthen

each other.

The model used by us cannot be extrapolated directly to the clinical

situation. First, the age of the experimental rat can be compared to that of a young

human adult when estimating on basis of physiological and behavioural

parameters.30

However, it should be noted that we chose the age of our test animals

based on the age at which neuropathy typically develops, which is around 20

weeks.24

Second, the precise interspecies difference between rats and humans

concerning neuropathy and toxicity of local anaesthetics is unclear. Nevertheless,

rodent models have been used to investigate diabetes and its neuropathy,24

and

determine functional and toxicological aspects of regional anaesthesia in the past.31

Lastly, we attest to the fact that the focus of behavioural testing after sciatic

nerve block was on the motor component of the nerve block, while DPN profoundly

affects sensory function as well.1 However, in a previous study using the same


197

model, we obtained comparable prolongations of both motor and sensory blockade

upon sciatic nerve blockade.11

Ethical considerations

We sought to minimize animal distress by conducting all invasive

procedures such as electrophysiology and nerve block under general anaesthesia. To

limit tissue injury and prevent post-interventional pain, we performed nerve blocks

percutaneously as described by Kroin et al.,10

and not open. At the same time, this

theoretically entails that the position of injection is less reliable, and intraneural

injection is possible. However, the only study directly comparing these two modes

of injection found comparable results for both approaches,10

such that we felt

confident to perform our blocks percutaneously. The same percutaneous approach

using thin needle electrodes was chosen for electrophysiological measurements. To

avoid repetitive injections of insulin for the animals randomised to the late group,

we used subcutaneous implants. To minimize the number of animals used in

experiments, we obtained approval to use the heart and the right sciatic nerve of our

test animals for pilot experiments investigating enzyme expression and epigenetic

markers in type II diabetes, respectively.

Future perspectives

We describe a novel comprehensive model to investigate toxicological and

functional consequences of diabetic neuropathy in vivo, combining behavioural,

electrophysiological and histopathology methods. Despite lidocaine being the most

widely used local anaesthetic for toxicity research, results obtained by Kroin et al.

suggest that longer-acting local anaesthetics such as bupivacaine and/or ropivacaine

may be more toxic with respect to neurohistopathology.10

We suggest to investigate

the neurotoxic potential of long-lasting local anaesthetics such as bupivacaine, and

the value of adjuvants, which may increasingly become relevant in clinical practice.7

26 32 Similarly, the pharmacokinetics and –dynamics of the diabetic nerve as relevant

to regional anaesthesia warrant further investigation.

Chapter 3.4

198

Conclusions

Our results suggest increased sensitivity of diabetic nerves for short-acting

local anaesthetics without adjuvants in vivo, as evidenced by prolonged block

duration in a rodent Type II diabetes mellitus model with longstanding diabetic

neuropathy. This sensitivity appears to increase with progression of neuropathy. We

observed very subtle changes suggestive of nerve injury after nerve block in general,

with no correlate in gross behavioural testing or neurohistopathology, and no

specific effect of neuropathy. Our results do not support the hypothesis that

neuropathy due to Type II diabetes mellitus increases the risk of nerve injury after

peripheral nerve block.

Acknowledgments

We acknowledge the expert technical assistance of Marian Slaney BSc in

preparing neurohistopathologic samples for analysis, of Shu-Hsien Sheu MD PhD

for digitalization of neurohistopathologic specimens, and of Susanne van Dieren

PhD for statistical advice.

References

1 Said G. Diabetic neuropathy--a review. Nat Clin Pract Neurol 2007; 3: 331-40

2 Dhatariya K, Levy N, Kilvert A, et al. NHS Diabetes guideline for the

perioperative management of the adult patient with diabetes. Diabet Med 2012; 29:

420-33

3 Malinzak EB, Gan TJ. Regional anesthesia for vascular access surgery. Anesth

Analg 2009; 109: 976-80



5 Lirk P, Rutten MH, Haller I, et al. Management of the patient with diabetic

peripheral neuropathy presenting for peripheral regional anesthesia: a European

survey and review of literature. Minerva Anestesiol 2013; 79: 1039-48

6 Hebl JR, Kopp SL, Schroeder DR, Horlocker TT. Neurologic complications after

neuraxial anesthesia or analgesia in patients with preexisting peripheral


199


9

7 Ibinson JW, Mangione MP, Williams BA. Local Anesthetics in Diabetic Rats

(and Patients): Shifting From a Known Slippery Slope Toward a Potentially Better

Multimodal Perineural Paradigm? Reg Anesth Pain Med 2012; 37: 574-6

8 Sertoz N, Deniz MN, Ayanoglu HO. Relationship between glycosylated

hemoglobin level and sciatic nerve block performance in diabetic patients. Foot

Ankle Int 2013; 34: 85-90

9 Cuvillon P, Reubrecht V, Zoric L, et al. Comparison of subgluteal sciatic nerve

block duration in type 2 diabetic and non-diabetic patients. Br J Anaesth 2013; 110:

823-30



50

11 Lirk P, Flatz M, Haller I, et al. In Zucker Diabetic Fatty Rats, Subclinical

Diabetic Neuropathy Increases In Vivo Lidocaine Block Duration But Not In Vitro

Neurotoxicity. Reg Anesth Pain Med 2012; 37: 601-6

12 Galley HF. Mice, men, and medicine. Br J Anaesth 2010; 105: 396-400

13 Brussee V, Guo G, Dong Y, et al. Distal degenerative sensory neuropathy in a

long-term type 2 diabetes rat model. Diabetes 2008; 57: 1664-73

14 Oh SS, Hayes JM, Sims-Robinson C, Sullivan KA, Feldman EL. The effects of

anesthesia on measures of nerve conduction velocity in male C57Bl6/J mice.

Neurosci Lett 2010; 483: 127-31

15 Price SA, Agthong S, Middlemas AB, Tomlinson DR. Mitogen-activated protein

kinase p38 mediates reduced nerve conduction velocity in experimental diabetic

neuropathy: interactions with aldose reductase. Diabetes 2004; 53: 1851-6

16 Verhamme C, King RH, ten Asbroek AL, et al. Myelin and axon pathology in a

long-term study of PMP22-overexpressing mice. J Neuropathol Exp Neurol 2011;

70: 386-98

17 Schaumburg HH, Zotova E, Raine CS, Tar M, Arezzo J. The rat caudal nerves: a

model for experimental neuropathies. J Peripher Nerv Syst 2010; 15: 128-39

Chapter 3.4

200

18 Thalhammer JG, Vladimirova M, Bershadsky B, Strichartz GR. Neurologic

evaluation of the rat during sciatic nerve block with lidocaine. Anesthesiology 1995;

82: 1013-25

19 Richardson EP, de Girolami U. Pathology of the Peripheral Nerve. Philadelphia:

WB Saunders, 1995

20 Schmidt RE, Dorsey DA, Beaudet LN, Peterson RG. Analysis of the Zucker

Diabetic Fatty (ZDF) type 2 diabetic rat model suggests a neurotrophic role for

insulin/IGF-I in diabetic autonomic neuropathy. Am J Pathol 2003; 163: 21-8





23 Kroin JS, Buvanendran A, Tuman KJ, Kerns JM. Effect of Acute Versus

Continuous Glycemic Control on Duration of Local Anesthetic Sciatic Nerve Block

in Diabetic Rats. Reg Anesth Pain Med 2012; 37: 595-600

24 Oltman CL, Coppey LJ, Gellett JS, Davidson EP, Lund DD, Yorek MA.

Progression of vascular and neural dysfunction in sciatic nerves of Zucker diabetic

fatty and Zucker rats. Am J Physiol Endocrinol Metab 2005; 289: E113-22

25 Etgen GJ, Oldham BA. Profiling of Zucker diabetic fatty rats in their progression

to the overt diabetic state. Metabolism 2000; 49: 684-8




27 Bierhaus A, Fleming T, Stoyanov S, et al. Methylglyoxal modification of

Na(v)1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic

neuropathy. Nat Med 2012; 18: 1445

28 Jain K, Asina S, Yang H, et al. Glucose control and long-term survival in

biobreeding/Worcester rats after intraperitoneal implantation of hydrophilic

macrobeads containing porcine islets without immunosuppression. Transplantation

1999; 68: 1693-700

29 Voelckel WG, Klima G, Krismer AC, et al. Signs of inflammation after sciatic

nerve block in pigs. Anesth Analg 2005; 101: 1844-6


201

30 Quinn R. Comparing rat's to human's age: how old is my rat in people years?

Nutrition 2005; 21: 775-7




32 Williams BA, Tsui BC, Hough K, Ibinson JW. In Vitro Cytotoxicity Evaluation

of Ropivacaine + Clonidine-Buprenorphine-Dexamethasone-Midazolam American

Society of Anesthesiologists Annual Meeting. San Diego, CA, 2010

Chapter 3.4

202

Table 1

Body weight (gr)

Blood glucose (mmol)

Weight and blood glucose levels of all groups over time.

Tested by ANOVA with posthoc Bonferroni correction. n.s. = not significant

Weeks 10 14 16 18

LC (n=10) 282 ± 14 364 ± 17 386 ± 23 402 ± 37

LD (n=10) 345 ± 19 419 ± 39 427 ± 38 435 ± 36

LDI (n=10) 350 ± 20 471 ± 25 484 ± 35 494 ± 41

LC vs. LD p < 0.001 p < 0.001 p < 0.05 n.s.

LC vs. LDI p < 0.001 p < 0.001 p < 0.001 p < 0.001

LD vs. LDI n.s. p < 0.01 p < 0.01 p < 0.01

Weeks 10 14 16 18

LC (n=10) 7.6 ± 1.9 6.0 ± 0.5 6.2 ± 0.5 8.1 ± 1.2

LD (n=10) 11.9 ± 5.3 24.2 ± 5.1 25.5 ± 3.2 28.4 ± 3.4

LDI (n=10) 11.5 ± 4.6 17.8 ± 7.4 21.6 ± 5.0 25.2 ±5.5

LC vs. LD n.s. p < 0.001 p < 0.001 p < 0.001

LC vs. LDI n.s. p < 0.001 p < 0.001 p < 0.001

LD vs. LDI n.s. p < 0.05 n.s. n.s.


203

Figure legends

Figure 1

Timeline of experimental interventions.

Figure 2

Development of sensible conduction velocity (2A) and minimal F-wave latency (2B)

over time in the three experimental groups. After nerve block, conduction velocity

tended to decline in all groups, but did not reach statistical significance even if all

groups were pooled (p = 0.15).

After the nerve block the minimal F-wave latency increased significantly, when the

data of all three groups were pooled.

Analysis of variance (ANOVA) with posthoc Bonferroni test, paired samples T test.

* P < 0.05; # P < 0.01; ## P < 0.001.

Figure 3

Sciatic nerve motor block duration in late diabetic animals. Analysis of variance

(ANOVA) between groups followed by posthoc Bonferroni correction. * P < 0.05; #

P < 0.01; ## P < 0.001.

Figure 4

Neurohistopathology of a healthy control nerve nerve (A), and a nerve from a

diabetic animal, showing focal areas of acute myelin injury and axonal damage,

associated with scattered chronic inflammatory cells (B).

Chapter 3.4

204

Figure 1


205

Figure 2: Neurophysiology of tibial nerve

2A

Chapter 3.4

206

2B


207

2C

Chapter 3.4

208

Figure 3

Management of the neuropathic patient

209

Chapter 3.5

Management of patients with diabetic neuropathy

Based on

Lirk P, Rutten MVH, Haller I, Stevens MF,

Laudolff-Birmingham J, Hollmann MW, Birmingham B

Survey of the management of the patient with diabetic peripheral

neuropathy presenting for regional anesthesia.

Minerva Anesthesiol 2013; 79: 1039-48.

Chapter 3.5

210

Introduction

Diabetes mellitus is one of the most common diseases worldwide, and its

prevalence is predicted to increase tremendously within a decade.1 Among other

end-organ complications, it may lead to diabetic peripheral neuropathy (DPN). It is

estimated that about 10% of diabetic patients presented with neuropathy at the time

of diagnosis, while more than 50% of diabetic patients will develop neuropathy

during the subsequent years, making it the most common neuropathy worldwide.2

The diagnosis of diabetic neuropathy may influence perioperative

management, especially the decision whether or not to perform regional anaesthesia.

Reasons to perform surgery under regional anaesthesia may include the wish to

avoid general anaesthesia in light of comorbidities. Some types of surgery for

diabetic complications are highly suitable for regional anaesthesia, e.g.,

arteriovenous fistula in end-stage renal disease.3 On the other hand, DPN may

increase risk of neurological injury following regional anaesthesia.4

Recommendations have been issued regarding the performance of peripheral

regional anaesthesia in patients with DPN arguing for, among others, decreased

doses of local anaesthetics to decrease the chance of adverse outcome.5

However, these recommendations rest on a very limited evidence-base.

There is limited epidemiological evidence suggesting that DPN may predispose

patients to new-onset neurological symptoms,6 and some case reports describe

associations between adverse outcomes of regional anaesthesia and pre-existing

neuropathy.4 7-12

Experimental evidence is equivocal, and there are studies to support

13 14 and to refute

15 16 increased toxicity of local anaesthetics in DPN. Recently,

increased attention has been paid to the possibility that DPN may prolong nerve

block duration.15 16

It has not been determined whether these controversial results and

recommendations have been incorporated into clinical practice. Therefore, this study

first sought to assess reported practice in the perioperative management of patients

with DPN presenting for peripheral regional anaesthesia among members of the

European Society of Regional Anaesthesia and Pain Therapy (ESRA). Second, we

wanted to contrast findings from our survey with a review of a current status of the

literature concerning peripheral regional anaesthesia in diabetic neuropathic patients.


211

We hypothesized that the majority of responders would consider DPN a potential

risk factor for nerve damage in peripheral regional anaesthesia, and would adapt

their technique.

Materials and Methods

Survey

The survey was approved by the ESRA Scientific Board. We developed a

web-based survey questionnaire in English using an online survey engine

(www.surveymonkey.com), validated in a pilot study by two study authors (PL,

MVR) and one independent anaesthetist. An anonymous e-mail containing an

invitation to participate in the survey was distributed to members of ESRA by the

society’s secretariat in June 2012, with an anonymous reminder email sent two

weeks later, and the survey remained open for two months. To ensure

confidentiality, survey responses did not contain personal information or data which

would allow identification of an individual or his institution. No free text input was

possible. The survey collected demographic data and assessed practitioners’ routine

methods for preoperative assessment and intraoperative management of patients

with suspected diabetic neuropathy presenting for regional anaesthesia. The

complete survey questionnaire can be found in the Appendix. To increase the

response rate, participants were given the opportunity to sign up for the drawing of

three textbooks of anaesthesia following the survey. All personal information

entered for this purpose was kept separate from survey answers at all times.

Literature review

For each section of the literature review (“local anaesthetic neurotoxicity”,

“regional anaesthesia adjuvants”, “nerve block duration” and “nerve stimulation”),

we performed a comprehensive literature search for reports in journals indexed in

MEDLINE covering manuscripts until November 2012, with reference lists of

retrieved articles searched for additional trials or reports. The initial search strings

were “diabetic neuropathy” and “regional anaesthesia”, and “neurotoxicity”.

Chapter 3.5

212

Statistics

All responses were stored on a secure database and were exported to IBM

SPSS Statistics 20 (Armonk, NY) for analysis. The data collected in this survey

were mostly categorical in nature. Demographic variables are strictly categorical,

while the variables coded on the Likert scale were considered ordered. We first

carried out single (uni-variate) analysis of the data collected for each variable.

Subsequently, we constructed contingency tables to study association between

answers related to our main hypothesis, and Pearson’s Chi-square test for

independence was used to discover if there is a relationship between two categorical

variables. To address the concern of small expected numbers of values in

crosstabulation, some of the variables were recoded combining levels of the variable

with low cell counts.

Results

Survey

A total of 3,343 emails were sent to members of the European Society of

Regional Anaesthesia (ESRA), of which 236 could not be delivered, resulting in a

final number of 3,107 emails delivered. We received 584 responses with fully

completed questionnaires, which represented an overall response rate of 19%.

Demographics - We received responses from European anaesthesiologists (34

countries, 509 responders), while 69 participants were located outside of Europe and

6 participants gave no information. The countries with high numbers of responders

were the United Kingdom (n = 164, 24 %), followed by France, Belgium,

Netherlands and Italy, each one accounting for more than 5 % of the respondents.

Anaesthesia training had been completed by 91% of responders. While more than

80% of responders stated the amount of regional anaesthesia performed at their

institution was below 40 %, the personal practice of our responders seemed slightly

above what was practiced at their institution. Demographic data are given in detail in

Table 1.


213

Block technique - When asked about the main techniques used for nerve localization,

ultrasound was preferred (51%), with double guidance (ultrasound, nerve stimulator;

41%) and nerve stimulator (30%) alone used less frequently. Other techniques were

used very seldom (Table 2A). Anaesthesiologists with more years of experience

tended to use nerve stimulator techniques more often and they were less likely to use

ultrasound. The same trend was observed for anaesthesiologists from institutions

with a higher utilization of peripheral regional anaesthesia. Similarly,

anaesthesiologists at smaller hospitals tended to use nerve stimulator more often,

and dual guidance less often than their colleagues at larger hospitals.

Neuropathy - When asked about the approach to the diabetic patient, a minority of

participants would order a neurologic consult and the same holds true for patients

with confirmed diabetic neuropathy (Table 2B). Anaesthesiologists from smaller

hospitals, with more years of experience or who performed more peripheral regional

anaesthesia were more likely to consult a neurologist in case of diabetes and/or

neuropathy. In neuropathic patients 17 % of participants stated they would be

inclined to avoid peripheral nerve blocks, yet 59 % of participants would counsel

patients about possibly increased risk of nerve damage.

In patients with confirmed neuropathy, approximately a quarter of participants

would modify the technique of regional anaesthesia, a third would do so sometimes,

and while 41 % rarely or never do so (Table 2C). When technique was to be altered,

43 % of responders stated they would usually or always decrease the dose of local

anaesthetic, almost 77 % would regularly decrease or omit epinephrine, and 40 %

would accept a higher nerve stimulation threshold (Table 2D).

Personal valuation - Figure 1 summarizes the respondents’ agreement or

disagreement with to statements regarding the risk of peripheral regional anaesthesia

in patients with diabetic neuropathy. Answers were not associated with years of

experience or site of practice.

Anaesthesiologists who agreed that performing peripheral nerve blocks on patients

with diabetic peripheral neuropathy increases their risk of postoperative neurologic

deficit (Figure 1A), and those who agreed that regional anesthesia in DPN patients

Chapter 3.5

214

would increase medical liability risk (Figure 1B) were more attentive to duration and

severity of diabetes and diabetic neuropathy, and more likely to inform the patient

about increased risk concerning peripheral regional anaesthesia. Moreover, they

were more likely to alter their technique or avoid regional anesthesia at all.

Anaesthesiologists who agreed to the statement that general anaesthesia is, in

principle, superior to regional anaesthesia in diabetic neuropathic patients (Figure

1C) tended to use a nerve stimulator alone, and were less likely to use ultrasound. In

addition to being more attentive to symptoms of diabetes and DPN, these colleagues

were more inclined to order neurology consults preoperatively, change technique, or

avoid nerve blocks altogether.

Anaesthesiologists who agreed with the statement that peripheral nerve

blocks can be performed safely in patients with diabetic neuropathy (Figure 1D)

tended to use ultrasound alone, and not nerve stimulation. They were also less likely

to order a neurologic consult for patients with diabetes or diabetic peripheral

neuropathy. In addition they were less inclined to counsel their patients of an

increased risk of nerve damage, and they did not alter their technique of peripheral

nerve blocks.

Regional practice differences – The only geographical sub-group of responders

lending itself to in-depth analysis was responders from the United Kingdom,

representing 24 % of all responses. In comparison to responses from colleagues

from continental Europe, UK anaesthetists tended to use less regional anaesthesia in

their daily practice, and tended to use the nerve stimulator technique less often. They

more often inquired about the symptoms of peripheral neuropathy but were less

likely to order neurologic consults. They also less likely to modify their technique

than their counterparts in Continental Europewere, and more likely to avoid

peripheral nerve blocks in neuropathic patients altogether. They were more likely to

counsel the patient about the increased risk of regional anaesthesia.

Future research - Finally, 82 % of responders agreed with the statement that

research regarding the safety of peripheral nerve blocks in patients with diabetic

peripheral neuropathy should be given high priority in the future.


215

Literature search

Results of the literature search are summarized in Tables 3 and 4. No

randomized controlled trial examining potential adverse effects of peripheral

regional anaesthesia in diabetic neuropathy was found. We retrieved one

epidemiological study, one prospective clinical trial examining spinal block duration

in diabetic patients, two clinical trials examining sciatic nerve block duration, one

retrospective investigation examining block success in diabetic patients, and 6 single

case reports (Table 3). Moreover, we found 7 experimental studies in three different

animal models of diabetes (Table 4).

Discussion

The results from our literature search reveal inconsistent findings

concerning a potential increase in local anaesthetic-induced neurotoxicity in diabetic

patients, but there is consensus that diabetic neuropathy will prolongs block duration

(Table 2). This view is mirrored in the survey. Few responders are inclined to

request a neurologic consult in patients with confirmed neuropathy prior to

performing peripheral regional anaesthesia, about a quarter of participants stated

they would avoid regional anaesthesia in patients with neuropathy, yet almost 60 %

would regularly counsel patients with neuropathy about increased risk of nerve

damage as a consequence of regional anaesthesia. When techniques are modified,

most participants decrease or omit epinephrine, while fewer respondents would

decrease dose of local anaesthetic or perform other adjustments.

In summarizing responses, two large groups became obvious: one smaller

group of regional anaesthetists who are more cautious concerning diabetic

neuropathy, more likely to use a nerve stimulator only, and who are inclined to use

general anaesthesia in diabetic neuropathic patients. The second, larger, group is

characterized by the predominant use of ultrasound, and a more optimistic approach,

less likely to order consults, and of the opinion that nerve blocks can safely be

performed in diabetic neuropathic patients. Stratification according to years of

experience or site of practice does not explain this difference in attitude. In

analyzing the subgroup of UK anaesthetists, a more conservative approach was

observed, with a higher likelihood of avoiding regional anaesthesia in neuropathic

Chapter 3.5

216

patients, and increased chance of counseling neuropathic patients about perceived

greater risks of regional anaesthesia.

Local anaesthetic neurotoxicity

The respondents were divided regarding the risk of new-onset neurologic

deficit in neuropathic patients. Concerning risk of regional anaesthesia in these

patients, strong agreement or disagreement was very rare. This reflects the scarce

data support in this field. Guidelines issued by the American Society of Regional

Anesthesia (ASRA) pay attention to patients with pre-existing neurological disease,

such as DPN. With regards to local anaesthetic neurotoxicity, “consideration may be

given” to avoiding more potent local anaesthetics, and reducing the dose or

concentration of local anaesthetic.5

The clinical evidence that diabetic neuropathy may be a risk factor for

nerve damage rests, to a large extent, on case reports and case report series

summarized in Table 3. The only respective epidemiological study was conducted

on diabetic neuropathic patients undergoing neuraxial anaesthesia. Hebl and

coworkers retrospectively analyzed the incidence of new-onset neuropathy, and

found an incidence of 0.4%.6 In contrast with other recent estimates of risk,

17 this

seems substantially increased, but the number of complications observed was very

small and therefore, extrapolation is not straightforward.4

Clinical evidence is underpinned by equivocal experimental evidence. In a

first landmark study, Kalichman and Calcutt demonstrated in streptozotocin diabetic

rats (as a model of type I diabetes) that lidocaine was more toxic in diabetic

nerves.13

Using the same model, Kroin and coworkers found only small increases in

neurotoxic effects when long-lasting local anaesthetics, or adjuvants such as

epinephrine and clonidine were used.14

Results obtained in dissociated neurons of

Zucker Diabetic Fatty (ZDF) rats (as a model of type II diabetes) similarly show

only a marginal increase in toxicity when exposed to lidocaine in vitro.16

Other

evidence was obtained in a spinal injection model in type I diabetic rats. Even

though these results cannot be directly extrapolated to peripheral regional

anaesthesia, it is notable that no increased pathologic findings were observed when

animals were subjected to 4 subsequent injections of high-dose lidocaine (2%) or


217

bupivacaine (0.75%) with or without epinephrine.15

In conclusion, experimental

results are conflicting. One observed trend is that higher concentrations of local

anaesthetic are more likely to cause neurotoxic effects in diabetic animals (see Table

4). However, results from animal studies, including very specific models such as

streptozotocin-induced DM, can only be extrapolated to the clinical situation with

great care.

Based on the available evidence, it has been suggested to lower the dose of

local anaesthetic in the presence of severe DPN.4 In diabetic animals, recovery of

nerve function from anoxia or ischemia-reperfusion was significantly delayed as

compared to healthy control animals, suggesting that diabetic nerves are, in general,

more sensitive to toxic challenges than are healthy nerves (Table 4). Further research

should include late diabetic neuropathic patients and compare the influence of early

versus late neuropathy on the toxicological safety profile of local anaesthetics. It is

notable that despite the uncertainty in contemporary literature, approximately sixty

percent of responders stated to counsel neuropathic patients about the potentially

increased risk of nerve damage with regional anaesthesia. Many colleagues perform

clinical exams to determine preoperative status in patients with DPN. From a legal /

forensic point of view, this is highly recommended. It should be noted, however,

that many new-onset neurologic symptoms following regional anaesthesia are due to

factors unrelated to the blockade, such as positioning.4

Regional anaesthesia adjuvants

Among those respondents who would change nerve block technique in

patients with confirmed DPN, almost eighty percent would decrease or omit

epinephrine as adjuvant to local anaesthetics. This coincides with several

recommendations to decrease 5 18

or omit 4 epinephrine in diabetic neuropathic

patients. Whether epinephrine is acceptable in the context of a test dose remains to

be determined, and the theoretical risk of increased neurotoxicity needs to be

weighed against the danger of an unrecognized intravascular injection with potential

deleterious effects in its own right. As mentioned above, limited evidence suggests

epinephrine may increase toxic effects of lidocaine and other local anaesthetics

Chapter 3.5

218

because nerve blood flow is impaired, and the concentration of local anaesthetic

remains elevated for longer periods.14

Nerve block duration

While the first study by Kalichman 13

did not show a prolongation of block

duration, subsequent studies by others 14 15 19

and us 16

demonstrate increased block

duration in diabetic versus non-diabetic animals. Recent reports in patients support

the concept of longer block duration in diabetic patients. Specifically, Sertoz

investigated diabetic patients undergoing popliteal sciatic nerve block stratified into

three groups according to their glycosylated hemoglobin levels, a surrogate marker

of recent glucose regulation. Notably, block onset was delayed, and duration

prolonged in patients with the highest glycosylation levels.20

Next, Cuvillon and

coworkers showed that in patients with diagnosed DPN, ropivacaine-induced

subgluteal sciatic nerve block onset time was similar, but sensory and motor block

duration was prolonged by four hours, respectively.20

In line with these clinical

reports, a recent study in diabetic rats showed that acute glycemic control did not

reverse the duration of anaesthesia to normal levels, indicating that long-term

changes rather than the current blood glucose level are most likely responsible for

this phenomenon.19

The reason for increased sensitivity may be pharmacodynamic

(altered pattern of ion channel composition or modification in diabetic neurons 21

),

or pharmacokinetic (decreased absorption due to angiopathy 22

). Moreover, a recent

study comparing pregnant diabetics versus non-diabetics receiving lidocaine

epidural anaesthesia revealed decreased clearance of lidocaine and its metabolite

monoethylglycinexylidine (MEGX), indicating that diabetes as such may impair the

function of cytochrome P450 enzymes responsible for local anaesthetic

breakdown.23

Finally, a recent retrospective study found a higher block success rate

in diabetic patients undergoing surgery under supraclavicular block, potentially

because nerves are already partially numb due to neuropathy, or more sensitive

toward effects of local anaesthetics.24


219

Nerve stimulation

We found that a relatively large number of responders use nerve

stimulation, and almost 40 % of responders stated they were inclined to accept a

higher stimulation threshold in neuropathic nerves. A quarter of participants stated

that they would tend to avoid the use of a nerve stimulator in diabetic patients. It

seems reasonable that a nerve which has undergone degenerative changes and does

not conduct normal stimuli properly might not be optimally suited for nerve

stimulator guided regional anaesthesia. Experimental evidence in a small number of

diabetic dogs showed that the possibility of intraneural injection was increased in

diabetic versus healthy dogs.25

Recently, using ultrasound combined with nerve

stimulation, Bigeleisen and colleagues demonstrated that stimulation thresholds

were increased in diabetic patients.26

Severe diabetic neuropathy may be an

indication to prolong the stimulating pulse or employ double guidance (simultaneous

use of ultrasound and nerve stimulation).4

Limitations

Some limitations of the present study should briefly be addressed. First, the

response rate was low. However, considering the open and non-personal manner in

which society members were recruited into the survey we feel that the response rate

was as expected. To maximize response rate, we drafted an invitation letter

explaining the aim and potential importance of the study, devised a survey that could

be completed in less than five minutes, sent a reminder two weeks later, and invited

participants to sign up for a prize draw of three major textbooks of Anaesthesia. In

addition, we attest to the fact that the survey population was a selected group of

colleagues, with membership in a dedicated specialist society. Because almost three

quarters of responders perform > 20 % of regional anaesthesia in daily practice, we

surmise that the survey was completed by a group of anaesthesiologists with a

dedicated interest in regional anaesthesia. Our survey was designed in English,

which may have constituted a language barrier for some recipients of the invitational

email, precluding them from answering. Preliminary analysis of regional differences

showed that participants from the United Kingdom had a more conservative and

cautious stance when dealing with diabetic neuropathic patients. No data are

Chapter 3.5

220

available on the approach to the diabetic neuropathic patient in other world regions.

Future surveys focused on types of hospitals and different regions will be required to

delineate differences in attitudes and practices in more detail. Moreover, it may be

necessary to differentiate between various forms of neuropathy, specifically

concerning onset, treatment, and localization. Finally, our survey did not take into

account the distinctive pathophysiology of neuropathy during diabetes mellitus Type

I (primarily due to insulin shortage) and Type II (due to insulin resistance). Whereas

glucose control is very crucial in the neuropathy of Type I diabetes, the disease

aetiology is more complex in Type II diabetes. In the latter form, other factors such

as dyslipidemia contribute to the pathogenesis of DPN.2 Future clinical and

experimental studies will need to take these factors into consideration.

Conclusions

In conclusion, we report the results of the first survey analyzing attitudes and

standards of care among European anaesthesiologists with regards to peripheral

regional anaesthesia in Diabetic Peripheral Neuropathy. While literature is divided

on the question whether pre-existing diabetic neuropathy is a risk factor for new

neurological deficit after regional anaesthesia, many of the respondents take

measures to reduce risks, counsel patients on a perceived greater risk of neurologic

complications, and only a minority avoids peripheral regional anaesthesia altogether.

References

1 Inzucchi SE, Bergenstal RM, Buse JB, et al. Management of hyperglycemia in

type 2 diabetes: a patient-centered approach: position statement of the American

Diabetes Association (ADA) and the European Association for the Study of

Diabetes (EASD). Diabetes Care 2012; 35: 1364-79

2 Callaghan BC, Cheng HT, Stables CL, Smith AL, Feldman EL. Diabetic

neuropathy: clinical manifestations and current treatments. Lancet Neurol 2012; 11:

521-34

3 Reynolds TS, Kim KM, Dukkipati R, et al. Pre-operative regional block

anesthesia enhances operative strategy for arteriovenous fistula creation. J Vasc

Access 2011; 12: 336-40


221



5 Neal JM, Bernards CM, Hadzic A, et al. ASRA Practice Advisory on Neurologic

Complications in Regional Anesthesia and Pain Medicine. Reg Anesth Pain Med

2008; 33: 404-15

6 Hebl JR, Horlocker TT, Schroeder DR. Neuraxial anesthesia and analgesia in

patients with preexisting central nervous system disorders. Anesth Analg 2006; 103:


7 Waters JH, Watson TB, Ward MG. Conus medullaris injury following both

tetracaine and lidocaine spinal anesthesia. J Clin Anesth 1996; 8: 656-8

8 Lena P, Teboul J, Mercier B, Bonnet F. [Motor deficit of the lower limbs and

urinary incontinence following peridural anesthesia]. Ann Fr Anesth Reanim 1998;

17: 1144-7

9 Horlocker TT, O'Driscoll SW, Dinapoli RP. Recurring brachial plexus neuropathy

in a diabetic patient after shoulder surgery and continuous interscalene block. Anesth

Analg 2000; 91: 688-90

10 Al-Nasser B. Toxic effects of epidural analgesia with ropivacaine 0.2% in a

diabetic patient. J Clin Anesth 2004; 16: 220-3

11 Blumenthal S, Borgeat A, Maurer K, et al. Preexisting subclinical neuropathy as

a risk factor for nerve injury after continuous ropivacaine administration through a

femoral nerve catheter. Anesthesiology 2006; 105: 1053-6

12 Angadi DS, Garde A. Subclinical neuropathy in diabetic patients: a risk factor

for bilateral lower limb neurological deficit following spinal anesthesia? J Anesth

2012; 26: 107-10





50



Chapter 3.5

222

16 Lirk P, Flatz M, Haller I, et al. In Zucker Diabetic Fatty Rats, Subclinical

Diabetic Neuropathy Increases In Vivo Lidocaine Block Duration But Not In Vitro

Neurotoxicity. Reg Anesth Pain Med 2012; 37: 601-6



965-74



19 Kroin JS, Buvanendran A, Tuman KJ, Kerns JM. Effect of Acute Versus

Continuous Glycemic Control on Duration of Local Anesthetic Sciatic Nerve Block

in Diabetic Rats. Reg Anesth Pain Med 2012; 37: 595-600

20 Sertoz N, Deniz MN, Ayanoglu HO. Relationship between glycosylated

hemoglobin level and sciatic nerve block performance in diabetic patients. Foot

Ankle Int 2013; 34: 85-90

21 Bierhaus A, Fleming T, Stoyanov S, et al. Methylglyoxal modification of

Na(v)1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic

neuropathy. Nat Med 2012; 18: 1445




23 Moises EC, Duarte Lde B, Cavalli Rde C, et al. Pharmacokinetics of lidocaine

and its metabolite in peridural anesthesia administered to pregnant women with

gestational diabetes mellitus. Eur J Clin Pharmacol 2008; 64: 1189-96

24 Gebhard RE, Nielsen KC, Pietrobon R, Missair A, Williams BA. Diabetes

mellitus, independent of body mass index, is associated with a "higher success" rate

for supraclavicular brachial plexus blocks. Reg Anesth Pain Med 2009; 34: 404-7




26 Bigeleisen PE, Moayeri N, Groen GJ. Extraneural versus intraneural stimulation

thresholds during ultrasound-guided supraclavicular block. Anesthesiology 2009;

110: 1235-43


223

Table 1 - Demographics of survey responders

Primary site of practice

Community hospital 53 %

University hospital 44 %

Outpatient surgery center 1 %

Office based anesthesia 2 %

Size of hospital / practice

≤ 250 beds 20 %

251 – 500 beds 28 %

501 – 750 beds 20 %

751 – 1000 beds 17 %

> 1000 beds 15 %

Anesthesia experience

In training 9 %

0 – 5 years 20 %

5 – 10 years 24 %

10 – 20 years 29 %

> 20 years 18 %

Regional anesthesia in institution and own practice

0 – 20 % 43 % 28 %

20 – 40 % 38 % 31 %

40 – 60 % 13 % 26 %

60 – 80 % 4 % 10 %

80 – 100 % 2 % 5 %

Chapter 3.5

224

Table 2 - Survey responses regarding Block technique and Neuropathy

A.

Localization aid employed always/usually sometimes seldom/never

Nerve stimulator alone 30 % 25 % 45 %

Ultrasound alone 51 % 23 % 26 %

Dual guidance 41 % 26 % 33 %

Others 3 % 19 % 78 %

B.

In diabetics, assess … always/usually sometimes seldom/never

… duration of diabetes 82 % 10 % 8 %

… symptoms of neuropathy 51 % 23 % 26 %

… neurologic assessment 41 % 26 % 33 %

… neurologic consultation 3 % 19 % 78 %

C.

In neuropathics … always/usually sometimes seldom/never

… request neuro consult 12 % 17 % 71 %

… avoid nerve blocks 17 % 37 % 46 %

… counsel about ↑ risk 59 % 19 % 22 %

… modify technique 28 % 31 % 41 %

D.

Modifying block by … always/usually sometimes seldom/never

… ↓ local anesthetic dose 43 % 24 % 33 %

… ↓ epinephrine 77 % 9 % 14 %

… ↓ other adjuvants 59 % 15 % 26 %

… avoid nerve stimluator 25 % 20 % 55 %

… accept higher st. thr. 40 % 25 % 35 %

st. thr. stimulation threshold


225

Table 3 - Clinical studies or case reports reporting comparisons among patients with

or without DPN.

Author/Year N Block LA Tox O

Epidemiological

Hebl 2006 6 R 567 Neuraxial Bupi + -

Case reports

Waters 1996 7 R 1 2 x Spinal Tetracaine 1%

Lidocaine 5%

+ -

Lena 1998 8 R 1 Epidural Lido 2%

Bupi 0.16%

+ +

Horlocker 2000 9 R 1 Interscal Bupi 0.25% + +

Al-Nasser 2004 10

R 1 Epidural Ropi 0.75% + +

Blumenthal 2006 11

R 1 Spinal /

Femoral

Ropi + +

Angadi 2012 12

R 1 Spinal Bupi 0.5% + -

Clinical trials

Echevarria 2008 18

P 88 Spinal Bupi 0.5% x D

Gebhard 2009 19

R 262 Supraclav Bupi 0.5% x S

Sertoz 2013 20

P 45 Popliteal Levo 0.5%

Prilocaine 2%

x D

Cuvillon 2013 21

P 72 Sciatic Ropi 0.475% x D

R retrospective P prospective. Tox Toxicity + increased x not reported. O Final

patient outcome + resolution - persistent deficits x not reported D duration increased

S success rate increased.

Chapter 3.5

226

Table 4 - Experimental studies reporting block duration and neurologic

symptoms in test animals with or without DPN.

Author / Year Animal DM Block LA Tox

Kalichman

1992 13

rat STZ I sciatic Lidocaine 2%

Lidocaine 4%

+

++

Lindstrom

1994 22

rat BB II sciatic Anoxia x

Baba 2006 23

rat STZ I sciatic Ischemia +

Kroin 2010 14

rat STZ I sciatic Lidocaine 1%

Lido / Epin

Lido / Clonid

Ropivacaine

-

≈

+

+

Kroin 2012 15

rat STZ I spinal Lidocaine 1%

Lido / Epin

Bupivacaine

Bupi / Epin

o

o

o

o

Kroin 2012 24

rat STZ I sciatic Lido / Epin x

Lirk 2012 16

rat ZDF II sciatic Lido 2% o

DM diabetes mellitus. LA local anesthetic. /epin with added epinephrine. /clonid

with added clonidine. STZ streptozotocin rat model. BB biobreeding Worcester rat

model. ZDF Zucker Diabetic Fatty rat model. + increased - decreased ≈ similar o no

difference x not performed/reported.


227

Figure 1 – Personal valuation of Neuropathy.

Legend ++ agree strongly, + agree somewhat, o neutral, - disagree somewhat, --

disagree strongly.

229

Section 4

Epigenetic effects of local anesthetics

Lidocaine demethylates DNA in breast cancer cells

231

Chapter 4.1

Lidocaine demethylates DNA in breast cancer cells in vitro

Based on

Lirk P, Berger R, Hollmann MW, Fiegl H

In vitro, lidocaine demethylates DNA in breast cancer cells

Br J Anaesth 2012; 109: 200-207

Chapter 4.1

232

Introduction

Surgical tumour removal remains a highly relevant treatment option for

cancer patients, despite concerns that the perioperative period may facilitate

progression of the underlying disease due to immunosuppression, surgical stress,

and administration of drugs suspected of promoting tumour spread.1 Regional

anaesthetic procedures, or the intravenous administration of local anaesthetics, have

both been demonstrated to reduce perioperative surgical stress.2 3

The ultimate clinical relevance of these effects is unclear, and currently

available evidence is undecided as to whether potential positive effects of regional

anaesthesia are relevant enough to influence patient outcome after cancer

surgery.e.g.,4-8

Current large-scale multicenter randomized controlled trials are

projected to last until the end of the decade.1

In the meantime, research is focused on potential mechanisms of local

anaesthetic-induced tumour suppression. Several pathways have been described in

literature. On one hand, local anaesthetics, at high doses, are cytotoxic in vitro,9 10

and on the other hand, they may induce sensitization of tumour cells to

chemotherapeutics 11

and heat.12

Another potential mechanism whereby local anaesthetics may influence

tumour growth is by interaction with the tumour epigenome.13

In malignancy,

increased methylation frequently leads to down-regulation of tumour suppressor

genes, favouring tumour progression.14

The prototype ester-type local anaesthetic, procaine, has been shown to

demethylate DNA and inhibit tumour growth and in the MCF-7 breast cancer cell

line,15

and similar results were later obtained in hepatoma 9 and leukaemia cell

lines.16

Epigenetic effects of amide-type local anaesthetics, such as lidocaine, have

not been examined. Given the structural differences between these two types of local

anaesthetics, differential effects on epigenetic features are possible. In addition,

lidocaine is very widely used in contemporary regional anesthesia, and increasingly

employed intravenously in the context of multimodal treatment regimens.3

We sought to examine the effects of the prototype amide-type local

anaesthetic, lidocaine, on DNA methylation in prototypical breast cancer cell line


233

cultures. Our working hypothesis was that exposure of cancer cells to lidocaine

would decrease DNA methylation.

Methods

Cell culture

Human breast cancer cell lines BT-20 (estrogen receptor-negative) and

MCF-7 (estrogen receptor-positive) were obtained from the American Type Culture

Collection (ATCC) and were cultured according to company recommendations.

Amplification of 15 short tandem repeat (STR) loci and the gender-specific locus

amelogenin was carried out in the Institute of Legal Medicine of the Medical

University Innsbruck to authenticate the cell lines, using 10 ng of template DNA

applying the Geneprint PowerPlex 16 System (Promega) following the

manufacturer’s recommendations as previously described.17

Drug treatments

The following drugs were purchased from Sigma Aldrich (Vienna,

Austria): lidocaine n-ethyl bromide (L5783) and procaine hydrochloride, (P9879),

both dissolved in distilled water. We treated BT-20 and MCF-7 breast cancer cell

lines with a final varying concentration lidocaine, procaine and the positive control

substance, 5 µM 5-aza-2’-deoxycytidine (DAC) for 72 and 96 hours respectively.

Twenty-four hours after seeding, the medium was removed and replaced with

medium containing the drug solutions at the desired final concentration. DAC was

dissolved in DMSO to a final concentration of 10 mM, aliquoted, and stored at -

20°C. Lidocaine and procaine were dissolved in water to a final concentration of 1

M and 0.5 M respectively, aliquoted, and stored at -20°C. Whenever needed, a fresh

aliquot was diluted to the desired final concentration.

Effect of lidocaine and procaine on cell proliferation.

We analyzed the effects of lidocaine and procaine on cell proliferation in

the human breast cancer cell lines BT-20 and MCF-7 during 72 hours and 96 hours

incubation by counting the cell number. To this end, Breast cancer cells were seeded

into medium size cell culture bottles in MEM medium with 10% FCS (MCF-7: 2.2

Chapter 4.1

234

Mio cells, BT-20: 1 Mio cells) and treated 24 hours later. After the indicated

incubation time, 72 hours or 96 hours respectively, the cells were trypsinized and

counted (Beckman coulter). Biotin-labeled POD TUNEL Apoptosis detection kit for

adherent cell was used according to manufacturer’s protocol (GenScript,

Piscataway, NJ). Cells were visualized using an Olympus 1X70 inverted Microscope

in conjunction with Kappa ImageBase software V2.7.2 (Gleichen, Germany).

Global Genomic DNA Hypermethylation

Global genomic 5-methylcytosine content was determined by quantitative

MethyLight assay specific for Chromosome 1 SAT2 repeat sequences.18

We

analyzed the effects of 1mM procaine, different lidocaine concentrations (0.01mM,

0.1mM, 1mM) and 5µM DAC on the global DNA methylation status in MCF-7 and

BT-20 breast cancer cells after 72, 96 and 120 hours (Fig. 2). Genomic DNA from

treated cells was extracted using the DNeasy tissue kid (Qiagen) method. Sodium

bisulfite conversion of genomic DNA and MethyLight was performed as described

previously.19

DNA methylation and RNA expression of several Tumour Suppressor Genes

We decided to test the effects of lidocaine in comparison to procaine on

particular hypermethylated loci in both cell lines. RASSF1A, GSTP1 and MYOD1

are known epigenetically inactivated genes in breast cancer.20 21

Primers and Probes

for COL2A1 (reference gene), RASSF1A, MYOD1 and GSTP1, and Chromosome 1

juxtacentromeric satellite 2 (SAT2) DNA sequences have recently been published.18

19 We compared the effects of 1mM lidocaine and procaine. Total cellular RNA was

extracted by the standard acid guanidium thiocyanate-phenol-chloroform method

and reverse transcription of RNA was performed as previously described.22

Primers

and probe for Real time quantitative PCR analysis (qPCR) for RASSF1A, MYOD1

and GSTP1 were purchased from Applied Biosystems (AB Assay ID:

Hs00200394_m1, Hs00159528_m1 and Hs00168310_m1). Primers and probes for

the TATA box-binding protein (TBP; a component of the DNA-binding protein

complex TFIID; was used as endogenous RNA control) were used according to

Bieche et al.23

Real-time PCR was performed using an ABI Prism 7900HT


235

Detection System (Applied Biosystems, Foster City, CA). The standard curves were

generated using serially diluted solutions of standard cDNA derived from the

Hs578T carcinoma cell-line.

Statistics

Results are expressed as mean ± standard deviation (SD). The unpaired

Student's t-test was used for the comparison of the various effects after the different

treatments in normally distributed data, and the Mann-Whitney U test for non-

normally distributed data. P-values less than 0.05 were considered as statistically

significant. SPSS 17.0 was used for the statistical analyses.

Results

Effect of lidocaine and procaine on cell proliferation.

Treatment with 1mM procaine or 1mM lidocaine resulted in significant

reductions in cell number, while lower concentrations of local anaesthetics did not

lead to significant cell number reduction (Fig. 1A). In both cell lines no increase in

the apoptosis rate was observed upon lidocaine (1mM, 0.1mM, 0.01mM) or

procaine (1mM) treatment respectively (Fig. 1B).


At baseline, methylation was 100-fold higher in BT-20 than in MCF-7

cells. At equal dose, lidocaine was a stronger demethylating agent than procaine

(Fig. 2). In MCF-7 cells, we observed demethylation after 96, but not 72, hours after

treatment with 1mM procaine(Fig. 2A; p=0.004). Treatment with 1 mM lidocaine

resulted in a statistically significant increase of global Sat2 DNA methylation after

72 hours, whereas the treatment with 0.1mM and 0.01mM lidocaine revealed a

significant demethylation after 72 and 96 hours (Fig. 2A; p<0.001, respectively). In

comparison to procaine, treatment with 0.1mM or 0.01mM lidocaine had a stronger

demethylating effect (Fig. 2A). Even in comparison to the demethylating agent,

DAC, the treatment with 0.1 or 0.01mM lidocaine revealed a more significant

decrease of DNA methylation (Fig. 2A).

Chapter 4.1

236

In BT-20 cells, the treatment with 1mM procaine revealed a significant

decrease of DNA methylation after 72 and 96 hours (Fig. 2B; p<0.001 or p=0.019,

respectively). We observed a dose-dependent decrease in DNA methylation in

response to lidocaine (1mM, 0.01mM, 0.01 mM) after 72 hours (Fig 2B; p<0.001,

p<0.001 or p=0.004, respectively) and 96 hours (Fig. 2B; p<0.001, p=0.034 or

p=0.38, respectively), similar to effects noted upon incubation with DAC.

Additionally we treated the breast cancer cells with 0.01mM, the clinically

relevant dose of lidocaine, for 48, 72, 96 and 120 hours. We observed a statistical

significant demethylating effect in MCF-7 after 72 to 120 hours, whereas in BT-20

cells the demethylating effect was seen only between 72 and 96 hours (Fig. 3).

DNA methylation and RNA expression of specific Tumour Suppressor Genes

In MCF-7 cells, after 72 hours or 96 hours respectively, neither DNA-

methylation nor mRNA expression changes in our three exemplary tumour

suppressor genes were observed (Fig. 4). Only for MYOD1 we observed a statistical

significant increase in DNA methylation after 72 hours in lidocaine treated MCF-7

cells (Fig. 4A; p=0.006), without effecting the MYOD1 mRNA expression. In

procaine treated cells we identified an increase in MYOD1 mRNA expression after

72 hours (Fig. 4B; p=0.036). DAC as control substance decreased methylation of the

three tumour suppressor genes (Fig. 4A), and resulted in an increased mRNA

expression only for MYOD1 after 72 hours (Fig. 4B).

In BT-20 cells we identified no decrease in methylation or mRNA

expression upon procaine or lidocaine treatment after 72 or 96 hours. The treatment

with DAC gave rise to a decreased RASSF1A, GSTP1 and MYOD1 DNA

methylation, and increased the mRNA expression of these three tumour suppressor

genes (Fig 4B).


237

Discussion

The present study demonstrates that lidocaine time- and dose-dependently

demethylates DNA in BT-20 breast cancer cells (estrogen receptor-negative cell

line), whereas in MCF-7 cells (estrogen-receptor-positive cell line) this

demethylating effect was only observed to a smaller, albeit statistically significant,

extent. This effect was noted at concentrations corresponding to those reached after

systemic application of local anaesthetics, or after systemic absorption from epidural

or paravertebral anaesthesia. These concentrations are, however, insufficient to

cause direct cytotoxicity. Elicitation of cytotoxic effects would necessitate

concentrations of local anesthetic that can only be reached after direct application of

local anesthetic on the tumour intraoperatively. Finally, epigenetic modification

appears to be transient, since demethylation was returned to non-significant levels

after 120 hours in BT-20 cells. While global methylation status was profoundly

influenced, the expression status of three exemplary tumour suppressor genes

(RASSF1A, GSTP1 and MYOD1) was not significantly changed, such that further

studies will need to elucidate which genes are affected by the demethylating effects

of lidocaine.

The potential clinical implication of these results is that a potent pathway

leading to malignancy seems to be influenced by local anaesthetics at clinically

relevant doses. This may, in part, explain beneficial effects of regional anaesthesia

on cancer recurrence observed in some studies. Furthermore, the magnitude of this

effect is dependent upon epigenetic features of the tumour type. Whereas we

observed statistically significant alterations in methylation status in both exemplary

cell lines, effects were much more pronounced in an ER-negative cell line, which is

associated with high methylation levels at baseline. Thus, beneficial effects relating

to epigenetic modulation of cancer biology may be limited to certain types of

tumours. The ultimate clinical relevance of our findings remains to be determined.

The issue whether the mode of anaesthesia influences outcome after cancer

surgery has been a topic of intense debate. In a retrospective study on melanoma

patients, Schlagenhauff et al. found that patients who had undergone melanoma

excision under general anaesthesia had a decreased survival as compared to those

Chapter 4.1

238

receiving local (infiltration) anaesthesia only.4 It should be noted that anaesthetic

infiltration of peri-neoplastic tissues leads to very high regional concentrations of

local anaesthetic in the milimolar range. These concentrations of local anaesthetics

are known to be cytotoxic when applied to tumour cell lines.10

In contrast, when

local anaesthetics are administered during epidural or paravertebral anaesthesia, low

micromolar concentrations are attained in the systemic circulation.24

In breast cancer

patients, a retrospective study revealed a substantial benefit with regards to

metastatic spread in patients in whom paravertebral anaesthesia had been used

during mastectomy.5 Corresponding data on abdominal cancer patients could,

however, not replicate these promising findings, except for a subgroup of older

patients in one study.8 25

27

In another study, all-cause mortality was reported to be

reduced after resection of rectal, but not colonic, malignancies when epidural

anaesthesia was part of anaesthetic technique.26

Results regarding prostate cancer

patients remain equivocal.6

7

28 Two studies in patients suffering from ovarian

carcinoma have linked intraoperative epidural analgesia to increased 3- and 5-year

survival,29

and increased recurrence-free interval,30

while epidural anesthesia during

brachytherapy had no beneficial survival effect in patients with cervical cancer.31

In

patients undergoing percutaneous radiofrequency ablation of hepatocellular

carcinoma, use of epidural anesthesia was associated with decreased survival.32

All

these equivocal results may indicate that either, regional anaesthesia has no effect,

or, they may be explained by the biological heterogeneity of tumours. Our results

indicate that lidocaine influences cell lines with different biological properties

differently, exerting strong demethylating effects on ER-negative, and less

pronounced effects on ER-positive, cell lines. Thus, the question of whether regional

anaesthesia or, for that sake, other components of the perioperative care process,

modulate cancer recurrence is perhaps not one that can be answered unequivocally.

Rather, our findings would indicate that tumour-suppressive effects of local

anaesthetics may only detectable in specific types of cancer.

The role of local anaesthetics in tumour progression may be indirect or

direct. Indirect actions of regional anaesthesia include attenuation of the

neuroendocrine response to surgery followed by improved preservation of


239

immunocompetence, and effects on the administration of drugs suspected of

modulating tumour growth, e.g., opioid-sparing effect.1 Direct actions are caused by

direct effects of local anaesthetic on tumour cells,10

or sensitizing effects towards

other therapeutic measures such as thermo-33

and chemotherapy.11

Local

anaesthetics have been shown to protect against tumour cell invasion in

concentrations attained clinically after local infiltration 34

and suppress the

proliferation of a number of tumour cell lines.e.g.,10

Our results suggest that next to

classic cytotoxic effects seen at doses exceeding those attained clinically, local

anaesthetics are also able to subtly modulate tumour biology. Modulation of tumour

epigenome was observed at far lower doses than those needed to elicit overt

cytotoxicity. Cancer treatment strategies based upon epigenetics are being

developed, and the ester-type local anaesthetic, procaine, had been suggested as a

candidate substance.15

Our results suggest that lidocaine is an even more potent

demethylating agent than procaine.

The stability and expression of the human genome is controlled by

methylation of specific regions of desoxyribonucleic acid (DNA).13

Next to the

classic four nucleotides adenine, thymine, guanine, and cytosine, a fifth base, 5-

methylcytosine, confers epigenetic features to control a variety of physiologic and

pathologic processes.13

Increasing evidence suggests epigenetic aberrations as a

major pathogenic factor in the development of malignancy.13

Modulation of tumour

suppressor or promoter genes has been described as a key event in cancer.14

In short,

increased methylation can lead to silencing of tumour suppressor genes, thereby

promoting tumour progression. In these cases, decreased methylation may be of

therapeutic benefit. Catalyzation of methylation is achieved by the family of DNA

methyltransferases (DNMT). DNMT1 seems to be the main pathogenic enzyme in

human cancer, while its suppression can significantly inhibit tumour growth.36

We

hypothesized that similar to procaine, lidocaine leads to demethylation by inhibiting

DNMT. In an early landmark study, Kennedy et al. postulated that sensitizing

effects of lidocaine for the chemotherapeutic, bleomycine, were potentially caused

by interference with DNA integrity.37

We show that in vitro, lidocaine is an even

more potent DNA-demethylating agent than procaine.

Chapter 4.1

240

The differential effect of lidocaine at a concentration of 1mM in BT-20 and

MCF-7 cell lines is a novel finding, but not unexpected given the heterogeneity of

human cancer types. In part, it confirms previous ex vivo experiments, in which

inhibitory effects of serum taken from patients undergoing regional anaesthesia

impeded ER-negative cells in vitro.35

For example, breast cancer, which we chose to

serve as the tumour paradigm for this study, has been subdivided into at least five

distinct subtypes based upon gene expression profiling, and immunohistochemical

markers.38

These are characterized by differential sensitivity to treatment, and

clinical outcome, and biological heterogeneity is only beginning to be fully

understood.38

In our case, BT-20 cells showed a much higher index of methylation

than MCF-7 cells, with PMR values 100-fold higher in BT-20 cells (Fig. 2).

Demethylating effects of local anaesthetics may depend on a high level of pre-

existing methylation to show effect. Conversely, in cancer types in which DNA

methylation is not a key pathogenic factor, the potential therapeutic effect seems

negligible.

Finally, some potential limitations of our study should be briefly

mentioned. First, we used lidocaine as a prototypical amide-type local anaesthetic,

whereas in clinical routine, long-acting local anaesthetics are more frequently used

for epidural anaesthesia and analgesia. We chose to investigate lidocaine as the first

prototypical substance since it constitutes the most widely investigated local

anaesthetic, and is the only local anaesthetic that is routinely administered locally

for infiltration, for epidural or paravertebral anaesthesia, or intravenously as part of

multimodal anaesthetic and analgesic regimens.3 Extrapolating from the fact that

both procaine and lidocaine exert demethylating effects, the same should be

anticipated for long-acting compounds such as bupivacaine and ropivacaine, even if

testing this hypothesis was beyond the scope of the present study. These results were

obtained in two cell lines representative of estrogen-positive and estrogen-negative

breast cancer, respectively. These cell lines have been extensively researched and

validated. Yet, they may feature genotypic and phenotypic drift over time. We

sought to minimize this potential bias, by authenticating cell lines using

amplification of several STR loci. Local anaesthetics have been demonstrated to


241

sensitize tumour cells towards cytotoxic effects of chemotherapeutics, and it is thus

possible that such sensitization may also take place towards demethylating effects of

novel chemotherapeutics.

Our findings suggest that demethylating tumour-suppressive effects of anaesthetic

interventions may only be detectable in specific types of cancer due to differential

methylation profiles. Lidocaine dose-dependently demethylates DNA of breast

cancer cell lines in vitro.

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short tandem repeat profiling: power and limitations. FASEB J 2005; 19: 434-6

18 Weisenberger DJ, Siegmund KD, Campan M, et al. CpG island methylator

phenotype underlies sporadic microsatellite instability and is tightly associated with

BRAF mutation in colorectal cancer. Nat Genet 2006; 38: 787-93

19 Eads CA, Danenberg KD, Kawakami K, et al. MethyLight: a high-throughput

assay to measure DNA methylation. Nucleic Acids Res 2000; 28: E32

20 Park SY, Kwon HJ, Lee HE, et al. Promoter CpG island hypermethylation

during breast cancer progression. Virchows Arch 2011; 458: 73-84

21 Widschwendter M, Apostolidou S, Raum E, et al. Epigenotyping in peripheral

blood cell DNA and breast cancer risk: a proof of principle study. PLoS One 2008;

3: e2656


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22 Muller HM, Fiegl H, Goebel G, et al. MeCP2 and MBD2 expression in human

neoplastic and non-neoplastic breast tissue and its association with oestrogen

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23 Bieche I, Onody P, Laurendeau I, et al. Real-time reverse transcription-PCR

assay for future management of ERBB2-based clinical applications. Clin Chem

1999; 45: 1148-56




25 Christopherson R, James KE, Tableman M, Marshall P, Johnson FE. Long-term

survival after colon cancer surgery: a variation associated with choice of anesthesia.

Anesth Analg 2008; 107: 325-32

26 Gupta A, Bjornsson A, Fredriksson M, Hallbook O, Eintrei C. Reduction in

mortality after epidural anaesthesia and analgesia in patients undergoing rectal but

not colonic cancer surgery: a retrospective analysis of data from 655 patients in

central Sweden. Br J Anaesth 2011; 107: 164-70

27 Myles PS, Peyton P, Silbert B, Hunt J, Rigg JR, Sessler DI. Perioperative

epidural analgesia for major abdominal surgery for cancer and recurrence-free

survival: randomised trial. BMJ 2011; 342: d1491

28 Wuethrich PY, Hsu Schmitz SF, Kessler TM, et al. Potential influence of the

anesthetic technique used during open radical prostatectomy on prostate cancer-

related outcome: a retrospective study. Anesthesiology 2010; 113: 570-6

29 Lin L, Liu C, Tan H, Ouyang H, Zhang Y, Zeng W. Anaesthetic technique may

affect prognosis for ovarian serous adenocarcinoma: a retrospective analysis. Br J

Anaesth 2011; 106: 814-22

30 de Oliveira GS, Jr., Ahmad S, Schink JC, Singh DK, Fitzgerald PC, McCarthy

RJ. Intraoperative neuraxial anesthesia but not postoperative neuraxial analgesia is

associated with increased relapse-free survival in ovarian cancer patients after

primary cytoreductive surgery. Reg Anesth Pain Med 2011; 36: 271-7

31 Ismail H, Ho KM, Narayan K, Kondalsamy-Chennakesavan S. Effect of

neuraxial anaesthesia on tumour progression in cervical cancer patients treated with

brachytherapy: a retrospective cohort study. Br J Anaesth 2010; 105: 145-9

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32 Lai R, Peng Z, Chen D, et al. The Effects of Anesthetic Technique on Cancer

Recurrence in Percutaneous Radiofrequency Ablation of Small Hepatocellular

Carcinoma. Anesth Analg 2011

33 Arai Y, Kondo T, Tanabe K, et al. Enhancement of hyperthermia-induced

apoptosis by local anesthetics on human histiocytic lymphoma U937 cells. J Biol

Chem 2002; 277: 18986-93

34 Mammoto T, Higashiyama S, Mukai M, et al. Infiltration anesthetic lidocaine

inhibits cancer cell invasion by modulating ectodomain shedding of heparin-binding

epidermal growth factor-like growth factor (HB-EGF). J Cell Physiol 2002; 192:

351-8

35 Deegan CA, Murray D, Doran P, Ecimovic P, Moriarty DC, Buggy DJ. Effect of

anaesthetic technique on oestrogen receptor-negative breast cancer cell function in

vitro. Br J Anaesth 2009; 103: 685-90

36 Laird PW, Jackson-Grusby L, Fazeli A, et al. Suppression of intestinal neoplasia

by DNA hypomethylation. Cell 1995; 81: 197-205

37 Kennedy KA, Hait WN, Lazo JS. Chemical modulation of bleomycin induced

toxicity. Int J Radiat Oncol Biol Phys 1986; 12: 1367-70

38 Tang P, Skinner KA, Hicks DG. Molecular classification of breast carcinomas

by immunohistochemical analysis: are we ready? Diagn Mol Pathol 2009; 18: 125-

32


245

Figure Legends

Figure 1: Effect of lidocaine and procaine on cell proliferation of MCF-7 and

BT-20 human breast cancer cells. (A) Cell numbers after lidocaine and procaine

treatment. Results of 8 independent experiments are shown. (B) TUNEL assay.

Results of 1 representative experiment out of 4 independent experiments are shown.

Results are expressed as mean ±SD. Statistical significance between control and

treated samples was assessed by the unpaired Student's t-test (*, p < 0.05; ***, p <

0.001).

Figure 2: Global Genomic SAT2 DNA methylation analysis. (A) SAT2 DNA

methylation levels in MCF-7 and (B) BT-20 breast cancer cells treated with 1 mM

procaine, 1mM lidocaine, 0.1mM lidocaine, 0.01mM lidocaine or 5 µM 5-aza-2’-

deoxycytidine (DAC) respectively. Results of 8 independent experiments are shown.

Results are expressed as mean ±SD. Statistical significance between control and

treated samples was assessed by the unpaired Student's t-test (*, p < 0.05; ***, p <

0.001).

Figure 3: Global Genomic SAT2 DNA methylation analysis: time series. (A)

SAT2 DNA methylation levels in MCF-7 and (B) BT-20 breast cancer cells treated

with 0.01mM lidocaine for 48, 72, 96 and 120 hours. Results are expressed as mean

±SD. Results of at least 3 independent experiments are shown. Statistical

significance between control and treated samples was assessed by the unpaired

Student's t-test (*, p < 0.05; ***, p < 0.001).

Figure 4: DNA methylation and RNA expression analysis of Tumour

Suppressor Genes. (A) DNA methylation (results of 8 independent experiments are

shown) and (B) mRNA expression (results of 5 independent experiments are shown)

of RASSF1A, GSTP1 and MYOD1 after a treatment with 1mM procaine, 1mM

lidocaine or 5µM DAC for 72 or 96 hours respectively. Results are expressed as

mean ±SD. Statistical significance between control and treated samples was assessed

Chapter 4.1

246

by the unpaired Student's t-test t or Mann-Whitney U test (for GSTP1 and MYOD1

mRNA expression) (*, p < 0.05; ***, p < 0.001).


247

Figure 1

Chapter 4.1

248

Figure 2


249

Figure 3

Chapter 4.1

250

Figure 4

Demethylating properties of lidocaine, bupivacaine and ropivacaine

251

Chapter 4.2

Lidocaine and ropivacaine, but not bupivacaine,

demethylate deoxyribonucleic acid in cancer cells in vitro

Based on

Lirk P, Hollmann MW, Hauck-Weber NC, Fleischer M, Fiegl H

Lidocaine and ropivacaine, but not bupivacaine, demethylate

deoxyribonucleic acid in cancer cells in vitro

Br J Anaesth 2014; in press.

Chapter 4.2

252

Introduction

More than 100 years ago, it was recognized that tumour surgery makes

metastasis more likely.1 The perioperative period of tumour surgery may well be a

crucial time when patient outcome can be decisively influenced.2 Numerous

perioperative factors which may explain tumour progression have now been

identified. For example, resecting a primary tumour may promote the progression of

distant metastases, or even induce tumour self-seeding, where circulating tumour

cells re-infiltrate the original site of a resected tumour.3 This concept is supported by

recent evidence that the number of circulating tumour cells increases dramatically in

the perioperative period.4 One very important determinant of metastatic potential is

the epigenetic signature of tumour cells.5

In a healthy body, epigenetic mechanisms are responsible for the stability

and expression of human deoxyribonucleic acid (DNA) as they regulate the

methylation of specific DNA regions.6 Epigenetic mechanisms are increasingly

recognized as pathogenic factors in several forms of cancer.6 Specifically, increases

in methylation levels can deactivate tumour suppressor genes and lead to the

progression of cancer.6 7

In these cases, decreasing methylation levels may be of

therapeutic benefit. So a new class of chemotherapeutics designed to demethylate

tumour DNA has been introduced into clinical practice.8

We have recently shown that the prototype local anaesthetic, lignocaine,

can reduce methylation of breast cancer cells at clinically relevant concentrations in

vitro.9 This study sought to determine whether similar demethylating effects could

also be observed for two prototype long-acting local anaesthetics typically used for

perioperative neuraxial anaesthesia: bupivacaine and ropivacaine. In addition, local

anaesthetics have been described as enhancing the tumoricidal effects of

conventional chemotherapeutics,10

and we wanted to investigate whether local

anaesthetics would also increase the demethylating effect of a prototypical

demethylating chemotherapeutic, 5-aza-2’-deoxycytidine (DAC).

The aim of this study was therefore to test two hypotheses: 1) that the local

anaesthetics lignocaine, bupivacaine, and ropivacaine decrease methylation levels in

tumour cells, and 2) that local anaesthetics enhance the demethylating effects of

DAC.


253

Methods

Cell culture

Human breast cancer cell lines BT-20 (estrogen receptor [ER]-negative)

and MCF-7 (ER-positive) were obtained from the American Type Culture

Collection (ATCC, Manassas, USA) and were cultured according to ATCC

recommendations. Amplification of 15 short tandem repeat (STR) loci and the

gender-specific locus amelogenin was carried out in the Institute of Legal Medicine

of the Medical University Innsbruck, Austria, to authenticate the cell lines. This was

done using 10 ng of template DNA, applying the Geneprint PowerPlex 16 System

(Promega, Madison, USA) according to the Manufacturer’s recommendations, as

previously described.11

Drug treatments

The following drugs were purchased from Sigma Aldrich (Vienna,

Austria): lignocaine N-ethyl bromide (L5783), bupivacaine hydrochloride

monohydrate (B5274) and ropivacaine hydrochloride monohydrate (R0283), all

dissolved in distilled water. We treated BT-20 and MCF-7 breast cancer cell lines

with these local anaesthetics first alone and then in combination with varying

concentrations of DAC for 72 hours. The following concentrations were used: 10

µM and 100 µM lignocaine, 2 µM and 20 µM bupivacaine, 3 µM and 30 µM

ropivacaine; 0.001 µM, 0.02 µM, 0.1 µM, 0.2 µM 0.5 µM and 1 µM DAC. Twenty-

four hours after seeding, the medium was removed and replaced with medium

containing the drug solutions at the desired final concentration. DAC was dissolved

in DMSO to a final concentration of 10 mM, aliquoted, and stored at -20°C.

Lignocaine, bupivacaine and ropivacaine were dissolved in water to a final

concentration of 1 M and 100 mM respectively, aliquoted, and stored at -20°C.

Whenever needed, a fresh aliquot was diluted to the desired final concentration.

Effect of local anaesthetics on cell viability.

We analysed the effects of lignocaine in combination with DAC and

bupivacaine and ropivacaine on cell viability in the human breast cancer cell lines

BT-20 and MCF-7 during 72 hours’ incubation by the colorimetric MTT assay

Chapter 4.2

254

(M5655; Sigma, Vienna, Austria). The tetrazolium dye MTT (3-[4,5-

dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) was dissolved in RPMI-

1640 without phenol red. The assay was performed according to Manufacturers’

instructions. Absorbance of converted dye was measured at a wavelength of 570 nm

with background subtraction at 630–690 nm.12


Global genomic 5-methylcytosine content was determined by quantitative

MethyLight assay, specific for Chromosome 1 SAT2 repeat sequences.13

We

analysed the effects of 10 µM and 100 µM lignocaine, 2 µM and 20 µM bupivacaine

and 3 µM and 30 µM ropivacaine alone or in combination with 0.001 µM, 0.02 µM,

0.1 µM, 0.2 µM, 0.5 µM or 1 µM DAC respectively on the global DNA methylation

status in BT-20 and MCF-7 breast cancer cells after 72 hours. Genomic DNA from

treated cells was extracted using the DNeasy tissue kit (Qiagen, Hilden, Germany).

Sodium bisulfite conversion of genomic DNA and MethyLight was performed as

described previously.14

Additivity

We sought to determine whether the interaction between DAC and

lignocaine as the prototype local anaesthetic was supra-additive. Our calculations

were based on the Loewe isobolographic additivity model, which has been described

as particularly useful when investigating the interplay between two toxic substances

in vitro.15

The half maximal DNA demethylation concentration (EC50) was

calculated in BT-20 cells for DAC and lignocaine from at least 7 independent

experiments at different concentrations of DAC and lignocaine, resulting in a

preliminary EC50 of 0.08 µM for DAC, and 77.3 µM for lignocaine (line of

additivity, Figure 4). We assumed supra-additive effects if 50% of demethylation

were achieved with a combination of concentrations significantly lower than those

representing the line of additivity.


255

Statistics

Results are expressed as mean ± standard deviation (SD). The Mann-

Whitney U test was used for the comparison of the various effects after the different

treatments. P-values less than 0.05 were considered statistically significant. SPSS

17.0 (IBM, Vienna, The Austria) was used for statistical analyses.

Results

Effect of lignocaine, bupivacaine and ropivacaine on cell viability

Treatment with 10 µM or 100 µM lignocaine alone had no cytotoxic effect.

Lignocaine at concentrations of 10 µM and 100 µM did not increase cytotoxicity of

DAC in either BT-20 (Fig. 1A) or MCF-7 (Fig. 1B) breast cancer cell lines.

Similarly, treatment with bupivacaine or ropivacaine at doses equipotent to

lignocaine showed no cytotoxic effect in either breast cancer cell line (Fig. 1C, 1D).

Effect of bupivacaine and ropivacaine on global genomic DNA-methylation

Treatment with bupivacaine at 2 µM and 20 µM revealed no significant

demethylating effect on global genomic DNA methylation in either breast cancer

cell line BT-20 (Fig. 2A) or MCF-7 (Fig. 2B).

Treatment with ropivacaine for 72 hours at concentrations of 3 µM or 30

µM decreased methylation in BT-20 cells (Fig. 2A; P=0.003 or P=0.023,

respectively). In MCF-7 cells, no demethylation was observed (Fig. 2B).

Effect of lignocaine, bupivacaine and ropivacaine in combination with DAC on

global genomic DNA-methylation.

To determine whether local anaesthetics could increase the

demethylating effect of DAC, we treated BT-20 and MCF-7 breast cancer cells for

72 hours with 10 µM and 100 µM lignocaine, 2 µM bupivacaine and 3 µM

ropivacaine, and combined these treatments with DAC at several concentrations. We

observed increased demethylation after the combined treatment of BT-20 cells with

0.1 µM DAC and 10 µM or 100 µM lignocaine respectively (Fig. 3A; P=0.014 or

P=0.001 respectively) and 0.5 µM DAC with 100 µM lignocaine (Fig. 3A;

P=0.008), in comparison to the DAC treatment alone. In MCF-7 cells, only the

Chapter 4.2

256

combined treatment with 0.5 µM DAC and 10 µM lignocaine revealed a stronger

demethylation in comparison to the mono-treatment with 0.5 µM DAC alone (Fig.

3B; P=0.006). All other combined treatments of bupivacaine and ropivacaine with

various concentrations of DAC revealed no increased demethylating effect in BT-20

or MCF-7 cells, as compared to treatment with DAC alone (Fig. 3).

Additivity

Since lignocaine was the most potent local anaesthetic agent as regards

demethylating properties in this study, additivity experiments were based upon

calculated EC50 in methylation for lignocaine and DAC. These theoretical results

were compared to demethylation using several concentrations of DAC (0.02, 0.04,

0.08, 0.16, and 0.32 µM), combined with lignocaine (19.3, 38.7, 77.3, 154.6, 309.2

µM, respectively, Table 1). We then compared demethylation levels with the

theoretical line of additivity (Figure 4), and found no supra-additivity (Figure 4,

Table 1).

Discussion

This study was designed to test the hypotheses that 1) the local anaesthetics

lignocaine, bupivacaine, and ropivacaine can decrease methylation levels in tumour

cells, and that 2) lignocaine as the strongest demethylating agent would enhance the

demethylating effects of DAC – the prototype epigenetic chemotherapeutic.

We found that 1) lignocaine and ropivacaine, but not bupivacaine, induce

DNA demethylation, and that 2) lignocaine showed no supra-additive effects when

combined with DAC.

The concentrations of local anaesthetics employed in the present

investigation are in the range of concentrations reached during epidural infusion of

local anaesthetics.16 17

Equipotent concentrations of lignocaine, ropivacaine, and

bupivacaine were calculated based on a previous electrophysiological study.18

Comparable doses of lignocaine (i.e. up to 10 µM) are observed after typical

regimens of perioperative intravenous lignocaine infusion.19


257

Epigenetic effects of local anaesthetics

Our results build upon and confirm previous results which indicated that at

clinically relevant doses, lignocaine demethylates DNA in breast cancer cells in

vitro.9 In addition, our results from individual drug treatments (Fig. 3), and Loewe

additivity experiments (Fig. 4) suggest that the methylating effects of lignocaine and

DAC are additive. We did not find evidence of supra-additivity. The mechanism by

which local anaesthetics may influence methylation was not directly investigated in

the present study, but procaine has previously been shown to inhibit DNA

methyltransferase-1, the driving force behind the methylation of cytosine.20

While local anaesthetics have been shown to make conventional

chemotherapeutics work better in selected experimental settings,10 21

no-one has yet

tested their effects on the performance of demethylating chemotherapeutics. Further

investigations are needed to determine the biological consequences of systemic

lignocaine on tumour progression in the perioperative setting. In contrast to

lignocaine, bupivacaine and ropivacaine seem less potent in inducing demethylation

in tumour cells. The reasons for this differential effect can only be speculated upon.

The equipotent doses chosen were based on sodium channel blockade,22

and so we

surmise that the demethylating effect of these substances is not related to sodium

channel blockade. This is not surprising since at least three alternative effects of

local anaesthetics are mediated by pathways independent of sodium channel

blockade. Firstly, different local anaesthetics show differential effects on G-protein-

mediated priming of human neutrophils ex vivo, which are not correlated with their

potency in blocking sodium channels.23

Secondly, given equipotent doses,

lignocaine is much more effective than bupivacaine in preventing thrombus

formation.24 25

And thirdly, Piegeler et al. demonstrated that the effects of lidocaine

and ropivacaine on the phosphorylation of Src, a key molecule conjectured in

tumour metastasis, were not related to potency at the sodium channel, and that ester-

type local anaesthetics had no effect.26

In contrast to the latter study on Src

phosphorylation however, the epigenetic effects of local anaesthetics are discernible

in both amide-type and ester-type compounds. In a landmark manuscript, Villar-

Garea showed that the prototype ester-type local anaesthetic, procaine, demethylated

DNA and inhibited tumour growth in MCF-7 breast cancer cells,27

and others found

Chapter 4.2

258

similar effects when investigating solid organ and haematopoietic tumour cell

lines.28

20

Relevance of perioperative epigenetic modulation

The most important epigenetic alterations are methylation of cytosine

residues in DNA to produce 5-methyl-cytosine, resulting in a change in the spatial

configuration of histones.29

Most frequently, the pathologic epigenetic changes in

malignancy involve increased methylation, which leads to the silencing of tumour

suppressor genes.6 29

Modifications in the epigenetic signature of tumour cells are

nowadays considered as important as genetic mutations themselves.29

In addition to

this increasingly appreciated role in primary oncogenesis, epigenetic alterations are

also increasingly understood to influence the prognosis and response to treatment of

malignancy, including the probability of metastasis, in many tumours.5 Current

treatments targeting the epigenome are associated with considerable side-effects. For

example, the paradigmatic anti-epigenetic drug, decitabine (DAC), a potent inhibitor

of DNA methylation, is associated with significant adverse effects such as

myelosuppression and organ toxicity.30

We have previously shown that lignocaine,

given at clinically relevant concentrations, acts as a demethylating agent. Here, we

show that lignocaine shows additive demethylating effects when combined with

DAC. Given the very good safety profile of systemic or regional administration of

lignocaine, this drug offers potentially beneficial epigenetic effects in the

perioperative period of tumour surgery.

Limitations

In larger concentrations, local anaesthetics can have direct cytotoxic effects

on tumour cells, and this may explain protective effects against tumour recurrence

observed after local anaesthesia for local superficial tumour excision.31

However,

the concentrations used in the present study were found insufficient to cause direct

cytotoxicity (Figure 1). Also, we note that the two tumour cell lines that we used

have different baseline methylation properties. The effects of demethylation are

largest in BT-20 cells, which have a high baseline methylation level.9 In the same

way, biological heterogeneity may explain why specific anaesthetic interventions


259

seem to affect outcome in some types of cancer 32 33

while no effect was found in

other studies,34

and some studies found effects only in defined subpopulations.35

Conclusions

Assessing our study alongside previous evidence, we conclude that, at

clinically relevant doses, lignocaine and ropivacaine exert demethylating effects on

breast cancer cells in vitro, but bupivacaine does not. When combined, lignocaine

and DAC exhibit additive demethylating effects.

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263

Table 1: Global Genomic SAT2 DNA methylation in BT-20 breast cancer cell

line after treatment with various concentrations of DAC, Lignocaine and a

combined treatment.

DNA-methylation is indicated as percentage of a fully methylated reference (PMR).

Data are presented as mean ± SD.

Tre

atm

ent

DAC

[µM]

Lignocaine

[µM]

SAT2 PMR value (+ SD)

after DAC

treatment

alone

after

Lignocaine

treatment

alone

after

combined

treatment

0 0 30 (± 7) 30 (± 7) 30 (± 7)

0.02 19.3 18 (± 5) 23 (± 1) 31 (± 7)

0.04 38.7 16 (± 4) 38 (± 7) 14 (± 1)

0.08 77.3 19 (± 1) 39 (± 26) 17 (± 4)

0.16 154.6 22 (± 7) 24 (± 1) 18 (± 3)

0.32 309.2 14 (± 3) 34 (± 1) 9 (± 0)

Chapter 4.2

264

Figure Legends

Figure 1: Effect of lignocaine in combination with DAC, and effects of

bupivacaine and ropivacaine on MCF-7 and BT-20 human breast cancer cells.

The cell viability was analyzed by MTT analysis after being cultured for 72 h with

the indicated drugs. (A) BT-20 cells and (B) MCF-7 cells treated with lignocaine

and various concentrations of DAC. (C) BT-20 cells and (D) MCF-7 cells treated

with bupivacaine and ropivacaine. Data are represented as the mean ± SD from three

independent experiments.

Figure 2: Global Genomic SAT2 DNA methylation analysis in breast cancer

cell lines after treatment with bupivacaine and ropivacaine. (A) SAT2 DNA

methylation levels in BT-20 breast cancer cells treated with 2 µM and 20 µM

bupivacaine, 3 µM and 30 µM ropivacaine or 1 µM DAC and (B) MCF-7 breast

cancer cells treated with 2 µM bupivacaine, 3 µM and 30 µM ropivacaine or 1 µM

DAC. Results from an average of 6 independent experiments are shown. Results are

expressed as mean ± SD. Statistical significance between control and treated

samples was assessed by Mann-Whitney U test (*, p < 0.05; **, p < 0.01;***, p <

0.001).

Figure 3: Global Genomic SAT2 DNA methylation analysis in breast cancer

cell lines after combined treatment of lignocaine, bupivacaine and ropivacaine

with various concentrations of DAC. (A) SAT2 DNA methylation levels in BT-20

breast cancer cells and (B) MCF-7 breast cancer cells. Results from an average of 6

independent experiments are shown. Results are expressed as mean ±SD. Statistical

significance between control and treated samples was assessed by Mann-Whitney U

test (*, p < 0.05; **, p < 0.01).

Figure 4: Line of additivity based on the preliminary calculated ED50 for DAC

and Lignocaine (0.08 µM and 77.3 µM, respectively). The marker denotes the first

concentration which leads to a near-50% demethylation (actual ED50).


265

Figure 1

Chapter 4.2

266

Figure 2


267

Figure 3

Chapter 4.2

268

Figure 4

269

Section 5

Conclusion and Discussion: Weighing risks and benefits

of Regional Anaesthesia

Based on

Lirk P, Hollmann MW

Regional anesthesia: weighing risks and benefits

Minerva Anesthesiol 2013; epub ahead of print Nov 6

270

Introduction

During the past 30 years, regional anaesthesia has undergone dramatic

changes in technique and indications, and has become a widely used method to

provide intraoperative anaesthesia, and postoperative analgesia. A considerable base

of scientific evidence has been achieved from which to estimate risks and benefits of

regional anaesthesia, including the insight that the failure rate of regional

anaesthesia in daily practice may be high.1 Evidence-based medicine seeks to

combine these scientific results with pathophysiological knowledge and the needs of

the individual patient to devise a judicious anaesthetic plan. This review seeks to

address the question whether patient outcomes are sufficiently improved by regional

anaesthesia to an extent that warrants its use as a routine perioperative method.

The benefits

In the past, the perceived ability of regional anaesthesia to provide excellent

management of acute postoperative pain combined with a good safety profile was

considered a sufficient reason in itself to perform regional anaesthesia. Indeed, many

authors continue to appreciate the value of good perioperative pain therapy as such,

even if it cannot be directly translated into improved overall long-term outcome.2

However, recent literature has advocated a more rigorous judgement of the value of

regional anaesthesia. With the advent of improved general anaesthesia regimens and

multimodal pain therapy, the superiority of regional anaesthesia in many types of

surgery has receded to levels that may be statistically, but not clinically, significant.3

The benchmarks against which the efficacy of regional anaesthesia should be

measured were summarized by Fischer (2010): optimal postoperative analgesia with

a minimum of adverse effects, promotion of rehabilitative efforts, and improvement

of long-term outcome including chronic pain.4 The problematic nature of these bars

was highlighted in a recent editorial.2 Specifically, regional anaesthesia is but one,

albeit important, therapeutic option during the perioperative course of an individual

patient. In the optimal evidence-based situation, all patient- and procedure-related

factors and interventions should be efficient in altering overall outcome. In an age

where increasing numbers of patients are in fast-track discharge programs and

Weighing risks and benefits

271

perioperative risk of major morbidity and mortality is low, respective

benefits of regional anaesthesia become harder to discern.

Acute and Chronic Pain

Poorly managed acute postoperative pain is, in general, thought to be

associated with the development of chronic pain.5 However, it is difficult to predict

why certain patients go on to develop chronic pain while in others, pain resolves.

Part of this risk is determined genetically. The respective concept is that of an

“estimated heritability score”. In the case of acute pain, the risk “share” of genetic

predisposition is between 22-55%. In contrast, the heritable component for the risk

of developing persistent pain is higher, in the range of 55-68%.6 Such data clearly

suggest a genetic predisposition for those who develop chronic pain following a

precipitating incident. Pain which is difficult to control perioperatively may reflect

greater sensitivity of patients based on genetic predisposition, with chronic pain as a

logical consequence. Strict regimens of pain therapy may help to reduce chronic

pain after prototypical high-risk, such as amputation.7 In the latter scenario, use of

epidural anesthesia 7 or peripheral nerve blocks

8 may substantially reduce the rate of

phantom limb pain, but the evidence-base is limited at this point. The prolonged use

of peripheral sciatic nerve block for amputation was shown in recent reports to have

a high success rate in preventing phantom limb pain.8-10

Potentially, future studies

will make it possible to identify individuals at high risk of acute and chronic pain,

and to tailor individual patient-specific treatment plans including the provision of

regional anaesthesia, according to both type of surgery and type of patient. In

patients at high risk of excessive pain perception undergoing major surgery, these

treatment plans may include the combination of a sufficient regional blockade

together with intravenous multimodal pain treatment (intravenous non-opiates,

combined with, e.g., systemic ketamine, clonidine, or gabapentin / pregabaline).

This would act to prevent the development of central sensitization via different

pathways.11

Section 5

272

Peripheral Nerve Blocks

An exhaustive analysis by Liu and Wu examined the beneficial effects of

regional anaesthesia when compared to systemic analgesia. In general, the authors

found an average acute pain score with regional anaesthesia which was only

marginally lower, and a lack of clear evidence showing improved long-term

outcome.3 Despite the fact that many studies report statistically significant

outcomes, a clear benefit in pain scores should only be assumed and taken as

clinically relevant when the absolute pain intensity is different by 2 points on a 10-

point scale, or pain differs by 33%.12

For specific surgeries, such as joint

replacement, regional anaesthetic techniques such as single-shot or continuous

peripheral nerve blocks are widely used, with an excellent safety profile and a

substantial reduction in opioid administration.13

This was confirmed in recent meta-

analyses and systematic reviews demonstrating superior analgesia, less opioid-

induced side-effects and sleep disturbances, and facilitated rehabilitation when

continuous nerve blocks were used for extremity surgery.14 15

The timepoint and

definition of analgesic endpoint is important, as well. For example, the meta-

analysis by Richman investigating continuous nerve blocks as compared to opioids

showed that the mean VAS score was reduced from 3.7 to 1.7, on the verge of

clinical significance.12 14

However, when maximal pain scores at breakthrough

points were considered, the reduction was more pronounced, from 5.9 to 3.2.14

In the future, alternative blockades such as truncal blocks 16

may replace

epidural anaesthesia for some indications, but the evidence is insufficient at this

moment. Early promising studies show that transversus abdominis plane block may

promote short-term recovery after abdominal surgery,17

and may be an alternative to

spinal morphine after caesarean section.16

Current literature regarding paravertebral

blockade shows a trend towards equally effective analgesia as compared to the

epidural route 18

with potentially less adverse effects.19

Epidural anaesthesia

Considering epidural anaesthesia for upper abdominal and thoracic surgery,

Andreae found a beneficial effect of regional anaesthesia on development of chronic


273

pain after open thoracotomy and breast cancer surgery but stated that in general,

studies were of suboptimal quality and had included few patients.20

The combination

of intraoperative epidural and multimodal analgesia has been found to prevent

chronic pain after major abdominal surgery more effectively than multimodal

analgesia alone.11

Moreover, patients undergoing major vascular surgery, and high-

risk patients, have been found to experience less perioperative complications when

general is combined with epidural anaesthesia, but the reported effect size is small.

For example, epidural administration of local anaesthetics protects against

postoperative ileus,3 but beneficial effects upon gastrointestinal motility have also

been published regarding the intravenous administration of local anaesthetics.21

The

same ambiguity also holds true for other outcomes. Whereas Liu did not observe a

difference in quality of recovery,3 another meta-analysis by Poepping, including

older studies, postulates that after upper abdominal and thoracic surgery, epidural

anaesthesia decreases the risk of pulmonary complications.22

Notably, in the latter

study, the incidence of pulmonary complications in patients receiving epidural

anesthesia remained steady at 8% throughout the meta-analysis inclusion range,

whereas the incidence in the systemic analgesia group decreased from 34% to 12

%,22

most likely reflecting improved supportive perioperative care, and higher

efficacy of multimodal systemic regimens. On a broader basis, it can be stated that

the quality of multimodal intravenous regimens and perioperative care is improving,

and it can be speculated that studies comparing the effects of epidural anaesthesia

against optimal intravenous regimens will demonstrate beneficial effects on a much

smaller scale. A recent Cochrane review failed to show a decrease in mortality in

patients undergoing open abdominal aneurysm surgery, but the use of epidural

analgesia reduced pain during the three first postoperative days (an arguable clinical

benefit if only seen by itself), decreased postoperative ventilation time by half, and

led to a reduction in postoperative myocardial infarction, acute respiratory failure,

and gastrointestinal and renal complications.23

It can be summarized that epidural

anesthesia and analgesia improve aspects of postoperative outcomes in selected

patient subgroups. Contemporary medicine is facing two distinct but opposing

trends. On one hand, evidence-based medicine relies on large-scale investigations

Section 5

274

investigating aspects of patient care, and when large patient collectives are

considered, the averaged effect size of epidural anaesthesia across all patients is

small to modest. On the other hand, the future trend is that of personalized,

individually tailored, medicine, based on, among others, the patient’s previous

medical and pain history, and genetic background. This trend is certain to

encompass the areas of perioperative and chronic pain medicine, and will lead to a

further refinement of the indications for neuraxial blockade.

Regional Anaesthesia and Cancer Medicine

Whether regional anaesthesia has the potential to improve outcome in

cancer patients has been the topic of intense discussion and research efforts ever

since a retrospective study demonstrated survival benefits in patients subjected to

perioperative paravertebral anaesthesia during mastectomy.24

Since then, some

studies have confirmed this promising trend, others have failed to detect beneficial

effects of regional anaesthesia, and some have found benefits only in specific patient

subgroups.25

The mechanisms by which regional anesthesia may theoretically affect

tumour progression are threefold.26

First, perioperative suppression of the surgical

stress response by regional anaesthesia improves host defence, and may protect

against circulating tumour cells. Second, opioids have arguably been linked to

promote tumour growth in some experimental models and regional anaesthesia

allows for dose-minimization of opiates, but it is unclear whether opiates clinically

affect cancer recurrence. Third, local anesthetics present in the systemic circulation

may directly limit viability of tumour cells, or sensitize them against

chemotherapeutics. If the large-scale studies currently ongoing do find a clinically

relevant effect of regional anaesthesia, it can be expected to be valid for certain

types of tumour only, and depend on the molecular biological background of the

specific malignancy.

Systemic Effects of Local Anaesthetics

Lastly, it should be mentioned that local anaesthetics have effects that reach

beyond “just” nerve blockade and ensuing pain control. Among others, they have


275

been shown to beneficially affect the postoperative surgical stress response 27

without impairing physiologic host response. Further, they have been shown to

prevent hypercoagulable states in patients after major vascular 28

and orthopaedic 29

surgery without increasing the risk of bleeding. When compared to placebo,

systemic administration of lidocaine has been reported to reduce opioid consumption

and related side effects, decrease duration of ileus, and decrease length of hospital

stay following abdominal surgery.21 30 31

Notably, these beneficial effects are

observed after both open and laparoscopic surgery.31 32

When comparing systemic lidocaine to epidural anesthesia as the

conventional “gold standard” for major abdominal surgery, the results are less

straightforward. Intravenous lidocaine started at the beginning of surgery has similar

effects as epidural analgesia started within one hour after surgery regarding duration

of ileus, pain, and length of hospital stay.33

However, in the latter study, initiation of

the epidural was performed only after surgery, and was not standardized, even

though other studies suggest that the main positive effect of epidural anesthesia and

analgesia is attained intraoperatively.11

Another study compared intraoperative

administration of thoracic epidural anesthesia, systemic lidocaine, or placebo.

Postoperatively, all patients were connected to patient-controlled epidural analgesia

(PCEA). Benefits on pain were short-lived after surgery as patients were allowed to

administer boluses of epidural solution themselves. However, beneficial effects on

duration of ileus, and the decrease in inflammatory mediators were more evident in

patients who had received epidural than in patients who had received intravenous

local anesthetics, and in both groups, functional outcome, but not length of hospital

stay, was improved as compared to intraoperative placebo.34

Adverse effects of intravenous lidocaine primarily stem from systemic

toxicity, and most studies report plasma levels well below the toxic threshold of 5

µM.31

Signs of central nervous system toxicity are seldomly reported.33

The systemic action of local anaesthetics is dependent on sufficient plasma

levels usually reached during neuraxial block, peripheral nerve blockade, and

intravenous administration. However, recent advances in the administration of

ultrasound-guided regional anaesthesia have led to descriptions of block success in

Section 5

276

the presence of dramatic reductions in the volume of administered local anaesthetic.

Some reported volumes are less than 10% of the “traditional” volume administered

when using nerve stimulation.35

Using very small volumes for peripheral nerve

block is theoretically advantageous because of the decreased danger of systemic

local anaesthetic toxicity, but at the same time, the systemic beneficial effects of

regional anaesthesia, whose contribution is only beginning to be fully understood,

will be lost because systemic absorption and resulting plasma levels are negligible.

In conclusion, beneficial effects of regional anaesthesia can be copied to a

large extent by the systemic administration of local anesthetics. Considering the

risk:benefit ratio, epidural anesthesia and analgesia should be considered in high risk

patients, thoracotomy, and major upper abdominal and vascular surgery, whereas

most cases of lower abdominal surgery and laparoscopic surgery can be adequately

treated using multimodal analgesia.

The risks

When considering the risk aspect of regional anaesthesia, neurologic

complications such as peripheral nerve injury or paralysis, and systemic

complications such as local anaesthetic intoxication are the most commonly cited

major adverse outcomes.

Permanent nerve injury

The incidence of perioperative peripheral nerve injuries has been assessed

in a variety of studies and in general, retrospective studies show a much lower

incidence than prospective studies. A summary of clinical studies by Brull et al.

found a rate of neurologic complications of 0.04 % after neuraxial, and 3 % after

peripheral regional anaesthesia, with the majority of neurologic deficits transient in

nature.36

In a recent analysis of 1000 ultrasound-guided peripheral nerve blocks, the

incidence of all-cause new-onset neurologic symptoms was 8.2%, with most cases

unrelated to the block after thorough analysis.37

. Since then, prospective data in

8,000 epidurals showed a complication rate for abscess formation of 0.1%.38

The

Third National Audit Project of the Royal College of Anaesthetists reported


277

permanent disability or death from central nervous blockade between 0.7 and 1.8 per

100,000 procedures.39

The question whether regional anaesthesia is more dangerous

in patients with pre-existing neurological disease has been controversially

discussed.40

Current evidence suggests that symptomatic diabetic neuropathy may

warrant a more careful weighing of the indication for regional anaesthesia, and may

necessitate adaptation of technique, such as a decrease in local anaesthetic dose, and

decreasing or omitting epinephrine from the injectate.40

Systemic Toxicity

Regional anaesthesia can further be complicated by systemic overdose of

local anaesthetic. This potentially life-threatening adverse event occurs after

inadvertent intra-arterial injection, intra-venous injection, or absorption following

large-volume blocks.41

In the landmark study by Auroy and colleagues, seizures as

indicators of systemic toxicity occurred in 1 / 10,000 patients undergoing epidural or

spinal anaesthesia, and between 2 and 5 / 10,000 patients undergoing peripheral

nerve blocks.42

Recently, ultrasound-guided peripheral regional anaesthesia was

reported to cause seizures in 8 / 10,000 patients, with no incidences of cardiac arrest

in a collective of more than 12,000 patients.43

Due to increased awareness and

multiple safety steps such as aspiration, careful titration of the local anaesthetic, and

the administration of test doses, the incidence of systemic toxicity is estimated to

have declined by a factor of 25.41

In addition, intralipid has been introduced as a

possible therapeutic adjunct for intoxication due to lipophilic local anaesthetics such

as bupivacaine.44

It should be briefly noted, however, that opiates as the main alternative

substance for postoperative pain relief, can cause severe adverse effects as well,

including respiratory depression and arrest. Together with anticoagulants and

insulin, opiates are among the three most common substances to cause serious

adverse drug events in hospitals.45

Section 5

278

Other adverse events

Several other potential adverse events need to be considered on a patient-

and block-specific basis. Pneumothorax has been reported after several upper

extremity nerve blocks, including interscalene, supraclavicular, and vertical

infraclavicular.46

Vascular puncture can occur, with hematoma formation or, very

rarely, pseudoaneurysm formation as sequelae.46

While this is perceived to be less of

a problem in peripheral regional anesthesia, epidural hematoma formation may lead

to compression of neural structures, and to permanent damage, with an incidence of

1:4000 – 1:6000 in the general surgical population, and less in obstetrical patients.47

48 Similarly, infection localized to the site of peripheral nerve block may be

troublesome, but development of an epidural abscess may precipitate permanent

neurologic dysfunction if occurring in the spinal canal.49

Moreover, excessive spread

of local anaesthetic can lead to inadvertent blockade of nerves other than the target

structure. Vocal cord paralysis can ensue due to recurrent laryngeal nerve block, and

hemidiaphragmatic paresis can occur due to phrenic nerve block after interscalene or

supraclavicular nerve block.46

These side-effects may potentially be avoided by

using small volumes during ultrasound-guided nerve blockade.35

Again, these latter

side-effects may be of limited clinical importance in healthy patients, but they may

be detrimental in patients with specific co-morbidities, e.g., contralateral recurrent

nerve palsy or severe chronic obstructive pulmonary disease, respectively.

Weighing the risks and benefits

At the moment, regional anaesthesia is undergoing a profound transition.

The current trend is to reconsider and reappraise indications and contraindications

for regional anaesthesia, often in the light of changing surgical techniques.50

One

paradigmatic example is analgesia for total knee arthroplasty (TKA). Regional

anaesthesia has been demonstrated to offer substantial benefits in the immediate

perioperative period of TKA as compared to systemic analgesia, including superior

pain control, reduced opioid consumption, and decreased opioid-related side-effects.

In the middle term, ambulation and range of motion is improved in patients

receiving regional anaesthesia groups, and rehabilitation goals are met faster.51

To


279

achieve this goal, some twenty years ago, it was considered good clinical practice to

perform epidural analgesia or combined spinal-epidural anaesthesia and analgesia.52

However, epidural analgesia is frequently associated with pruritus, urinary retention

and motor block, hindering optimal and early mobilization.3 Subsequent evidence

demonstrated that a combined femoral / sciatic nerve block would provide the same

degree of postoperative analgesia, but with a more favourable side-effect profile,

especially concerning early mobilization.53

Currently, the two-block approach is

undergoing scrutiny, and in many patients, continuous femoral nerve block (possibly

combined with a single-shot sciatic nerve block) seems to provide satisfactory

analgesia.54

Newer techniques such as saphenous nerve block 55

seem even more

tailor-made for analgesia following TKA. This nerve block provides analgesia for

the anterior portion of the knee joint, while avoiding blockade of motor fibers such

as those supplying the quadriceps muscle. In theory, mid-thigh saphenous block will

combine the analgesia of a femoral nerve block but without the motor weakness

potentially interfering with early rehabilitative efforts, providing targeted analgesia

as peripherally as possible.

However, in the long term, outcome after one year following TKA is not as

clear-cut. Whereas immediate postoperative benefits, including improved pain

control and less adverse opioid-induced adverse effects can readily be detected in

trials,54

functional patient outcome one year after surgery is not superior.56

With the

current cost-driven trend to fast-track TKA patients, it becomes increasingly

difficult to demonstrate outcomes such as reduced length of stay.51

It is debatable

whether the facilitation of rehabilitation, which has been clearly demonstrated 51

is a

sufficient reason to perform regional anaesthesia if one-year functional outcome is

not superior.56

The authors think that the combination of good perioperative pain

therapy, decrease of opioid-related adverse events, and facilitated rehabilitation

should be considered sufficient grounds to continue offering peripheral regional

anaesthesia to patients undergoing TKA.

Section 5

280

Future directions

Evolving research fields may alter the way we look at perioperative

interventions, including regional anaesthesia. For example, epigenetics is concerned

with the methylation of desoxyribonucleic acid (DNA), which determines gene

expression. An increasing number of pathological conditions, among them

persistence of experimental pain,57

has recently been linked to epigenetic

irregularities. Interestingly, chronic opioid use in patients leads to increased DNA

methylation correlating with increased pain, at least in part explaining opioid-

induced hyperalgesia.58

The effect of clinically relevant concentrations of lidocaine

has the opposing effect on methylation in vitro, i.e. de-methylation.26

Whether this

molecular effect can be translated into strategies to decrease acute pain and prevent

chronic pain needs to be determined at this point.

In parallel, the tools used to deliver Regional Anesthesia are enhanced. One

very prominent example is the new liposomal formulation of bupivacaine, which is

entering the United States market, and may extend pain relief to 48-72 hours after a

single bolus injection.59

The efficacy and safety of this treatment modality will

undoubtedly be the subject of a large number of studies.

Conclusion

In conclusion, the question whether regional anaesthesia generally

improves relevant indices of outcome in all patients has to be answered with no.

Research has led to the reconsideration of indications and contraindications for

regional anaesthesia in specific settings. The administration of regional anaesthesia

necessitates a judicious weighing of risks and benefits. In general, the indications for

epidural anaesthesia have decreased, and are limited to major upper abdominal and

major vascular surgery, and thoracotomy. In addition, patients might benefit from

regional anaesthesia on an individualized specific risk:benefit deliberation. It should

be noted that when broad patient collectives are considered, the benefits of epidural

anaesthesia are modest. Peripheral nerve blockade remains a mainstay of anaesthetic

management for extremity surgery. At this time, there is insufficient evidence to

ascribe a specific role to alternative blockades such as truncal blocks, but in the


281

future, they may replace epidural anaesthesia for some indications. Paravertebral

anaesthesia may evolve into an alternative to epidural anaesthesia for thoracotomy.

A substantial share of the alternative effects of regional anaesthesia on the immune

system, postoperative ileus, and haemostasis are due to systemic absorption of local

anaesthetics, and can be duplicated using intravenous administration of local

anaesthetics. Low-volume peripheral nerve blocks may lack these beneficial effects

because plasma levels are negligible. Multimodal analgesia has the potential to

replace regional anaesthesia in many types of surgery. Regional anaesthesia

continues to play a major role in perioperative medicine, but its role keeps getting

more defined and less noncommittal.

Key messages

• Every regional blockade should be preceded by a weighing of potential

risks and proven benefits, taking into account procedure- and patient-based

factors.

• At least in part, many beneficial effects of Regional Anaesthesia can be

duplicated by intravenous administration of local anaesthetics.

• Regional anaesthesia continues to play a major role in perioperative

medicine, but its role keeps getting more defined and less noncommittal.

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44 Weinberg GL. Treatment of local anesthetic systemic toxicity (LAST). Reg


45 Winterstein AG, Hatton RC, Gonzalez-Rothi R, Johns TE, Segal R. Identifying

clinically significant preventable adverse drug events through a hospital's database

of adverse drug reaction reports. Am J Health Syst Pharm 2002; 59: 1742-9

Section 5

286

46 Neal JM, Hebl JR, Gerancher JC, Hogan QH. Brachial plexus anesthesia:

essentials of our current understanding. Reg Anesth Pain Med 2002; 27: 402-28

47 Volk T, Wolf A, Van Aken H, Burkle H, Wiebalck A, Steinfeldt T. Incidence of

spinal haematoma after epidural puncture: analysis from the German network for

safety in regional anaesthesia. Eur J Anaesthesiol 2012; 29: 170-6

48 Bateman BT, Mhyre JM, Ehrenfeld J, et al. Brief report: the risk and outcomes

of epidural hematomas after perioperative and obstetric epidural catheterization: a

report from the multicenter perioperative outcomes group research consortium.

Anesth Analg 2013; 116: 1380-5

49 Reynolds F. Neurological infections after neuraxial anesthesia. Anesthesiol Clin

2008; 26: 23-52, v

50 Rawal N. Epidural technique for postoperative pain: gold standard no more? Reg


51 Macfarlane AJ, Prasad GA, Chan VW, Brull R. Does regional anesthesia

improve outcome after total knee arthroplasty? Clin Orthop Relat Res 2009; 467:

2379-402

52 Joshi GP, McCarroll SM. Evaluation of combined spinal-epidural anesthesia

using two different techniques. Reg Anesth 1994; 19: 169-74

53 Fowler SJ, Symons J, Sabato S, Myles PS. Epidural analgesia compared with

peripheral nerve blockade after major knee surgery: a systematic review and meta-

analysis of randomized trials. Br J Anaesth 2008; 100: 154-64

54 Wegener JT, van Ooij B, van Dijk CN, Hollmann MW, Preckel B, Stevens MF.

Value of single-injection or continuous sciatic nerve block in addition to a

continuous femoral nerve block in patients undergoing total knee arthroplasty: a

prospective, randomized, controlled trial. Reg Anesth Pain Med 2011; 36: 481-8

55 Andersen HL, Gyrn J, Moller L, Christensen B, Zaric D. Continuous Saphenous

Nerve Block as Supplement to Single-Dose Local Infiltration Analgesia for

Postoperative Pain Management After Total Knee Arthroplasty. Reg Anesth Pain

Med 2012

56 Wegener JT, van Ooij B, van Dijk CN, et al. Long-term pain and functional

disability after total knee arthroplasty with and without single-injection or


287

continuous sciatic nerve block in addition to continuous femoral nerve block: a

prospective, 1-year follow-up of a randomized controlled trial. Reg Anesth Pain Med

2013; 38: 58-63

57 Zhang Z, Cai YQ, Zou F, Bie B, Pan ZZ. Epigenetic suppression of GAD65

expression mediates persistent pain. Nat Med 2011; 17: 1448-55

58 Doehring A, Oertel BG, Sittl R, Lotsch J. Chronic opioid use is associated with

increased DNA methylation correlating with increased clinical pain. Pain 2013; 154:

15-23

59 Candiotti K. Liposomal bupivacaine: an innovative nonopioid local analgesic for

the management of postsurgical pain. Pharmacotherapy 2012; 32: 19S-26S

Summary

289

Section 6

Summary

Section 6

290

The present thesis sought to investigate and review the mechanism and

management of side-effects or failures of neuraxial anesthesia; investigate nerve

injury related to the performance of regional anesthesia, with special focus on

patients with pre-existing neuropathy; investigate potential novel actions of local

anesthetics on the epigenetic signature of tumour cells; and conclude with a

weighing of risks and benefits of regional anesthesia using local anesthetics.

In Section 1, an introduction was given to local anesthetics. While Chapter

1.1 described the aims of this thesis, Chapter 1.2 reviewed current literature which

shows that conventional local anesthetics in contemporary use block the voltage-

gated sodium channel by binding to a specific site on the inner facet of the channel

pore. This causes a conformational change, and the positive charge of the local

anaesthetic further obstructs the channel’s lumen. Only 2-3% of local anaesthetic is

thought to participate in nerve blockade, the rest is absorbed into surrounding tissues

or the systemic circulation. While small systemic levels of local anaesthetic can

have a beneficial effect on some outcomes and vital functions, excessive amounts of

free local anaesthetic will cause systemic toxicity.

In Section 2, focusing on the clinical application of local anaesthetics for

regional anesthesia, two studies concentrated on the use for spinal anesthesia for

cesarean section, while one review article described causes and management of

failed neuraxial blockade. In Chapter 2.1, we present a survey investigating the

strategies to prevent or treat hypotension following administration of spinal

anaesthesia for caesarean delivery. In comparison to previous surveys in the United

States and United Kingdom, we showed a trend towards increased use of

phenylephrine to treat hypotension, in keeping with a growing evidence-base

suggesting its side-effect profile is superior to ephedrine in most cases. In Chapter

2.2, we investigated pulmonary function indices following administration of spinal

anesthesia for caesarean section when using bupivacaine, ropivacaine or

levobupivacaine. We found that decreases in maternal pulmonary function tests

were similar following spinal anaesthesia with bupivacaine, ropivacaine, or

levobupivacaine for caesarean delivery. The clinical maternal and neonatal effects of

these alterations appeared negligible. This study does not support the clinical

Summary

291

superiority of any of the three investigated long-acting local anaesthetic for spinal

anesthesia. In Chapter 2.3, the evidence surrounding failures and their management

during neuraxial analgesia is reviewed. Failed epidural anaesthesia or analgesia is

more frequent than generally recognized. Reasons for an insufficient epidural

include primary erroneous placement, secondary migration of a catheter after correct

placement, and suboptimal dosing of drugs. As hinted in Chapter 2.2., when using

equipotent doses, the clinical difference between bupivacaine and the newer

isoforms levobupivacaine and ropivacaine appears minimal. During continuous

infusion, dose is the primary determinant of epidural anaesthesia quality, with

volume and concentration playing a subordinate role. Addition of adjuvants,

especially opiates and epinephrine, may substantially increase the success rate of

epidural analgesia. The method of delivery for postoperative analgesia most

supported by literature is patient controlled epidural analgesia with background

infusion.

In Section 3, the focus was on nerve damage during regional anesthesia,

with a special focus on patients with diabetic neuropathy. In Chapter 3.1, we sought

to answer the question whether intraneural needle placement is detrimental. In a

large animal model of sciatic nerve block, neurophysiological parameters following

nerve trauma and intraneural injection. Needle trauma as well as intraneural

injection of small volumes of saline produced a significant decline in cMAP

amplitude, corresponding to severely impaired nerve function. Although the degree

of long-term neurological dysfunction in our model is unclear, it could be

demonstrated that in the short term, nerve conduction is highly vulnerable to needle

trauma and intraneural injection, arguing against intraneural injection as proposed

by some authors. In Chapter, 3.2, current knowledge on regional anesthesia in

patients with diabetic neuropathy was summarized. Patients with pre-existing

neuropathies or neurological disease may be at increased risk of iatrogenic nerve

injury. This line of thought was developed further in Chapter 3.3., where in vitro and

in vivo investigations were conducted to investigate effects of local anesthetics on

nerve block duration and neurotoxicity in diabetic rats. We found that in vitro, local

anesthetic neurotoxicity was more pronounced on neurons from diabetic animals,

Section 6

292

but the survival difference was small. In vivo, subclinical neuropathy leads to

substantial prolongation of block duration. We concluded that early diabetic

neuropathy increases block duration, while the observed increase in toxicity was

small. In a subsequent experiment (Chapter 3.4), we refined this methodology by

introducing neurophysiologic monitoring to diagnose diabetic neuropathy before

administering sciatic nerve block and measuring nerve block duration as well as

neurotoxic effects. We found that nerve blocks last longer in late diabetic animals.

Sensitivity of diabetic nerves for local anesthetics seems to correlate with severity of

neuropathy. Our results, using a short-acting local anesthetic without additives, do

not support the hypothesis that nerve block in diabetic patients increases nerve

injury after peripheral nerve block. Lastly, in Chapter 3.5, we sought to assess the

patterns of practice in the management of diabetic neuropathic patients presenting

for regional anesthesia in Europe. While literature is divided on the question

whether pre-existing diabetic neuropathy is a risk factor for new neurological deficit

after regional anaesthesia, most of the responders of this survey take measures to

reduce risks, counsel patients on a possible greater risk of neurologic complications,

but only a minority of responders would avoid peripheral regional anaesthesia

altogether.

Section 4 concentrates on a novel mechanism of action of local anesthetics,

the modification of the epigenetic signature of human cells. While preliminary

evidence had suggested that procaine can modify epigenetics in tumour cells, we

could show in Chapter 4.1 that this was also the case for the classic local anesthetic,

lidocaine, at clinically relevant concentrations attained after epidural anesthesia or

intravenous administration as part of multimodal pain treatment regimens. In

Chapter 4.2, we show that lidocaine and ropivacaine, but not bupivacaine, at

clinically relevant doses, exert demethylating effects on certain breast cancer cells,

and seem to enhance demethylating properties of prototype epigenetic

chemotherapeutic, 5-aza, in an additive fashion. At equipotent doses, the

demethylating effects of local anesthetics seem to be most pronounced for lidocaine,

with decreased demethylating capacity by ropivacaine, and no detectable effect of

Summary

293

bupivacaine. These effects could be of substantial importance in perioperative

medicine, with focus on tumour surgery and pain medicine.

In Section 5, the potential benefits and risks of regional anesthesia were

compared. Regional anaesthesia has become a widely used method to provide

intraoperative anaesthesia, and postoperative analgesia. This review seeks to address

the question whether patient outcomes are improved to an extent that justifies using

regional anaesthesia as a routine method. During the past decade, a very critical

appraisal of risks and benefits of regional anaesthetic procedures has taken place. In

general, the indications for epidural blockade have decreased, and are limited to

individual high-risk patients, major upper abdominal and major vascular surgery,

and thoracotomy. We review the changing role of central and peripheral regional

anaesthesia in the perioperative management of total knee arthroplasty. Immediate

perioperative outcome after knee arthroplasty concerning function and pain is

improved, and rehabilitation facilitated, by peripheral nerve blockade, but this does

not translate into superior functional outcome one year later. A substantial share of

the beneficial effects of regional anaesthesia on the immune system, haemostasis,

pain, and the duration of ileus can be duplicated using intravenous administration of

local anaesthetics. In general, the use of regional anaesthesia should always be

preceded by a weighing of potential risks and proven benefits. Regional anaesthesia

continues to play a major role in perioperative medicine, but its role keeps getting

more defined and less noncommittal.

Summary

295

Section 7

Appendices

Appendix 1

297

Appendix 1

Dutch summary / Nederlandse samenvatting

Dutch Summary

298

Dit proefschrift getiteld “Local anesthetics – new insights into risks and

benefits” heeft als primaire doelstelling om specifieke bijwerkingen van de regionale

anesthesie te onderzoeken. Daarnaast wordt de invloed van diabetische neuropathie

op toxiciteit en functioneren van een perifeer zenuwblok omschreven, en het

potentiele effect van lokaal-anesthetica op de epigenetische markering van

tumorcellen bepaald. In het laatste hoofdstuk zullen de risico’s en baten van de

regionale anesthesie worden beschreven.

Lokaal-anesthetica blokkeren het spanningsafhankelijke natrium-kanaal.

Daarnaast komt echter een groot deel van het lokaal-anestheticum in de systemische

circulatie terecht, waar het bijdraagt aan de gewenste effecten van regionale

anesthesie. Men weet bijvoorbeeld dat intraveneus toegediende lokaal-anesthetica

een sterk anti-inflammatoir effect uitoefenen waardoor het herstel na specifieke

operaties wordt bevorderd. Maar lokaal-anesthetica kunnen ook bijwerkingen

veroorzaken, zoals blokkade van ademhalingsspieren, bloeddrukdaling, en

systemische toxiciteit.

De huidige discussie in de anesthesie heeft betrekking op de invloed van

een preexistente neuropathie op het functioneren van lokaal-anesthetica. Ons

onderzoek suggereert dat het risico op zenuwschade door regionale anesthesie bij

reeds bestaande diabetische neuropathie niet significant toe lijkt te nemen. Maar ons

onderzoek laat ook zien dat de zenuwblokkade in dit geval veel langer duurt dan bij

gezonde zenuwen. De zenuwen van diabetische patiënten zijn dus toch gevoeliger

voor lokaal-anesthetica ook al lijkt het risico op permanente beschadiging door

regionale anesthesie niet verhoogd. In de klinische praktijk is er veel onduidelijkheid

hoe er met diabetische patiënten met preexistente zenuwschade om moet worden

gegaan. Een van de conclusies in dit proefschrift is dat een regionaal-blok

verantwoord is mits een goede afweging van risico’s en baten plaats heeft gevonden.

Lokaal-anesthetica hebben naast de blokkade van het natrium-kanaal nog

andere, alternatieve, effecten. Wij zijn de eersten die aantonen dat lokale

verdovingsmiddelen in klinisch relevante concentraties ook in staat zijn om in vitro

de epigenetische markering in cellen te veranderen. Verder onderzoek naar dit

fenomeen is noodzakelijk.

Appendix 1

299

Als laatste bespreken wij de risico’s en baten van regionale anesthesie in de

gehele populatie. Regionale anesthesie ondergaat momenteel een belangrijke

verandering. Steeds meer indicaties voor specifieke technieken worden herzien of

komen te vervallen. Andere indicaties worden juist sterker. Al in al is het gebruik

van regionale anesthesie niet meer zo vrijblijvend als tien jaar geleden. Een

duidelijke afweging van risico’s en baten moet aan elke regionale techniek

voorafgaan.

Appendix 2

301

Appendix 2

Curriculum vitae of the candidate

Curriculum vitae

302

Curriculum vitae

Date of birth February 22nd

, 1977 in Salzburg, Austria.

Nationality Austrian.

Marital status Married to Petra Lirk-Heinrich, 2 sons Ferdinand and Leopold.

Languages spoken German, Dutch, English, French.

• 1995: Reifepruefung, Matura, Hoehere Internatsschule des Bundes, Saalfelden,

Austria

• 1995-1996: Army service, Austrian Federal Army, Reserve Officer Training

Course, current rank Lieutenant, commissioned with Territorial Infantry

Batallion “Erzherzog Rainer”, Salzburg, Austria.

• 1996-2002: M.D. degree, Leopold-Franzens Universität Innsbruck. Title of

dissertation: Psoas Compartment Block: Anatomic basis for ultrasound

guidance.

• 2001-2006: M.Sc. in Anesthesiology (Pain Management), University of Wales

College of Medicine, United Kingdom. Title of dissertation: Role of calcium

channels in a rodent model of chronic neuropathic pain.

• July 2003 – August 2004 Postdoctoral Research Fellowship at the Dept. of

Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.

• November 2002 – April 2010 Residency in Anesthesiology and Critical Care

Medicine, Innsbruck Medical University, Austria.

• April 2010 Austrian Board certification in Anesthesiology and Critical Care

Medicine.

• May 2010 Venia docendi (Postgraduate degree, Privatdozent), Innsbruck

Medical University, Austria.

• August 2010 – present Universitair Medisch Specialist (Attending

Anaesthesiologist) at the Academic Medical Center, University of Amsterdam.

• July 2011 – July 2016 Associate Professor at the University of North Florida

Brooks College of Health, Jacksonville, FL, USA.

Appendix 2

303

Grants, Awards and Prizes

• Medical Research Foundation Grant 103a (Innsbruck Medical University) 2004

• Midwest Anesthesia Resident Conference Second Best Poster Prize 2004

• Swarovski Research Award (Innsbruck Medical University) 2005

• ESRA Annual Meeting Best Free Paper Prize 2005 (First author)

• ESRA Annual Meeting 2nd

Best Free Paper Prize 2006 (Senior author)

• ESRA Annual Meeting Best Poster Prize 2006 (Presenting author)

• ESRA Annual Meeting 3rd

Best Free Paper Prize 2007 (Co-author)

• ESRA Annual Meeting Best Poster Prize 2011 (First author)

• Academic Medical Center Patient Care Quality Improvement Grant 2011

• Dutch Society of Anaesthesiology Young Investigator’s Award 2012

• European Society of Anaesthesiology Clinical Trial Network Award 2012

• Dutch Society of Anaesthesiology Ritsema van Eck Award 2nd

Prize 2013

• European Society of Regional Anaesthesia Research Grant 2013

Publications - Book chapter

Lirk P, Hogan Q. Spinal and Epidural Anatomy. In: Wong CA. 2007; Spinal and

Epidural Anesthesia. McGraw Hill, New York, NY.

Publications - Peer-reviewed

• Number of publications n = 74, 2 in press, 2 in revision.

• Number of times cited: 1070 (ISI Web of Knowledge, accession date

02.05.2014)

• H-index (Hirsch JE, Proc Natl Acad Sci USA 2005): 19

Appendix 3

305

Appendix 3

List of publications of the candidate

Publication list

306

Publications in revision

1 Kirchmair L, Voelckel W, Loescher W, Lirk P. Short-term neurophysiologic and

morphologic effects of experimental needle trauma and intraneural injection. Br J

Anaesth 2014; in revision

2 Lirk P, Verhamme C, Boeckh R, Stevens MF, ten Hoope W, Gerner P,

Blumenthal S, de Girolami U, van Schaik IN, Hollmann MW, Picardi S. Effects of

early and late diabetic neuropathy on sciatic nerve block duration and neurotoxicity

in Zucker Diabetic Fatty rats. Br J Anaesth 2014; in revision

Publications in press

1 Lirk P, Hollmann MW, Hauck-Weber NC, Fleischer M, Fiegl M. Lignocaine and

ropivacaine, but not bupivacaine, demethylate deoxyribonucleic acid in breast

cancer cells in vitro. Br J Anaesth 2014; in press

2 Lirk P, Picardi S, Hollmann MW. Local anaesthetics – 10 essentials (invited

review). Eur J Anaesthesiol 2014; in press

Peer-reviewed publications

1 Picardi S, Stevens MF, Hahnenkamp K, Durieux ME, Lirk P, Hollmann MW.

Time-dependent modulation of muscarinic m1/m3 receptor signalling by local

anaesthetics. Br J Anaesth 2014; 112: 370-9

2 Lirk P, Hollmann MW. Ondansetron: a new therapeutic option to attenuate

postoperative cognitive dysfunction and delirium? Minerva Anestesiol 2014; 80:

407-9

3 Ridderikhof ML, Lirk P, Schep NW, et al. The PanAM study: a multi-center,

double-blinded, randomized, non-inferiority study of paracetamol versus non-

steroidal anti-inflammatory drugs in treating acute musculoskeletal trauma. BMC

Emerg Med 2013; 13: 19

4 Picardi S, Cartellieri S, Groves D, et al. Local anesthetic-induced inhibition of

human neutrophil priming: the influence of structure, lipophilicity, and charge. Reg


Appendix 3

307

5 Meuwese JD, van Loon AM, Scholte HS, et al. NMDA receptor antagonist

ketamine impairs feature integration in visual perception. PLoS One 2013; 8: e79326

6 Lirk P, Stadlbauer KH, Hollmann MW. ESA Clinical Trials Network 2012:

PLATA--Prevention of Phantom Limb Pain After Transtibial Amputation:

randomised, double-blind, controlled, multicentre trial comparing optimised

intravenous pain control versus optimised intravenous pain control plus regional

anaesthesia. Eur J Anaesthesiol 2013; 30: 202-4

7 Lirk P, Rutten MV, Haller I, et al. Management of the patient with diabetic

peripheral neuropathy presenting for peripheral regional anesthesia: a European

survey and review of literature. Minerva Anestesiol 2013; 79: 1039-48


benefits. Minerva Anestesiol 2013

9 Gemes G, Koopmeiners A, Rigaud M, et al. Failure of action potential

propagation in sensory neurons: mechanisms and loss of afferent filtering in C-type

units after painful nerve injury. J Physiol 2013; 591: 1111-31

10 Lirk P, Haller I, Wong CA. Management of spinal anaesthesia-induced

hypotension for caesarean delivery: a European survey. Eur J Anaesthesiol 2012;

29: 452-3

11 Lirk P, Flatz M, Haller I, et al. In Zucker diabetic fatty rats, subclinical diabetic

neuropathy increases in vivo lidocaine block duration but not in vitro neurotoxicity.




Br J Anaesth 2012; 109: 200-7





15 Ong CK, Seymour RA, Lirk P, Merry AF. Combining paracetamol

(acetaminophen) with nonsteroidal antiinflammatory drugs: a qualitative systematic

Publication list

308

review of analgesic efficacy for acute postoperative pain. Anesth Analg 2010; 110:

1170-9

16 Lirk P, Kleber N, Mitterschiffthaler G, Keller C, Benzer A, Putz G. Pulmonary

effects of bupivacaine, ropivacaine, and levobupivacaine in parturients undergoing

spinal anaesthesia for elective caesarean delivery: a randomised controlled study. Int

J Obstet Anesth 2010; 19: 287-92

17 Schmutzhard J, Rieder J, Deibl M, et al. Pilot study: volatile organic compounds

as a diagnostic marker for head and neck tumors. Head Neck 2008; 30: 743-9




19 Lirk P, Poroli M, Rigaud M, et al. Modulators of calcium influx regulate

membrane excitability in rat dorsal root ganglion neurons. Anesth Analg 2008; 107:

673-85



neurotoxicity. Anesth Analg 2008; 106: 1456-64, table of contents

21 Kirchmair L, Lirk P, Colvin J, Mitterschiffthaler G, Moriggl B. Lumbar plexus

and psoas major muscle: not always as expected. Reg Anesth Pain Med 2008; 33:

109-14

22 Hogan Q, Lirk P, Poroli M, et al. Restoration of calcium influx corrects

membrane hyperexcitability in injured rat dorsal root ganglion neurons. Anesth

Analg 2008; 107: 1045-51

23 Ong CK, Lirk P, Tan CH, Seymour RA. An evidence-based update on

nonsteroidal anti-inflammatory drugs. Clin Med Res 2007; 5: 19-34



not by inhibiting caspase activity. Anesth Analg 2007; 105: 1657-64, table of

contents

Appendix 3

309

25 Hung YC, Chen CY, Lirk P, et al. Magnesium sulfate diminishes the effects of

amide local anesthetics in rat sciatic-nerve block. Reg Anesth Pain Med 2007; 32:

288-95

26 Haller I, Lirk P, Keller C, Wang GK, Gerner P, Klimaschewski L. Differential

neurotoxicity of tricyclic antidepressants and novel derivatives in vitro in a dorsal

root ganglion cell culture model. Eur J Anaesthesiol 2007; 24: 702-8

27 Fridrich P, Colvin HP, Zizza A, et al. Phase 1A safety assessment of intravenous

amitriptyline. J Pain 2007; 8: 549-55





mediated by apoptosis and are effectively blocked by inhibition of caspase activity.

Anesth Analg 2006; 102: 1728-33

30 Haller I, Hausott B, Tomaselli B, et al. Neurotoxicity of lidocaine involves

specific activation of the p38 mitogen-activated protein kinase, but not extracellular

signal-regulated or c-jun N-terminal kinases, and is mediated by arachidonic acid

metabolites. Anesthesiology 2006; 105: 1024-33

31 Almeling M, Schega L, Witten F, Lirk P, Wulf K. Validity of cycle test in air

compared to underwater cycling. Undersea Hyperb Med 2006; 33: 45-53

32 von Goedecke A, Keller C, Moriggl B, et al. An anatomic landmark to simplify

subclavian vein cannulation: the "deltoid tuberosity". Anesth Analg 2005; 100: 623-

8, table of contents

33 Sapunar D, Ljubkovic M, Lirk P, McCallum JB, Hogan QH. Distinct membrane

effects of spinal nerve ligation on injured and adjacent dorsal root ganglion neurons

in rats. Anesthesiology 2005; 103: 360-76

34 Rieder J, Lirk P, Bodrogi F, Sawires M, Gruber G, Hoffmann G. Cisatracurium,

but not mivacurium, induces apoptosis in human umbilical vein endothelial cells in

vitro. Eur J Anaesthesiol 2005; 22: 16-9

35 Pinggera GM, Lirk P, Bodogri F, et al. Urinary acetonitrile concentrations

correlate with recent smoking behaviour. BJU Int 2005; 95: 306-9

Publication list

310

36 Ong KS, Seymour RA, Yeo JF, Ho KH, Lirk P. The efficacy of preoperative

versus postoperative rofecoxib for preventing acute postoperative dental pain: a

prospective randomized crossover study using bilateral symmetrical oral surgery.

Clin J Pain 2005; 21: 536-42

37 Ong CK, Lirk P, Tan JM, Sow BW. The analgesic efficacy of intravenous versus

oral tramadol for preventing postoperative pain after third molar surgery. J Oral

Maxillofac Surg 2005; 63: 1162-8

38 Ong CK, Lirk P, Seymour RA, Jenkins BJ. The efficacy of preemptive analgesia

for acute postoperative pain management: a meta-analysis. Anesth Analg 2005; 100:


39 Moser B, Bodrogi F, Eibl G, Lechner M, Rieder J, Lirk P. Mass spectrometric

profile of exhaled breath--field study by PTR-MS. Respir Physiol Neurobiol 2005;

145: 295-300

40 Lirk P, Colvin JM, Biebl M, et al. [Evaluation of a cadaver workshop for

education in regional anesthesia]. Anaesthesist 2005; 54: 327-32

41 Lirk P, Colvin J, Steger B, et al. Incidence of lower thoracic ligamentum flavum

midline gaps. Br J Anaesth 2005; 94: 852-5

42 Lechner M, Lirk P, Rieder J. Inducible nitric oxide synthase (iNOS) in tumor

biology: the two sides of the same coin. Semin Cancer Biol 2005; 15: 277-89

43 Lechner M, Karlseder A, Niederseer D, et al. H. pylori infection increases levels

of exhaled nitrate. Helicobacter 2005; 10: 385-90

44 Fuchs A, Lirk P, Stucky C, Abram SE, Hogan QH. Painful nerve injury

decreases resting cytosolic calcium concentrations in sensory neurons of rats.


45 Friesenecker BE, Peer R, Rieder J, et al. Craniotomy during ECMO in a severely

traumatized patient. Acta Neurochir (Wien) 2005; 147: 993-6; discussion 6

46 Smolle M, Keller C, Pinggera G, Deibl M, Rieder J, Lirk P. Clear hydro-gel,

compared to ointment, provides improved eye comfort after brief surgery. Can J

Anaesth 2004; 51: 126-9

47 Moser B, Lirk P, Lechner M, Gottardis M. General anaesthesia in a patient with

motor neuron disease. Eur J Anaesthesiol 2004; 21: 921-3

Appendix 3

311

48 Lirk P, Moriggl B, Colvin J, et al. The incidence of lumbar ligamentum flavum

midline gaps. Anesth Analg 2004; 98: 1178-80, table of contents

49 Lirk P, Messner H, Deibl M, et al. Accuracy in estimating the correct

intervertebral space level during lumbar, thoracic and cervical epidural anaesthesia.


50 Lirk P, Longato S, Rieder J, Klimaschewski L. Cisatracurium, but not

mivacurium, inhibits survival and axonal growth of neonatal and adult rat peripheral

neurons in vitro. Neurosci Lett 2004; 365: 153-5

51 Lirk P, Keller C, Colvin J, Rieder J, Wulf K. Anaesthetic management of the

Prader-Willi syndrome. Eur J Anaesthesiol 2004; 21: 831-3

52 Lirk P, Keller C, Colvin J, et al. Unintentional arterial puncture during cephalic

vein cannulation: case report and anatomical study. Br J Anaesth 2004; 92: 740-2

53 Lirk P, Bodrogi F, Deibl M, et al. Quantification of recent smoking behaviour

using proton transfer reaction-mass spectrometry (PTR-MS). Wien Klin Wochenschr

2004; 116: 21-5

54 Keller C, Brimacombe J, von Goedecke A, Lirk P. Airway protection with the

ProSeal laryngeal mask airway in a child. Paediatr Anaesth 2004; 14: 1021-2

55 Keller C, Brimacombe J, Moriggl B, Lirk P, von Goedecke A. In cadavers,

directly measured mucosal pressures are similar for the Unique and the Soft Seal

laryngeal mask airway devices. Can J Anaesth 2004; 51: 834-7

56 Keller C, Brimacombe J, Lirk P, Puhringer F. Failed obstetric tracheal intubation

and postoperative respiratory support with the ProSeal laryngeal mask airway.

Anesth Analg 2004; 98: 1467-70, table of contents

57 Keller C, Brimacombe J, Bittersohl J, Lirk P, von Goedecke A. Aspiration and

the laryngeal mask airway: three cases and a review of the literature. Br J Anaesth

2004; 93: 579-82

58 Hinteregger M, Zschiegner F, Lirk P, et al. A new guidance device facilitates

percutaneous puncture of the foramen ovale in human cadavers. Can J Anaesth

2004; 51: 990-2

Publication list

312

59 Dunser MW, Ohlbauer M, Rieder J, et al. Critical care management of major

hydrofluoric acid burns: a case report, review of the literature, and recommendations

for therapy. Burns 2004; 30: 391-8

60 Summer G, Lirk P, Hoerauf K, et al. Sevoflurane in exhaled air of operating

room personnel. Anesth Analg 2003; 97: 1070-3, table of contents

61 Rieder J, Lirk P, Hoffmann G. Neopterin as a potential modulator of tumor cell

growth and proliferation. Med Hypotheses 2003; 60: 531-4

62 Rieder J, Keller C, Hoffmann G, Lirk P. Moscow theatre siege and anaesthetic

drugs. Lancet 2003; 361: 1131

63 Rieder J, Gruber G, Bodrogi F, Lirk P, Hoffmann G. Anaphylactoid reaction to

cisatracurium may be explained by atracurium metabolites. Anesth Analg 2003; 96:

301; author reply

64 Lorenz IH, Schubert HM, Lirk P, et al. [Evaluation of an established course of

hospital management through structured telephone survey of former participants].

Anasthesiol Intensivmed Notfallmed Schmerzther 2003; 38: 349-58

65 Lirk P, Rieder J, Schuerholz A, Keller C. Anaesthetic implications of Robinow

syndrome. Paediatr Anaesth 2003; 13: 725-7

66 Lirk P, Kolbitsch C, Putz G, et al. Cervical and high thoracic ligamentum flavum

frequently fails to fuse in the midline. Anesthesiology 2003; 99: 1387-90

67 Lirk P, Bodrogi F, Raifer H, Greiner K, Ulmer H, Rieder J. Elective

haemodialysis increases exhaled isoprene. Nephrol Dial Transplant 2003; 18: 937-

41

68 Rieder J, Lirk P. Neonatal atracurium toxicity may be explained by atracurium

moieties. Paediatr Anaesth 2002; 12: 656-7

69 Rieder J, Keller C, Brimacombe J, et al. Monitoring pollution by proton-transfer-

reaction mass spectrometry during paediatric anaesthesia with positive pressure

ventilation via the laryngeal mask airway or uncuffed tracheal tube. Anaesthesia

2002; 57: 663-6

70 Lirk P, Hoffmann G, Rieder J. Inducible nitric oxide synthase--time for

reappraisal. Curr Drug Targets Inflamm Allergy 2002; 1: 89-108

Appendix 3

313

71 Gruber G, Lirk P, Amann A, et al. Neopterin as a marker of immunostimulation:

an investigation in anaesthetic workplaces. Anaesthesia 2002; 57: 747-50

72 Rieder J, Prazeller P, Boehler M, Lirk P, Lindinger W, Amann A. Online

monitoring of air quality at the postanesthetic care unit by proton-transfer-reaction

mass spectrometry. Anesth Analg 2001; 92: 389-92

73 Rieder J, Lirk P, Summer G, et al. Exposure to sevoflurane in

otorhinolaryngologic operations. Can J Anaesth 2001; 48: 934

74 Rieder J, Lirk P, Ebenbichler C, et al. Analysis of volatile organic compounds:

possible applications in metabolic disorders and cancer screening. Wien Klin

Wochenschr 2001; 113: 181-5

Appendix 4

315

Appendix 4

Research portfolio of the candidate

Research portfolio

316

Grants, Prizes and Awards during Graduate Studies

• ESRA Annual Meeting Best Poster Prize 2011 (First author)

• Academic Medical Center Patient Care Quality Improvement Grant 2011

• Dutch Society of Anaesthesiology Young Investigator’s Award 2012

• European Society of Anaesthesiology Clinical Trial Network Award 2012

• NVA Ritsema van Eck Award 2nd

Prize 2013 (First author)

• European Society of Regional Anaesthesia Research Grant 2013

Courses during Graduate Studies

• Good Clinical Practice (GCP) course 2011

• Animal experimental course FELASA-C (Art 9 cursus) 2011

• Basiskurs Regelgeving en Organisatie voor Klinische Onderzoekers (BROK)

2013

• Reviewer workshop, British Journal of Anaesthesiology, 2013

Invited lectures (selection) during Graduate Studies

• Reasons not to block: diabetic neuropathy? European Society of

Anaesthesiology Annual Congress, Paris, France, June 2012

• Management of the elective cesarean section. Austrian Congress of

Anesthesiology, Resuscitation and Intensive Care Medicine, Klagenfurt,

Austria, September 2012

• Refresher course: local anesthetics. European Society of Regional Anesthesia

and Pain Therapy (ESRA) Annual Congress, Bordeaux, France, September

2012

• Intraneural injection: no! European Society of Regional Anesthesia and Pain

Therapy (ESRA) Winter Week, Grindelwald, Switzlerland, January 2013

• Local anesthetic neuro- and myotoxicity. European Society of Regional

Anesthesia and Pain Therapy (ESRA) Winter Week, Grindelwald,

Switzlerland, January 2013

Appendix 4

317

• Intralipid: basics of our guidelines. University of North Florida Patient Safety

Conference, Jacksonville, FL, USA, March 2013

• Ultrasound and what it has taught us about Regional anesthesia. University of

North Florida Patient Safety Conference, Jacksonville, FL, USA, March 2013

• Peripheral neuropathy and Peripheral nerve block. Americal Society of

Anesthesiology Annual Congress, San Francisco, CA, October 2013

• Local anesthetics: from basic research to new clinical applications.

Vinzenztage Congress, Vienna, Austria, November 2013

• Neuro- et myotoxicite des anesthesiques locaux. French University Diploma of

Regional Anesthesia, Paris, France, January 2014

• Local anesthetics demethylate DNA in breast cancer cells in vitro. Clincial

Epigenetics Society, Dusseldorf, Germany, March 2014

• Regional Anesthesia in Diabetic Neuropathy. University of North Florida

Patient Safety Conference, Jacksonville, FL, USA, March 2014

• Peripheral Nerve block in Diabetic Neuropathy. Morning Rounds, Hospital for

Joint Diseases, New York University Medical Center, New York, NY, USA,

March 2014

• Epigenetics and local anesthetics. Symposium Neural Therapy, University of

Heidelberg, Germany, May 2014

• Big data. Dutch Society of Anesthesiology Annual Congress, Maastricht, May

2014

• Local anaesthetics: the essentials. European Society of Anaesthesiology

Annual Congress, Stockholm, Sweden, June 2014

Editorial services

Regular reviewer for the journals Anesthesia & Analgesia, Minerva anestesiologica,

and the British Journal of Anaesthesia.

Service on committees

Member, European Society of Anaesthesiology (ESA) Scientific Subcommittee 8

(Regional Anesthesia) 2014-2016

Appendix 5

319

Appendix 5

Acknowledgment / Dankwoord

Acknowledgment

320

The last “opdracht” of this Thesis is a truly pleasurable one: to express

gratitude to the many people who each contributed such important pieces to the

Work as it stands now.

The first thanks traditionally go out to the Promotor, and that is especially

fitting for this Thesis. I would like to thank Prof. Markus Hollmann for welcoming

me into the Department of Anesthesiology at the Academic Medical Center (AMC),

and for giving me so much inspiration, support and liberty on my Academic path.

My second gratitude is expressed to my Department Chair Prof. Wolfgang Schlack,

for giving me the opportunity to come to the AMC, and for having the vision that

Amsterdam would be a good place for my family and me. Thank you so much. It is.

To the two co-promotores on this Thesis, Nina Hauck-Weber and Markus

Stevens, I would like to express my gratitude for their enormous and invaluable

support in all things laboratory, experimental, academic and Dutch. Their advice and

knowledge of Holland, Amsterdam and the AMC contributed greatly to making the

start back in 2010 a very positive and rewarding one.

I would like to thank all the members of the Graduation Committee, Prof.

Wolfgang Schlack, Prof. Ivo van Schaik, Prof. Marcus Schultz, Prof. Peter Gerner,

Prof. Jim Rathmell, dr. Geert-Jan van Geffen, and Dr. Lukas Kirchmair, for their

willingness to grade the Thesis, and to come to the Agnietenkapel on June 11th

2014

to enter into an academic discussion.

This work has been the product of a fruitful and productive collaboration

with many creative, diligent and at the same time “gezellig” colleagues from

different disciplines, Universities and countries. The research leading up to this

thesis was conducted in Innsbruck, and I am still grateful for all the support given to

me by the Dept. of Anesthesiology and Critical Care Medicine, Innsbruck Medical

University, especially by Volker Wenzel, Arnulf Benzer, Guenther Putz, Gottfried

Mitterschiffthaler and Christian Keller. Furthermore, I am grateful to Ingrid Haller,

Lars Klimaschewski and Barbara Hausott for their help and support on our studies

on Local-anesthetic-induced neurotoxicity. At the AMC, I would like to express my

thanks to Ivo van Schaik and Camiel Verhamme at the Dept. of Neurology, who

introduced me to the Neurophysiology of the Primary Sensory Neuron and Diabetic

Appendix 5

321

Neuropathy, and thereby laid the groundwork for hopefully many interesting

Regional Anesthesia projects to come. My thanks go out to Heidelinde Fiegl,

Innsbruck Medical University, and Martin Widschwendter, University College

London, for introducing me to the fascinating world of Epigenetics.

Along my academic career, I have been fortunate to encounter colleagues

who have influenced and inspired me. Among them, I would especially thank Peter

Gerner, Jim Rathmell and Quinn Hogan for being such exemplary role models as

Clinican Scientists and in the care they afford to their patients.

I am grateful to John McDonough and Juergen Osterbrink for giving me

insight into the discipline of Nurse anesthesia, for our many creative and fruitful

discussions, and the good collaboration on our mutual projects.

To my good friends Lukas Kirchmair and Josef Rieder (in alphabetical

order), I am grateful for your friendship and for our many common steps in

Research, in Anesthesia, and as friends.

Coming to Amsterdam also led to a wonderful collaboration with Susanne

Picardi at the University Hospital in Heidelberg. My gratitude for our interesting

mutual projects is linked to the hope that we will collaborate on many topics

surrounding local anesthetics in the years to come.

My two “paranimfen”, Holger Baumann and Jan Eshuis, I am grateful for

your support even from before the first day in Amsterdam, and that you will be at

my side on this important day.

Any good anesthesia department needs a well-balanced mix of clincians,

clinician-scientists and scientists. I think that our Department of Anaesthesiology

fits this description very very well and I am grateful to be working in such a

supporting and supportive environment.

Moving to Amsterdam almost four years ago was, naturally, a life event for

my family and me, but our arrival was facilitated and soothed by incountable

colleagues and friends who helped, supported, listened, gave advice, and made us

feel welcome. Hartelijk bedankt voor jullie steun en hulp.

Acknowledgment

322

The famous last words and words of gratitude belong to my family.

Specifically, I would like to thank my parents for supporting me all my life and for

affording me the liberty to pursue the study of my dreams, Medicine. Most

importantly, I would love to thank you, Petra, for our wonderful life together, and

you, Ferdinand and Leopold, for being such wonderful sons. Without you three (all

being well soon to be four), this Thesis would not be here, and that is literally the

case from front-page art to this very last page. And even if something was there,

without you it would not make sense. Ich lieb Euch, sehr sehr.

Local anestheticsNew insights into risks and benefits

Philipp Lirk

Local anesthetics P

hilipp Lirk

Hierbij nodig ik u uitvoor het bijwonen

van de openbare verdedigingvan mijn proefschrift getiteld

Local anestheticsNew insights into risks

and benefits

De verdediging vindt plaats woensdag 11 juni 2014

om 14:00 in de

AgnietenkapelUniversiteit van AmsterdamOudezijds Voorburgwal 231

1012 EZ Amsterdam

Receptie na afloop

Philipp LirkVan Breestraat 91/21071ZH [email protected]

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