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Article Title: Transcerebral net exchange of vasoactive peptides and catecholamines during lipopolysaccharide-induced systemic inflammation in healthy humans

Authors: Berg, R. M. G., Taudorf, S., Bailey, D. M., Dahl, R. H., Lundby, C., Møller, K

Journal: Canadian Journal of Physiology and Pharmacology

Citation: Berg, RMG, Taudorf, S, Bailey, DM, Dahl, RH, Lundby, C & Møller, K 2017, 'Transcerebral net exchange of vasoactive peptides and catecholamines during lipopolysaccharide-induced systemic inflammation in healthy humans' Canadian Journal of Physiology and Pharmacology. DOI: 10.1139/cjpp-2017-0266

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Transcerebral net exchange of vasoactive peptides and catecholamines during

lipopolysaccharide-induced systemic inflammation in healthy humans

Ronan M. G. Berg1,2; Sarah Taudorf1,3; Damian M. Bailey4; Rasmus H. Dahl5; Carsten Lundby6;

Kirsten Møller1,5

1Centre of Inflammation & Metabolism, University Hospital Rigshospitalet, Copenhagen, Denmark; 2Department of Clinical Physiology & Nuclear Medicine, Bispebjerg and Frederiksberg Hospitals,

Copenhagen, Denmark; 3Department of Neurology 2082, University Hospital Rigshospitalet,

Copenhagen, Denmark; 4Neurovascular Research Laboratory, Faculty of Life Sciences and

Education, University of South Wales, Pontypridd; 5 Department of Neuroanaesthesiology,

University Hospital Rigshospitalet, Copenhagen, Denmark; 6Zürich Center for Integrative Human

Physiology (ZIHP), University of Zürich, Zürich, Switzerland

Running head: Vasoactive peptides and catecholamines after LPS

Word count: ~1340; Figures: 2; Table: 1; References: 19

Target journal: Canadian Journal of Physiology and Pharmacology

Contact information:

Dr. Ronan M. G. Berg, MD, PhD

Department of Clinical Physiology & Nuclear Medicine

Nordre Fasanvej 57

Frederiksberg Hospital

DK-2000 Frederiksberg

Denmark

Phone: (+45) 38 16 47 71

E-mail: ronan@dadlnet.dk

Conflicts of interest: None

2

Abstract

The systemic inflammatory response triggered by lipopolysaccharide (LPS) is associated with

cerebral vasoconstriction, but the underlying mechanisms are unknown. We therefore examined

whether a four-hour intravenous LPS infusion (0.3 ng kg-1) induces any changes in the transcerebral

net exchange of the vasoactive peptides endothelin-1 (ET-1) and calcitonin-gene related peptide

(CGRP) and catecholamines in human volunteers. Cerebral blood flow was measured by the Kety-

Schmidt technique, and paired arterial-to-jugular venous blood samples were obtained for

estimating the transcerebral exchange of ET-1, CGRP and catecholamines by the Fick principle in

twelve volunteers before and after LPS. The cerebrovascular release of ET-1 was enhanced, while

the transcerebral net exchange of CGRP and catecholamines was unaffected. Our findings thus

point towards locally produced ET-1 within the cerebrovasculature as a contributor to cerebral

vasoconstriction after LPS.

Keywords:

Calcitonin-gene related peptide; cerebral blood flow; cerebral oxidative metabolism, epinephrine,

dopamine, endothelin-1; endotoxemia, norepinephrine, sepsis, sepsis-associated encephalopathy

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Introduction

Intravenous administration of the bacterial endotoxin lipopolysaccharide (LPS) elicits an acute

systemic inflammatory response similar to that encountered during the very early stages of sepsis

(Calvano & Coyle 2012). This is associated with cerebral vasoconstriction in both animals and

humans, and while the hyperventilatory response characteristic of acute systemic inflammation is

likely important to this (Brassard et al. 2012; Emerson & Parker 1977; Møller et al. 2002), a degree

of cerebral vasoconstriction ensues even when isocapnia is maintained (Ekström-Jodal et al. 1982a;

Emerson & Parker 1976). These LPS-induced cerebral hemodynamic changes may have clinical

implications, since sepsis-associated encephalopathy, a frequent complication that sets in from the

very early stages of sepsis, has been proposed as manifestation of a cerebrovascular dysfunction

triggered by the systemic inflammatory response (Gofton & Young 2012).

In the present study, we investigated whether LPS infusion enhances the cerebrovascular release of

the vasoconstrictor endothelin-1 (ET-1), which has previously been implicated as a contributor to

the cardiovascular dysfunction associated with LPS infusion and sepsis (Arnalic et al. 1996; Soop

Weitzberg 1993). Given that the vascular effects of ET-1 may be overruled by the vasodilator

calcitonin-gene related peptide (CGRP) (Meens et al. 2011), which is present in perivascular

sensory nerves of pial vessels (Russell et al. 2014), we furthermore investigated whether any

concomitant changes occur in the cerebrovascular net exchange of CGRP. Lastly, we assessed the

cerebrovascular net exchange of norepinephrine, epinephrine, and dopamine, since several now

classical studies have highlighted circulating catecholamines as putative triggers of both cerebral

vasoconstriction and increased cerebral oxidative metabolism following LPS (Ekström-Jodal et al.

1982b; Westerlind et al. 1991).

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Materials & Methods

Ethics

The study was approved by the Scientific Ethical Committee of Copenhagen and Frederiksberg

Municipalities, Denmark (file number [KF] 01 290011) and was performed in accordance with the

Helsinki Declaration with oral and written informed consent from participants prior to inclusion.

Data on the systemic inflammatory response, CBF and metabolism, and the transcerebral exchange

of amino acids in this study have previously been published elsewhere (Berg et al. 2010, 2012).

Participants and design

Twelve healthy male volunteers aged 26 (mean; SD 4) with an unremarkable medical history

participated in the study. Before inclusion, volunteers underwent a thorough physical examination

with appropriate laboratory tests, which were all normal.

After an overnight fast, subjects were catheterized with an antecubital vein catheter, and following

local anesthesia with lidocaine, a peripheral arterial line and a retrograde jugular bulb catheter.

After 30 minutes of rest, CBF was measured by the Kety-Schmidt technique as described elsewhere

(Taudorf et al. 2009), and paired arterial-jugular venous blood samples were concurrently obtained

for the measurement of vasoactive peptides, metabolites, and catecholamines. This was repeated

one hour after the cessation of a four-hour continuous intravenous infusion of purified E. coli LPS

(infusion rate, 0.075 ng/kg/h; total dose, 0.3 ng/kg; Batch G2 B274, US Pharmacopeial Convention,

Rockville, MD, USA) (Taudorf et al. 2007).

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Laboratory analyses

Samples for ET-1 and CGRP were collected in tubes prepared with aprotinin (Trasylol; Bayer,

Germany), while catecholamines were drawn into ice-chilled tubes containing glutathione (1.3

mg/mL blood) and EDTA (1.5 mg/mL blood). All samples were immediately centrifuged (3000

rpm at 4oC), and stored at -80oC until analysis. Radioimmunoassays were used to measure ET-1

(Peninsula, cat. no. RAS6901) and CGRP. Norepinephrine and epinephrine were measured by high-

performance liquid chromatography (Hewlett-Packard, Wald-bronn, Germany), while dopamine

was determined with a RIA kit (Biotech-IgG, Copenhagen, Denmark). Blood samples for the

determination of arterial and jugular venous blood gases, acid-base status, glucose and lactate were

analyzed on a blood-gas analyzer (ABL 605, Radiometer, Brønshøj, Denmark). White blood cell

and platelet counts determined in arterial blood samples by an automated analyzer (Sysmex XE-

2100, Sysmex Europe GmbH, Hamburg, Germany)

Calculations

The global cerebral metabolic rates (CMR; of oxygen, glucose, or lactate) and global transcerebral

net exchange (J) values (ET-1, CGRP, norepinephrine, epinephrine, and dopamine) were calculated

according to the Fick principle, that is by multiplying arterial-to-jugular venous whole-blood

concentration differences with CBF (for CMR values) or arterial-to-jugular venous plasma

concentration differences with cerebral plasma flow (CBF ×(1−Hct )). Cerebral intermediary

metabolism was further evaluated by calculating the cerebral oxygen-glucose (OGI=C aO2

−C jvO 2

C aglc−C jv glc

), lactate-glucose (LGI=C alac−C jv lac

C aglc−C jv glc), and lactate-oxygen (LOI=

C alac−C jvlac

C aO2−C jvO 2

) indices.

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Statistics

Normality of data was confirmed by visual inspection of normality plots and by means of the

Shapiro-Wilk W-test. Paired-samples t-tests were used to evaluate differences between baseline and

LPS. Data are presented as mean (SD), and within-subject differences are presented as mean (95%

CI). Significance was established at P < 0.05. All analyses were performed using SAS statistical

software version 9.2 (SAS Institute Inc., Cary, NC, USA).

Results

LPS induced a systemic inflammatory response with fever, leukocytosis, and flu-like symptoms.

This involved a 1.7 (95 % CI: 1.1-2.4) oC increase in tympanic temperature from 36.2 (0.5) to 38.1

(0.9) oC (p < 0.01), a 5 (95 % CI 3-7) mmHg reduction in PaCO2 from 44 (3) to 39 (5) mmHg

(<0.01), and an increase in pH of 0.03 (0.01-0.04) from 7.39 (0.01) to 7.41 (0.04) (p < 0.05).

CBF did not change after LPS, while CMRO2 increased with no changes in the indices of cerebral

intermediary metabolism. Data on cerebral hemodynamics and metabolism are summarized in

Table 1.

ET-1 levels increased after LPS infusion, (Figure 1A). A net cerebral release of ET-1 was present

at baseline, and this was increased after LPS infusion (Figure 1B). In contrast, CGRP levels were

unaffected (Figure 2A), and no net cerebral release of CGRP was present either at baseline or after

LPS infusion (Figure 2B).

The arterial levels of epinephrine increased after LPS infusion (182 [101] vs. 492 [292] pmol l -1; p <

0.01), while norepinephrine (757 [427] vs. 782 [359] pmol l-1) and dopamine (253 [95] vs. 201 [13]

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pmol l-1) levels were unaffected (p = 0.84 and 0.12, respectively). There were no changes in the

transcerebral net exchange of any of the catecholamines (epinephrine: 0.02 [0.05] vs. 0.02 [0.08]

pmol g-1 min-1; p = 0.91; norepinephrine: -0.01 [0.09] vs. -0.03 [0.04] pmol g-1 min-1; p = 0.58;

dopamine: 0.02 [0.06] vs. -0.01 [0.02] pmol g-1 min-1; p = 0.30).

Discussion

Our findings demonstrate that LPS-induced systemic inflammation enhances the release of ET-1

from the cerebrovasculature, while CGRP levels are unaffected. Furthermore, we found no changes

in the transcerebral exchange of catecholamines; our findings thus practically rule out circulating

catecholamines as mediators of the relative cerebral hypoperfusion (maintained CBF despite

increased CMRO2) observed after LPS.

The observed net cerebral release of ET-1 expectedly represents a minor fraction of the total

cerebrovascular ET-1 production. Hence, while circulating ET-1 is unlikely to pass the blood-brain

barrier, ET-1 produced in the endothelium is predominantly released to the abluminal side to trigger

local vasoconstriction (Weitzberg 1993), and the enhanced net cerebral release of ET-1 after LPS is

thus likely associated with a substantial perivascular ET-1 increase within the brain. The

cardiovascular effects of ET-1 during LPS-induced systemic inflammation have previously been

found to involve pulmonary, renal, and splanchnic vasoconstriction (Weitzberg 1993). Given that

ET-1 principally acts in a paracrine fashion, our findings suggest that it also contributes to cerebral

vasoconstriction.

In a previous study, we found no significant changes in the transcerebral exchange of either ET-1 or

CGRP 90 minutes after an intravenous bolus infusion of LPS (Berg et al. 2009). However, we

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probably failed to detect any changes, because the systemic inflammatory response to a bolus

infusion is highly dynamic and short-lived; in the present study, we therefore assessed the

transcerebral exchange of ET-1 and CGRP following a four-hour continuous endotoxin infusion,

where the systemic inflammatory response is sustained for several hours, and thus likely more

representative of systemic inflammation in the clinical setting (Taudorf et al. 2007).

In conclusion, our findings highlight ET-1 as a contender that acts in concert with a reduction in

PaCO2 to exert cerebral vasoconstriction after LPS, changes that may potentially also apply to the

very early stages of clinical sepsis. In contrast, our findings lend no support for changes in the

transcerebral exchange of CGRP or catecholamines as mechanisms of the cerebrovascular changes

in this context.

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Author contributions

RMGB conducted the study, acquired, analyzed and interpreted the data, performed statistical

analyses, and drafted the manuscript. ST, DMB, and CL conducted the study acquired, analyzed

and interpreted the data. RHD analyzed and interpreted the data and prepared figures and tables.

KM conceived and designed the research, conducted the study, acquired, analyzed and interpreted

the data, and handled funding and supervision. All authors made critical revisions and read and

approved the final manuscript.

Acknowledgements

We thank Ruth Rousing and Hanne Villumsen for their outstanding technical assistance.

Funding

This study was supported by grants from the Danish National Research Council (#22-04-0413 and

#504-14), the Copenhagen Hospital Corporation, the Lærdal Foundation, the AP Møller

Foundation, the Jensa la Cour Foundation, the Larsen Foundation, the Højmosegaard Foundation,

and the P. Carl Petersen Foundation. The Centre of Inflammation and Metabolism was supported by

a grant from the Danish National Research Foundation (#DG 02-512-555). This study was further

supported by the Research Board at Rigshospitalet, the Danish Medical Research Council (#09-

064930) and the Commission of the European Communities (contract no. LSHM-CT-2004-005272

EXGENESIS).

Conflicts of interest

None

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Legends

Table 1. Cerebral hemodynamics, metabolism, and oxygenation. Measurements were performed

in twelve volunteers at baseline and one hour after the cessation of a four-hour intravenous

lipopolysaccharide (LPS)-infusion (total dose: 0.3 ng kg-1). CBF: cerebral blood flow; CMRO2:

cerebral metabolic rate of oxygen; CMRglc: cerebral metabolic rate of glucose; CMRlac: cerebral

metabolic rate of lactate; CPF: cerebral plasma flow; LGI: lactate-glucose index; LOI: lactate-

oxygen index; OGI: oxygen-glucose index.

Figure 1. Endothelin 1 (ET-1). Measurements were performed at baseline and one hour after the

cessation of a four-hour intravenous lipopolysaccharide (LPS)-infusion. A. Arterial plasma levels of

ET-1 (PET-1). B. Transcerebral net exchange of ET-1 (JET-1). Different from baseline, *p < 0.05.

Figure 2. Calcitonin-gene related peptide (CGRP). Measurements were performed at baseline

and one hour after the cessation of a four-hour intravenous lipopolysaccharide (LPS)-infusion. A.

Arterial plasma levels of CGRP (PCGRP). B. Transcerebral net exchange of CGRP (JCGRP).

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Table 1

Baseline LPS 95 % CI p value

CBF (mL 100 g-1 min-1) 76 (13) 81 (16) 4 ([-3]–12) 0.32

CPF (mL 100 g-1 min-1) 44 (8) 47 (10) 3 ([-2]–8) 0.27

CMRO2 (µmol g-1 min-1) 1.95 (0.47) 2.33 (0.53) 0.38 (0.11–0.64) 0.02

CMRglc (µmol g-1 min-1) 0.36 (0.07) 0.39 (0.08) 0.03 ([-0.01]–0.07) 0.21

CMRlac (µmol g-1 min-1) -0.04 (0.05) -0.03 (0.05) 0.01 ([-0.02]–0.04) 0.63

OGI 5.45 (0.77) 6.05 (0.94) 0.60 ([-0.03]–1.22) 0.10

LGI 0.11 (0.15) 0.09 (0.13) -0.02 ([-0.12]–0.08) 0.73

LOI 0.02 (0.03) 0.01 (0.03) -0.01 ([-0.03]–0.01) 0.50

Figure 1 Figure 2

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