From the Department of Physiology and Pharmacology

Karolinska Institutet, Stockholm, Sweden

BEHAVIOURAL AND NEUROCHEMICAL EFFECTS OF

LONG-LASTING INFLAMMATORY PAIN

Umut Heilborn

Stockholm 2007

All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by ReproPrint AB. © Umut Heilborn, 2007 ISBN 978-91-7357-175-3

Cover image U. Heilborn, adapted from: Tractatus de Homine, by René Descartes (1596-1650) 1677 edition , Publisher D.Elsevir, illustration Ludwig de la Forge, M.D.

The French philosopher and scientist René Descartes wrote Tractatus de Homine (Eng. Treatise of Man) in 1637, but he did not dear to make it public during his lifetime due to fear that the Church would find it heretical. Therefore the book was published posthumously in 1662, first in the Latin translation and two years later in the French original version.

be the first textbook of Physiology and it introduces many ideas new to that time such as a mechanistic vue of the human body as a machine which is directed by a rational soul located in the brain.

The picture used on the cover of this thesis illustrates of pain transmission. In the figure legend Descartes emphasises that the sensation of pain is not perceived in the injured limb, but in the brain. Descartes recognized that the pain signal was transmitted by nerves via the spinal cord to the brain. The drawing also suggests that the author or illustrator had some notion of the signal being relayed at several points on the way to the brain.

(the body) were integrated with higher spiritual aspects (the soul) in the pineal gland of the brain. This almost 400 year old theory has obvious similarities to the present view of pain as an incorporation of nociceptive signals from the body with the emotional functions of higher CNS levels. One of the brain regions considered to be of importantce for this integration between nociception and emotion, or -philosophically expressed - the body and the mind, is the anterior cingulate cortex.

Ove Hagelin and Gertie Johansson, curators of rare och important books, the Hagströmer Library, Karolinska Institutet, are gratefully acknowledged for providing original copies of the book.

Till Johan

PUBLICATIONS

This thesis is based on the following articles, which are referred to in the text by their Roman numerals:

I. Effects of morphine on pain-related behaviour when given before or after

induction of monoarthritis and pharmacokinetics of morphine in rats

Umut Heilborn, Eva Schött, Olof Beck, Lotta Arborelius, Ernst Brodin.

Submitted

II. Spontaneous noci

adjuvant- and carrageenan-induced monoarthritis

Umut Heilborn, Odd-Geir Berge, Lotta Arborelius, Ernst Brodin.

Brain Research 2007, 1143 (1) 143-149

III. Increased cholecystokinin release in the rat anterior cingulate cortex during

carrageenan-induced arthritis.

Umut Erel, Lotta Arborelius, Ernst Brodin.

Brain Research 2004; 1022 (1-2) 39-46

IV. Arthritis-induced increase in cholecystokinin release in the rat anterior

cingulate cortex is reversed by diclofenac

Umut Heilborn, Benjamin Rost, Lotta Arborelius, Ernst Brodin.

Brain Research 2007; 1136 (1) 51-58

ABSTRACT

The treatment of chronic, inflammatory pain in humans is still unsatisfactory. In order to develop new anti-inflammatory and analgesic drugs, studies on the mechanisms underlying inflammatory pain in valid animal models, are of considerable importance. In the present thesis, we have investigated behavioural and neurochemical alterations in animal models of long lasting inflammatory pain. We have also addressed this field from a methodological perspective. In addition, we have investigated a pharmacological approach in animals which has been suggested to prevent the generation of chronic pain in humans.

Treatment with morphine prior to surgical interventions has been suggested to prevent the development of subsequent pain conditions. However, previous animal and human studies have shown contradictory results. We therefore examined the antinociceptive effect of morphine given before or after the induction of monoarthritis in rats. In order to investigate a possible correlation to the behavioural response, the plasma concentrations of morphine were followed. In the present preclinical setting, our data do not support an advantageous effect of administration of morphine prior to the induction of arthritis compared to morphine treatment after the onset of inflammatory pain.

The development of transgenic techniques has accentuated the need for valid disease models in mice. We therefore developed a model for joint inflammation in mice: adjuvant-induced monoarthritis. We estimated pain-related behaviour by scoring of gait and stance and we validated the model pharmacologically by administrating two different analgesic drugs, morphine and the non-steroid anti-inflammatory drug (NSAID) diclofenac. We conclude that adjuvant-induced monoarthritis, which has a limited effect on the general state of health of the animals compared to polyarthritic models, constitutes a robust and reproducible model in mice. This model is likely to be useful in patophysiological and pharmacological studies on inflammatory pain in transgenic mice. However, the choice of mouse strain is of importance for the results obtained.

Several human and animal studies indicate that the anterior cingulate cortex (ACC) plays an important role in the affective component of pain. The neuropeptide cholecystokinin (CCK) has been suggested to be involved in anxiety and in the modulation the antinociceptive effects of opioids in the spinal cord and medulla oblongata. However, its possible role in pain transmission or modulation in the brain is far less clear. CCK is particularly abundant in the ACC and we hypothesised that CCK in this brain region may play a role in pain, possibly in the affective component. Using a model for subchronic inflammatory joint pain in rats, we studied the effect of pain on the release of CCK in the ACC. We found that animals with inflammatory pain had a significantly increased release of CCK in the ACC compared to controls. We then proceeded by investigating whether the increased CCK release could be affected by morphine and diclofenac. Morphine, unlike diclofenac, is known to relieve the affective component of pain. Surprisingly, we found that diclofenac but not morphine reduced the increased CCK release in the ACC of rats with inflammatory pain. Thus, our results argue against the hypothesis that CCK in the ACC is involved in the affective component of pain, but rather indicates that the observed increase in CCK could be mediated by an elevation in prostaglandin levels. Further studies on the interaction between prostaglandins and CCK in the brain may provide a future basis for the treatment of inflammatory pain in humans.

ABBREVIATIONS

Neuroanatomical ACC anterior cingulate cortex IC insular cortex NRM nucleus raphe magnus NRPC nucleus reticularis paragigantocellullaris PAG periaqueductal grey PFC prefrontal cortex RVM rostral ventromedial medulla VP ventroposterior nucleus (of the thalamus) VPL ventroposterior lateral nucleus VPM ventroposterior medial nucleus Neurochemical CCK cholecystokinin CGRP calcitonin-gene related peptide GABA gamma amino butyric acid Glu glutamic acid NKA neurokinin A NKB neurokinin B NMDA N-methyl-D-Aspartate SP substance P Other ad lib. ad libitum (freely, as desired, Latin) ANOVA analysis of variance CNS central nervous system COX cyclo-oxygenase et al. et alii (and others, Latin) FCA HPLC high performance liquid chromatography i.t. intrathecal i.v. intravenous -LI - like immunoreactivity RIA radio immuno assay s.c. subcutaneous

CONTENTS

1 INTRODUCTION ......................................................................................................................................... 1

1.1 PAIN MECHANISMS ANATOMY AND PHYSIOLOGY ....................................................... 1

1.1.1 The primary afferent............................................................................................................. 2

1.1.2 The spinal cord ascending pathways ............................................................................. 2

1.1.3 Brain regions in pain perception ..................................................................................... 3

1.1.4 The anterior cingulate cortex ............................................................................................ 5

1.1.5 Modulation of nociceptive transmission ...................................................................... 5

1.1.6 Long lasting pain ............................................................................................................. ....... 6

1.2 NEUROTRANSMITTERS IN PAIN SIGNALLING AND PAIN MODULATION .............. 7

1.2.1 Neuropeptides ......................................................................................................................... 7

1.2.2 Endogenous opioids and opioid-modulating peptides ........................................... 7

1.2.3 Cholecystokinin ............................................................................................................... ........ 8

1.3 ANALGESIC DRUGS ...................................................................................................................... 10

1.3.1 Opioids ..................................................................................................................................... 10

1.3.2 NSAIDS ..................................................................................................................................... 12

1.3.3 Pre-emptive analgesia ....................................................................................................... 13

1.4 ANIMAL MODELS OF PAIN ....................................................................................................... 14

1.4.1 Methods for inducing pain in rodents ........................................................................ 14

1.4.2 Methods for assessing pain in rodents ....................................................................... 15

1.4.3 Ethical aspects ...................................................................................................................... 16

2 AIMS ............................................................................................................................................................. 17

3 METHODS AND METHODOLOGICAL REMARKS........................................................................ 18

3.1 LABORATORY ANIMALS ............................................................................................................ 18

3.2 INDUCTION OF ARTHRITIS ...................................................................................................... 18

3.3 PHARMACOLOGICAL TESTING ............................................................................................... 19

3.3.1 Stance scoring ................................................................................................................ ....... 19

3.3.2 Weight bearing ..................................................................................................................... 20

3.3.3 Ankle oedema ....................................................................................................................... 21

3.3.4 Administration of drugs ................................................................................................... 21

3.4 MICRODIALYSIS ............................................................................................................................ 22

3.4.1 Surgical Procedure.............................................................................................................. 22

3.4.2 Dialysis ..................................................................................................................................... 22

3.5 RADIOIMMUNOASSAY ................................................................................................................ 23

3.6 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ..................................................... 24

3.6.1 Characterisation of CCK-LI by HPLC ............................................................................ 24

3.6.2 Quantification of Morphine and M3G by HPLC ....................................................... 25

3.7 CALCULATIONS OF RESULTS AND STATISTICAL ANALYSIS ..................................... 25

3.7.1 Biochemical data .................................................................................................................. 25

3.7.2 Behavioural data .................................................................................................................. 26

4 RESULTS AND DISCUSSION ................................................................................................................ 27

4.1 INVESTIGATION OF A POSSIBLE PRE-EMPTIVE ANALGESIC EFFECT ....................... OF MORPHINE IN RATS WITH MONOARTHRITIS (PAPER I) .................................... 27

4.2 SPONTANEOUS NOCICEPTIVE BEHAVIOUR IN MICE WITH MONO- ........................... ARTHRITIS (PAPER II) ............................................................................................................... 29

4.3 MONOARTHRITIS INCREASES CHOLECYSTOKININ RELEASE IN ................................. THE RAT ANTERIOR CINGULATE CORTEX, AN EFFECT REVERSED ......................... BY NSAID BUT NOT MORPHINE (PAPER III AND IV) ................................................... 31

5 GENERAL DISCUSSION ......................................................................................................................... 33

5.1 EVIDENCE OF PRE-EMPTIVE ANALGESIA .......................................................................... 33

5.2 ANIMAL MODELS OF INFLAMMATORY PAIN ................................................................... 35

5.3 THE POTENTIAL ROLE OF CCK IN PAIN MODULATION .............................................. 37

6 CONLUSIONS ............................................................................................................................................. 41

7 SVENSK SAMMANFATTNING ............................................................................................................. 42

8 ACKNOWLEDGEMENTS ....................................................................................................................... 43

9 REFERENCES ............................................................................................................................................ 45

Introduction

1

1 INTRODUCTION

Pain is one of the most important functions of the nervous system, providing information about the presence or threat of injury. From this perspective, pain is a necessary consequence of and prerequisite for life. However, large parts of the human population are afflicted with chronic and disabling pain. Some of the major causes of such pain are musculoskeletal diseases such as rheumatoid arthritis, osteoarthritis and back pain. At any moment, one third of an adult Western society population will report back, hip or shoulder pain (Scott, 2006). These conditions not only generate large costs to society in economic terms, but also constitute a major source of suffering (Akesson, 2003; Brooks, 2006).

The symptomatic treatment of chronic arthritis in man is still unsatisfactory, especially regarding pain relief and a better understanding of the basic mechanisms underlying chronic inflammatory pain could offer a basis for the development of new therapeutic approaches. In the present thesis we have by using preclinical animal models, studied neurochemical mechanisms in the brain during long-lasting inflammatory pain. Following a general outline on pain mechanisms in the nervous system, the introduction will focus on the brain regions and neurotransmitters of interest for our studies.

1.1 PAIN MECHANISMS ANATOMY AND PHYSIOLOGY

The perception of noxious, that is, high-intensity, potentially damaging stimuli, is termed nociception (Sherrington, 1906; Burke, 2007). This needs to be distinguished from the term pain, which in addition to nociception includes a pronounced emotional (affective) component of discomfort (DeLeo, 2006; Treede, 2006). Nociception frequently occurs without pain being felt and can be below the level of consciousness. Even though it triggers pain and suffering, nociception is a critical component of the body's defence system. It is part of a rapid warning relay instructing the central nervous system to initiate motor neurons in order to minimize detected physical harm. Pain too is part of the body's defence system; it activates mental problem solving strategies that seek to end the painful experience and maintain homeostasis. Thus, pain promotes learning in order to reduce the risk of repetition of the painful situation. A schematic illustration of the pain transmission from the periphery to the cerebral cortex is shown in figure 1.

Figure 1. Schematic illustration of the transmission of pain signals from the periphery to the cerebral cortex (adapted from Descartes, 1677).

Umut Heilborn

2

1.1.1 THE PRIMARY AFFERENT

Under normal conditions, a stimulus, given that its intensity is high enough, can activate so-called nociceptors which are the peripheral endings of primary afferents. These in turn, are sensory small-diameter, slow conductance, predominantly unmyelinated (C-fibres) or myelinated ( -fibres) neurons. The majority of nociceptors can be activated by several types of sensory input (thermal, mechanical and chemical) and are therefore termed polymodal nociceptors (Julius and Basbaum, 2001; Julius and McCleskey, 2006). Tissue injury, which is commonly the immediate cause of pain, results in a local release or synthesis of a range of substances such as bradykinin and prostaglandins. These compounds either activate the nociceptors directly or affect them indirectly by increasing the sensitivity of the nociceptors to other activating ligands.

Nociceptors not only receive input from the surrounding tissue, but they also contribute to the local inflammatory reaction by releasing transmitters such as substance P (SP) and calcitonin-gene-related peptide (CGRP) (Meyer et al., 2006) and they thereby interact with adjacent immune cells and blood vessels in a complex manner.

1.1.2 THE SPINAL CORD ASCENDING PATHWAYS

The cell bodies of the primary afferents, which are located in the dorsal root ganglia, project their proximal nerve terminals mainly to the laminae I- II, IV-V and VII-VIII of the spinal cord dorsal horn (Julius and McCleskey, 2006).

The stimulation induced by the primary afferent transmission brings about a postsynaptic excitation of spinal cord ascending neurons, of which a major part project to the thalamus (for rev see Dostrovsky and Craig, 2006). The main types of neurons in this pathway cells are nociceptive-specific (NS) neurons that respond mainly to high-intensity stimuli, and wide dynamic range (WDR) neurons which respond both to innoxious and noxious input (Willis and Westlund, 1997; Price et al., 2003). In addition, there are cell types specific for temperature- , itch- and visceral input. This direct spinothalamic tract is the classical pathway most closely associated with pain, temperature and itch sensation. The spinothalamic tract projects to various nuclei of the thalamus which, in turn, project to diverse brain areas. Thereby the thalamus contributes to different aspects of pain processing. The most well known targets of spinothalamic tract are the ventral posterior (VP) nuclei of the thalamus. The VP can be divided into the medial (VPM) and the lateral (VPL) nuclei which receive sensory input from the trigeminal nuclei and the spinal cord (i.e. the face and the rest of the body), respectively. VP neurons project to the somatosensory cortex (S1/S2) (Price et al., 2003), and it has been known for long that the VP plays a key role in pain perception. Of late, it has been revealed that a large part of the spinothalamic projections in humans terminate in the posterior part of the ventral medial nucleus (VMpo) (Blomqvist et al., 2000; Willis et al., 2002). This nucleus is rudimentary in non-primates and most developed in humans (Craig, 2006). The VMpo projects to the insular cortex (IC) and mediates interoceptive sensory information contributing to the maintenance of homeostasis. Another thalamic region, the medial dorsal (MD) nucleus, projects partly to the orbitofrontal and prefrontal cortex (PFC) and partly to the fundus of the anterior cingulate cortex (ACC) (Dostrovsky and Craig, 2006). Further on, there are thalamic nuclei involved in pain that project to the motor cortex and the

Introduction

3

basal ganglia/substantia nigra. These are associated with pain-related orientation and motor activity.

In addition to the spinothalamic pathway, there are direct projections from the dorsal horn to brainstem areas of importance for pain. Spinal nociceptive pathways project to the parabrachial (PB) area, which, by connections to the amygdala, hypothalamus and the insular cortex, contributes to forebrain autonomic, neuroendocrine and emotional control of pain. Furthermore, there are nociceptive projections to catecholaminergic cell groups which mediate cardio-respiratory reactions and homeostatic control in response to nociceptive input (Card et al., 2006) as well as descending nociceptive modulation (Pertovaara, 2006). A spinal pathway of significance in this context is the projection to the periaqueductal gray (PAG) (see also 1.1.5). Spinal input to the PAG is integrated with descending anti-nociceptive modulation of the spinal cord through projections to the brain stem reticular formation (Sandkühler, 1996; Fields et al., 2006). There is also a direct spinal projection to the reticular formation which seems to be involved in descending pain modulation.

1.1.3 BRAIN REGIONS IN PAIN PERCEPTION

Consistent evidence indicate that the most commonly activated regions during pain in humans are the S1/S2, ACC, IC, PFC, and the cerebellum, as illustrated in figure 2 (for rev. see Bushnell and Apkarian, 2006). Several of these regions seem to be responsible for the perception of pain and processing related to pain suffering. These aspects of pain are difficult to study in animals, not necessarily because animals are devoid of such higher cerebral functions, but because our methods for assessing them are insufficient. Investigations of the importance of these regions for various aspects of pain, have therefore to a large extent been based on case studies of pathological lesions in humans. However, the development of modern neural-imaging techniques such as positron emission tomography (PET) and functional magnetic tomography (fMRI), currently provides a basis for more experimental strategies in the study of brain mechanisms of pain (Price, 2000). Still, these techniques are restricted to measurement of blood flow, metabolic activity or receptor binding and do not provide information regarding the neurochemical events during pain.

Figure 2. Schematic illustration of supraspinal regions of importance for pain perception. Black arrows indicate primary sensory-discriminative and gray arrows indicate the affective-motivational pathways. ACC, anterior cingulate cortex; Amy, amygdala; HT, hypothalamus; IC, insular cortex; MD, medialdorsal nucleus; PFC, prefrontal cortex; PPC, posterior parietal cortex; S1/S2, primary and secondary sensory cortex; SC, spinal cord, VP ventral posterior nucleus; Vmpo, posterior part of the ventromedial nucleus (adapted from Price, 2000).

MD

ACC

S1/S2

PFC

PPC

IC

Amy

PB

SC

HT

VMpoVP

Umut Heilborn

4

The temporal and hierarchical relationship between nociception, pain unpleasantness and pain affect, is illustrated in figure 3. The S1/S2, which receives input from the VP nuclei of the thalamus (Shi and Apkarian, 1995) is the main brain region responsible for identifying the localisation, intensity and modality of the nociceptive input, in other words the sensory-discriminative aspect of pain. There are connections from S1/S2 and posterior parietal cortices to limbic areas and this corticolimbic pathway may subserve sensory memory and learning (Friedman et al., 1986). The IC is considered to be the limbic sensory cortex associated with autonomic activity (Dostrovsky and Craig, 2006). It processes interoceptive sensory information regarding the physiological conditions of the body such as temperature, muscle- and visceral sensations, itch and pain. The IC may be a critical interface between the sensory-discriminative and the immediate affective-motivational dimension of pain. Electrical stimulation of several different sites within the posterior insula has been shown to evoke pain with both sensory and unpleasant qualities (Ostrowsky et al., 2002). Patients with damage to large parts of the insula can perceive nociception but do no longer appreciate the destructive significance of it sand do not withdraw from pain stimuli or potentially harmful stimuli, so-called pain asymbolia (Berthier et al., 1988). The PFC has been shown to be activated during acute pain in a number of imaging studies but not as commonly as the other cortical regions described above. The PFC receives input from the ACC, but there is no evidence that it receives

direct nociceptive input from the thalamus. The PFC is considered to be involved in complex cognitive and evaluative aspects of pain and hence pain-related suffering. This is supported by observations of patients subjected to prefrontal lobotomy. These patients were emotionally indifferent to painful stimuli, which, although perceived, evoked few emotional reactions (Davis et al., 1994). A classical statement exemplifying this condition is

(Hardy et al., 1952).

Figure 3. Interactions between pain sensation, painunpleasantness, and secondary pain-affect. Neural structureslikely to have a role in these dimensions are shown. Black arrows indicate nociceptive or endogenous physiological factors that influence pain sensation and unpleasantness. ACC, anterior cingulate cortex; PCC, posterior cingulate cortex; IC, insular cortex; HT, hypothalamus; S1/S2,somatosensory cortices; PPC, posterior parietal complex; Amy, amygdala; PFC, prefrontal cortex (adapted from Price,2000).

Autonomic and somatomotor

activation(HT)

Second orderappraisal

(PFC)

Perceived threat

(Amy, IC, PPC)

Extended pain affect

Immediate pain unpleasantness

(ACC)

Nociceptivesensations

(S1/S2)

Psychosocial context

Nociceptive input

Introduction

5

1.1.4 THE ANTERIOR CINGULATE CORTEX

An increasing body of evidence from studies in animals (Treede, 2006) and humans (for rev. see for rev. see Apkarian et al., 2005; Casey, 1999; Rainville, 2002; Villemure and Bushnell, 2002) suggest that the ACC, which forms one of the largest parts of the limbic system, plays a crucial role in pain processing. The ACC receives more afferents from diverse thalamic nuclei than any other cortical region and there are strong and reciprocal connections between dorsal, middle and inferior prefrontal cortices and the ACC in primates (Petrides and Pandya, 2006).

A large amount of data from behavioural and functional imaging studies in humans indicate that the ACC is involved in the emotional, rather than the sensory-discriminative, component of pain (Rainville et al., 1997; Coghill et al., 1999; Rolls et al., 2003). For example, the involvement of the ACC in the emotional component of pain in humans has been investigated by assessing subjectively perceived pain following positive or negative expectations (placebo analgesia or nocebo) (Kupers et al., 2005; Herwig et al., 2007). For instance, Sawamoto et al. (2000) found that the expectation of pain amplified the reported unpleasantness of an innocuous thermal stimulation, and that this negative anticipation was correlated to enhanced brain responses in the ACC measured by PET. Conversely, expectations of decreased pain reduced activation in the ACC (Koyama et al., 2005). Interestingly, the degree of pain expectation and the intensity of the noxious stimulus acted in an additive manner on the activation of pain-related brain areas such as the ACC (Keltner et al., 2006). The possible involvement of the ACC in pain processing, has also been investigated in animals. In the rat, nociceptive neurons with body receptive fields have been found in the ACC (Yamamura et al., 1996; Kuo and Yen, 2005) and there are interconnections between the ACC and other brain regions involved in pain modulation such as the PAG and the somatosensory cortices (Schnitzler and Ploner, 2000; Vogt, 2005). Moreover, prolonged noxious stimulation (Liu et al., 1998) as well as pain-related aversion (Lei et al., 2004) in rats, induces expression of the immediate early gene c-fos in the ACC, suggesting that this brain region is activated during pain. The findings obtained from humans studies regarding the involvement of the ACC in the affective, rather than the sensory discriminative component of pain, is further supported by behavioural studies on animals (LaGraize et al., 2004; Treede, 2006). For example, lesions in the ACC of rodents (Johansen et al., 2001; Kung et al., 2003) or primates (Koyama et al., 2001), nociceptive stimuli, without reducing the acute nociceptive thresholds.

1.1.5 MODULATION OF NOCICEPTIVE TRANSMISSION

Pain transmission in the spinal cord dorsal horn is subject to modulatory control by several routes (for examples, see figure 5, the chapter on opioids). In 1965 Wall & Melzack described the so called gate-control mechanism, i.e. the concept that innocuous sensory input

(substantia gelatinosa) which have an inhibitory effect on pain transmission in laminae I and V. Later it was demonstrated that from the PAG via the rostral ventromedial medulla (RVM) and that the PAG in turn, receives input from higher CNS areas such as the amygdala, hypothalamus, thalamus and cortex (for rev. see Fields et al., 2006). However, the descending pathways have also been demonstrated to facilitate nociception at least in some pain conditions such as opioid-induced hyperalgesia (Vanegas and Schaible, 2004). The RVM is a region of the medulla including the nucleus raphe

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magnus (NRM) and the nucleus reticularis paragigantocellularis (NRPGC). Being a critical region for nociceptive processing and modulation, it receives inputs from the spinal dorsal horn as well as from rostral sites (Basbaum and Fields, 1984). Electrophysiological studies on the responses of RVM neurons to noxious thermal stimulation have identified the existence of

- -cells (Heinricher et al., 1987; Mason, 2005). Simply expressed, the off-cells tonically inhibit noxious input, whereas the on-cells are activated by nociceptive signalling and facilitate the firing of ascending dorsal horn neurons (Fields and Heinricher, 1985).

1.1.6 LONG LASTING PAIN

Chronic pain is in humans defined as ongoing pain for longer than three months (Merskey and Bogduk, 1994), while the definition of chronic pain in animals varies. However, a hallmark for chronic pain in all species, is that while acute pain has a physiological protective function by warning the body of damaging stimuli, chronic pain has a pathological character since it may outlast the precipitating tissue injury (Julius and Basbaum, 2001) and sometimes even worsen the underlying condition (Verbunt et al., 2003). Possible mechanisms for the development of chronic pain include sensitization, a process involving a reduction in the threshold for activation, an increase in response to a given stimulus and the appearance of spontaneous activity in the nociceptors. These states are associated with aberrations of the normal physiological pathway, resulting in clinical manifestations such as allodynia (sensation of pain in response to a previously non-noxious stimulus), hyperalgesia (enhanced response to noxious stimuli) or spontaneous pain (Merskey and Bogduk, 1994).

The above described events are examples of the neural plasticity by which the nervous system responds to peripheral tissue injury and inflammation (Woolf and Salter, 2000). Sensitization can occur at both peripheral and central levels of nociceptive processing (for rev. see Campbell and Meyer, 2006). For example, stimulation by primary afferents leads to a postsynaptic excitation of ascending neurons. If the nociceptive stimulus in the spinal cord is frequently repeated, the postsynaptic electrophysiological response may increase with every stimulation, a mechanism called wind-up (Zimmermann, 2001). This feature, although shorter in duration, has similarities to long term potentiation, a mechanism attributed to the facilitation of NMDA-receptor stimulation due to repetitive high-intensity stimulation (Sandkühler, 2007). Such electrophysiological phenomena are believed to have a connection to the development of chronic pain conditions in humans and animals (Rygh et al., 2005).

Introduction

7

1.2 NEUROTRANSMITTERS IN PAIN SIGNALLING AND PAIN MODULATION

In the periphery, painful stimulation and inflammation leads to the local production or release of a number of chemical substances. Metabolites such as ATP, protons and K+ as well as inflammatory mediators e.g. histamine, bradykinin, serotonin and prostaglandins, affect nociceptive nerve terminals (for rev. see Meyer et al., 2006). Several of these molecules are agonists on G-protein-coupled receptors which indirectly increase the phosphorylation of the capsaicin-sensitive vanilloid receptor1, thereby sensitising it (see Krause et al., 2005). This brings about a depolarisation of the nociceptor, leading to the conduction of impulses to the dorsal horn and the release of predominantly substance P (SP), calcitonin gene related peptide (CGRP) and glutamate (Glu) from the proximal terminal of the primary afferent into the spinal cord dorsal horn. Adenosine plays a dual role in nociceptive transmission; activation A2 receptors in the periphery mediate pain whereas A1 receptors in the periphery and spinal cord mediate analgesia (Liu and Salter, 2005). The amino acids GABA (gamma-amino butyric acid) and glycine which are released by spinal cord interneurons inhibit transmitter release from the primary afferent terminals in the dorsal horn. There are also interneurons containing neuropeptides that have an inhibitory, (e.g. neuropeptide Y and galanin) or excitatory (e.g. neurotensin and somatostatin) function in the dorsal horn (for rev. see Todd and Koerber, 2006).

1.2.1 NEUROPEPTIDES

The abundance of neuropeptides in the brain and elsewhere became evident in the 1970-80s, and new examples are still emerging (for rev. see Hökfelt et al., 2000). Neuropeptide-mediated transmission resembles monoamine or amino acid signalling in many aspects, the most pronounced difference being the absence of re-uptake mechanisms for neuropeptides. Furthermore, while other transmitters act on ion channels or G-protein coupled receptors, neuropeptides act exclusively on the latter type of receptors, thereby mediating slower,

- effects. The main neuropeptides known to be involved in pain transmission are the tachykinin SP, the endogenous opioids, and the opioid-modulating peptides.

1.2.2 ENDOGENOUS OPIOIDS AND OPIOID-MODULATING PEPTIDES

The six known endogenous ligands in mammalian brain that bind to the opioid receptors are -endorphin, endomorphin-1, endomorphin-2, met-enkephalin, leu-enkephalin and dynorphin (Fichna et al., 2007), - - - receptors (for rev. see Mollereau et al., 2005). In addition, two more recently discovered peptides, nociceptin (Chiou et al., 2007) and nocistatin (Okuda-Ashitaka and Ito, 2000), act on receptors with similarities to opioid-receptors, although the evidence for their effects on pain transmission have been contradictory (see Heinricher, 2003). In addition to the endogenous opioids, the peptides galanin, neuropeptide FF and CCK, all acting on their own separate receptors, seem to either affect pain transmission by themselves or to modulate the effects of opioids (Mollereau et al., 2005). Galanin appears to exercise mainly inhibitory effects on nociceptive transmission (Wiesenfeld-Hallin et al., 2005), while neuropeptide FF has been reported to act bimodally with pro- or anti-opioid effects depending on the CNS-level (Panula et al., 1999).

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1.2.3 CHOLECYSTOKININ

The peptide cholecystokinin (CCK) was first isolated from the gastrointestinal tract in 1928 by Ivy and Oldberg and almost 50 years later, it was shown to also be present in the central nervous system (Vanderhaeghen et al., 1975; Dockray, 1976). The protein was sequenced in 1968 by Mutt and Jorpes and was later found to be synthesised as prepro-CCK, a protein which is cleaved into peptides with lengths of 83, 58, 39, 33, 22, 8, 5 and 4 residues that share a common carboxyl-terminal sequence (Beinfeld, 1997). During posttranslational processing, pro-CCK undergoes modifications that include tyrosine sulphatation, endoproteolytic cleavage, basic residue removal, and C-terminal amidation (for rev. see Beinfeld, 2003). The major bioactive form of the peptide in the human brain is sulphated CCK-8 (Rehfeld, 1978).

Being one of the most abundant peptides in the CNS, CCK has been shown to be present in many areas related to pain transmission, such as the spinal cord dorsal horn, the PAG, the thalamus and the cortex (for rev. see for rev. see Hökfelt et al., 2002; Dockray, 1980; Hebb et al., 2005). It is particularly abundant in the ACC in both humans (Emson et al., 1982; Savasta et al., 1990) and rodents (Beinfeld et al., 1981; Hökfelt et al., 1991).1 The localisation of CCK-like immunoreactivity (CCK-LI) containing nerve terminals largely parallel the distribution of the terminals of endogenous opioids as well as opioid receptors, suggestive of functional interactions between CCK and opioids (see see Noble and Roques, 2003; Stengaard-Pedersen and Larsson, 1981; Skinner et al., 1997).

The cloning of the CCK receptor (Wank et al., 1992) has led to the identification of two distinct G protein-coupled receptors, previously named CCK-A and CCK-B (A for alimentary and B for brain) now termed CCK1 and CCK2 (for rev. see Noble and Roques, 1999). Both receptor subtypes increase cAMP and open calcium channels (Wu and Wang, 1996) and thereby their main effect is an increase in neuronal activity. The main receptor type in the CNS of the mammalian species investigated so far, is the CCK2 receptor, but also the CCK1 receptor is present in a few regions of the CNS in primates. In rodents, most of the CCK receptors in the brain and spinal cord are of the CCK2 type (see Wank, 1995). A large body of evidence supports that the physiological role of the CCK in pain modulation, is mediated by the CCK2 receptor (for rev. see Noble and Roques, 1999). A variety of CCK-receptor antagonists, termed

affinity (for rev. see Herranz, 2003). -peptide ligands which pass the BBB and are used for pharmacological characterisation of the physiological function of CCK. Although several of these CCK antagonists are candidates for the clinical treatment of anxiety (Bradwejn and Koszycki, 2001), overweight (Little et al., 2005) or pain (see below) (Kalso, 2005; Hruby et al., 2006), none are yet used in clinical practice (de Tullio et al., 2000).

CCK opioid interactions

The involvement of CCK in pain processing is well established especially regarding its opioid-modulating effects (for rev. see Wiesenfeld-Hallin and Xu, 2001; Hebb et al., 2005).

1 In addition to areas of importance for pain transmission, CCK is present in nigrostriatal

dopamine neurons and corticostriatal and thalamocortical glutamatergic neurons, which all belong to a circuitry essential for the control of motor behaviour (see Hökfelt et al., 2002).

Introduction

9

Particularly at spinal level, interactions between CCK and opioids have been extensively studied (Ossipov et al., 2000; Wiesenfeld-Hallin et al., 2002). A possible pain-regulating function for CCK on the cortical level is, on the other hand, less well explored. CCK is well established as a functional opioid antagonist although it is not considered to alter the basal pain threshold on its own. For instance systemic, i.t. or i.c.v. administration CCK agonists reduce (Itoh et al., 1982; Faris et al., 1983; Wang et al., 1990), while CCK antagonists enhance (Watkins et al., 1984; Dourish et al., 1990) the antinociceptive effect of exogenous opioids such as morphine. In addition, CCK antagonists can enhance the analgesic effect of experimentally induced release of endogenous opioids in rats (Watkins et al., 1985; Han et al., 1986; Valverde et al., 1994). Interestingly, CCK antagonists not only potentiate the analgesic effect of morphine, but also the analgesic effect of placebo treatment (Benedetti, 1996), suggesting that CCK is involved in the affective component of pain.

In the spinal cord, anti-opioid effects of CCK2 receptor activation seem to involve the (Wang et al., 1990; Benoliel et al., 1991). At supraspinal sites

including the posterior nucleus accumbens, CCK2 receptor antagonists seem to potentiate opioid- - opioid receptor activity (Vanderah et al., 1996). Whether CCK also has pro-nociceptive effects by itself has been a subject of debate, but there is evidence that CCK directly facilitates nociception in the RVM (Heinricher and Neubert, 2004).

Cellular mechanisms of anti-opioid action

There are several potential cellular mechanisms by which CCK may reduce morphine analgesia. First, on neurons that express both CCK- and opioid-receptors, CCK may counteract the intracellular events following opioid-receptor activation since CCK and opioids have opposite effects on mobilisation of intracellular Ca+2 (Wang et al., 1992). Secondly, the binding of CCK to the CCK2 receptor has been suggested to cause a reduced binding of morphine to the

- - - receptors (Wang and Han, 1990). Thirdly, CCK may via inhibitory interneurons tonically inhibit the release of enkephalins. This can reduce the antinociceptive effect of morphine since enkephalins normally, by

-receptors, potentiate the effect of morphine (Vanderah et al., 1996). A fourth possible mechanism for the anti-analgesic effect of CCK, is to enhance the tonic inhibition of descending -cells in the RVM (Heinricher et al., 2001).

Regulation of CCK release

The mechanisms regulating the release of CCK are far from clear. There is evidence that CCK release in many brain regions is under the tonic inhibition by GABAA-receptors (Yaksh et al., 1987). In several brain regions, CCK is released by dopamine (D1) (Brog and Beinfeld, 1992) or serotonin (5-HT3) receptors (Nevo et al., 1996). Interestingly, it has been demonstrated that

Figure 4. Illustration of a possible

mechanism for the regulation of the CCK-releaseand the anti-opioid effect of CCK on descendingpain modulatory pathways in the prefronal cortex (PFC) and the anteior cingulate cortex(ACC). Via delta receptors, opioids decrease the tonic inhibition of CCK release mediated by GABA. The released CCK counteracts the opioid-mediated analgesia via the CCK2 receptor.

Umut Heilborn

10

morphine and endogenous opioids increase the release of CCK, possibly serving as a self-regulatory function for opioid effects (for rev. see Wiesenfeld-Hallin et al., 1999). Different subtypes of opioid receptor agonist seem to affect the CCK release differentially in a complex way although the delta-opioid receptor seems to be of particular importance (Benoliel et al., 1991; Ruiz-Gayo et al., 1992). For example, systemic and local administration of morphine increases the spinal release of CCK-LI and this increase is mediated by delta-opioid-receptors (de Araujo Lucas et al., 1998; Gustafsson et al., 2001). In addition, data from the frontal cortex show that acute (Becker et al., 1999) or subchronic (Becker et al., 2000) administration of morphine increases the release of CCK-LI and, as observed in the spinal cord, this effect can be blocked by delta-opioid antagonists (as illustrated in figure 4). Although the ACC has one of the highest densities of CCK-containing neurons as well as opioid receptors in the brain, the regulation of the CCK-release in this region, has to our knowledge, not yet been investigated.

CCK in other conditions

In addition to pain modulation, central CCK has also been implicated in anxiety and stress (van Megen et al., 1996; Rotzinger and Vaccarino, 2003). For instance, administration of CCK2 receptor agonists induces anxiety in humans (Benkelfat et al., 1995; Koszycki et al., 1998; Radu et al., 2002) and increases anxiety-like behaviour in animals (Wang et al., 2005), whereas CCK2 receptor antagonists or antisense appear to have anxiolytic effects, at least in rodents (Tsutsumi et al., 2001; Herranz, 2003). Stressful stimuli have been shown to affect the tissue levels (Radu et al., 2001) and release (Nevo et al., 1996) of CCK-LI in limbic brain regions and prefrontal cortex. Moreover, stress-induced increase of CCK-LI release in the frontal cortex can be attenuated by anxiolytic drugs (Becker et al., 2001). Rats subjected to chronic stress in a social-defeat model, have been reported to display significantly more pain-related behaviour as compared to control animals and this was accompanied by a significant increase in CCK-LI release in the frontal cortex (Andre et al., 2005). Recently it was shown that transgenic mice overexpessing the CCK2 receptor were more susceptible to stress-induced anxiety-like behaviour, and this susceptibility could be reversed by blocking the receptor overexpression (Chen et al., 2006). This indicates a close interaction between nociceptive and emotional functions with a possible involvement of CCK in this brain region.

1.3 ANALGESIC DRUGS

1.3.1 OPIOIDS

Morphine, which is an alkaloid derived from opium, the juice extract of the poppy Papaver somniferum, has been used for centuries to produce euphoria, analgesia and sleep (Dale and Rang, 2007). The term opioid applies to any endogenous or synthetic substance producing morphine-like effects that can be blocked by antagonists such as naloxone (Adler and Herz, 1993). The opioid group of substances comprises morphine analogues, synthetic derivatives and the endogenous opioids, which were discovered in 1973 (Pert and Snyder, 1973; Terenius, 1973).

(for rev. see Ossipov et al., 2004) and

pharmacological effects of opiates such as analgesia, sedation and bradycardia. All three receptor subtypes belong to the family of G-protein-coupled receptors and inhibit adenylyl cyclase, thereby reducing intracellular cAMP and secondarily affect protein phosphorylation

Introduction

11

pathways (see Dhawan et al., 1996). There is also a direct G-protein coupling to ion channels, a mechanism by which opiates promote the opening of potassium channels and inhibit the opening of voltage-gated calcium channels. The overall effect is thus a reduction of excitability and neuronal release of transmitters. Yet, in certain neuronal pathways (see below), opiates increase postsynaptic activity, possibly by suppressing the firing of inhibitory interneurons. All three receptor subtypes mediate similar effects at the cellular level, although the diverse distribution of the receptor subtypes mediate the differential effects of selective agonists (see Waldhoer et al., 2004). Studies on knock-out mice demonstrate that the major pharmacological effects of morphine, including -receptor (see Law et al., 2000). A simplified illustration of the sites of action for morphine is displayed in figure 5.

Opioid receptors are widely distributed in the CNS and have been demonstrated in the spinal cord, the frontal cortex, the nucleus accumbens, hippocampus, thalamus, hypothalamus the PAG and the RVM (Mansour et al., 1995). A considerable amount of evidence exists to establish that opioid-induced antinociception is mediated in part by descending pathways that originate from the PAG and the RVM (Yaksh and Noueihed, 1985). Opioids can cause an activation of a population of cells in the PAG that, in turn, excites neurons of the RVM (for rev. see Fields et al., 1988; Ossipov et al., 2004). In brief, the firing of neurons in the descending inhibitory pathways is increased by morphine, confirming that there is a significant supraspinal component of the analgesic effect of opioids. In addition to its antinociceptive effects, morphine has been shown to reduce the affective component of pain and a part of this effects seems to be mediated by opioid receptors in the ACC of humans (Zubieta et al., 2003; Sprenger et al., 2005) and rodents (LaGraize et al., 2006). This is an important component of its analgesic effect, since the agitation and anxiety associated with a painful illness or injury may aggravate the underlying condition.

Figure 5. Schematic illustration of the descending control system, showing the main sites of action of opioids on pain transmission. Very simplified, morphine can be described to have an inhibitory effect on ascending pain transmission pathways and an activating effect on descending inhibitory systems (exceptions from this rule are mentioned below). Opioids excite neurons in the periaqueductal grey matter (PAG) and in the nucleus reticularis paragigantocellularis (NRPG), which in turn project to the rostroventral medulla, which includes the nucleus raphe magnus (NRM). From the NRM, serotonin- and enkephalin-containing neurons run to the substantia gelatinosa (SG) of the dorsal horn, and exert an inhibitory influence on transmission. Opioids also act directly on the dorsal horn, and possibly on the peripheral terminals of nociceptive afferent neurons. The effect of opioids on cortical levels, is less well known (adapted from Fields, Basbaum and Heinricher, 2006)

Nociceptor

Spinal corddorsal horn

Cerebralcortex

NRM

PAG

SG

Lam. I, II, V

Mechanoreceptor

Morphine

Morphine

+

+

-

Morphine

-?

Thal.

NRPG

Umut Heilborn

12

1.3.2 NSAIDS

The non steroid anti-inflammatory drugs (NSAIDs) have been used for over 100 years and are among the most widely used of all drugs (Dale and Rang, 2007). They provide symptomatic relief from pain and swelling in chronic joint disease such as osteo- and rheumatoid arthritis, and in more acute inflammatory conditions, for instance soft tissue injuries and postoperative pain. The three main therapeutic effects are anti-inflammatory, antipyretic and analgesic. The primary mechanism of action of NSAIDs is considered to be reduction of the production of prostaglandins (PG) and thromboxanes from arachidonic acid by inhibition of the enzyme cyklo-oxygenase (COX) (for rev. see Burian and Geisslinger, 2005).

There are three known isoforms of COX (1-3), of which COX-1 and COX-2 have been detected in man and rodents. COX-1 is a constitutive enzyme expressed in most tissues and is responsible for the production of prostaglandins involved in many physiological organ functions. COX-2 on the other hand, is induced in inflammatory cells when they are activated by inflammatory cytokines. Thus, the COX-2 isoform is responsible for the production of the prostanoid mediators of inflammation (for rev. see Vane et al., 1998). In addition to its peripheral inflammatory function, there is a considerable pool of COX expressed in the CNS (further discussed below), although its function is not completely clear. The prostaglandin considered most important for the development of oedema and pain during peripheral inflammation, is prostaglandin E2 (PGE2) (Schaible et al., 2002). It activates the G-protein coupled receptors EP2 EP4 EP3 and EP1 which increase cAMP, decrease cAMP or induce cellular Ca2+-influx, respectively.

Anti-inflammatory action

The inhibition of COX by NSAIDs leads to a decrease in PGE2 and prostacyclin which reduces vasodilatation and, indirectly, oedema. In contrast, the accumulation of inflammatory cells, is not reduced.

Analgesic action

Prostaglandins do not produce nociception in themselves, but especially PGE2 causes peripheral sensitization by facilitating the excitatory effect of inflammatory mediators such as histamine, bradykinin and serotonin on the nociceptors in the periphery (see Burian and Geisslinger, 2005). PGs also lower the excitatory threshold for ion channels on the nociceptors (Schaible et al., 2002).

Of late, increasing attention has been directed to the central regulation of the COX isoenzymes, since it has been shown that peripheral nociceptive stimulation brings about an increase in COX-2 and PGE2 production in the spinal cord (Samad et al., 2001; Svensson and Yaksh, 2002) 2. Furthermore, pain hyperalgesia in various inflammatory models is associated

2 The linkage between peripheral inflammation and the up-regulation of COX-2 in the CNS is unclear, but both neuronal and humoral mechanisms may be important. Prolonged activity in small primary afferents results in a spinal release of Glu and SP. The subsequent stimulation of NK1, AMPA and NMDA receptors in dorsal horn neurones (Dickenson et al., 1997) may produce a synaptic activity-dependent increase in the transcription of a variety of enzymes, including spinal COX-2 (ref). Humorally, peripheral inflammation results in the release of proinflammatory cytokines such as TNF-a and IL-1b. Although these cytokines cannot pass the blood brain barrier, they may induce COX-2 and specific

Introduction

13

with an increase in PGE2 in the spinal cord (Vanegas and Schaible, 2001) and in supraspinal structures including the pons, ventral midbrain, hypothalamus, thalamus and cortex (Samad et al., 2001). This central sensitization by prostaglandins is believed to be mediated by facilitation of neural transmission in the spinal cord, however, the hyperalgesic effects of PGE2 on higher CNS-levels, are still very unclear.

Traditionally, NSAIDs have been considered to mediate their analgesic effect by decreasing the peripheral production of PGE2, thereby reducing the peripheral sensitization of nociceptors. However, during the last decades there has been growing evidence that the anti-inflammatory and the antinociceptive effects of NSAIDs may be independent of each other (McCormack and Brune, 1991) and there are findings supporting a central antinociceptive effect of NSAIDs (Vanegas, 2002). For instance, NSAIDs inhibit prostaglandin synthesis in the CNS and intrathecal administration of NSAIDs has an antinociceptive effect in inflammation induced by formalin (Malmberg and Yaksh, 1995), carrageenan (Dirig et al., 1998) or FCA (Seybold et al., 2003). In humans, very low concentrations of systemically administered NSAIDs which are insufficient to reduce peripheral PG-production are analgesic (Gordon et al., 2002), suggesting that central mechanisms involved in pain are much more sensitive to the effects of NSAIDs than peripheral anti-inflammatory mechanisms.

1.3.3 PRE-EMPTIVE ANALGESIA

Treatment with analgesics prior to surgical interventions that cause peripheral inflammation, has been suggested to prevent central and peripheral mechanisms leading to neuronal sensitization and subsequent chronic pain (Wall, 1988; Woolf and Chong, 1993; Melzack et al., 2001). Thus, during the last decade there has been a change in anaesthetic practice that includes administration of morphine, NSAIDs or local anaesthetics before invasive procedures, so-called pre-emptive analgesia . The evidence of the presumed beneficial effect for this practice in postoperative pain management has, however, been very contradictory (for rev. see Bromley, 2006). Although some clinical (Richmond et al., 1993; Rockemann et al., 1996) and preclinical (Woolf and Wall, 1986; Chapman et al., 1994; Field et al., 1997; Perrot et al., 1999; Locher-Claus et al., 2005) data support this theory, other studies do not (Yamamoto and Yaksh, 1992; Wilson et al., 1994; Collis et al., 1995; Sevostianova et al., 2003). Further on, most of the preclinical investigations of the presumed pre-emptive analgesic effect of morphine have been performed using short-lasting (a few seconds up to an hour) nociceptive tests of hyperalgesia and allodynia. Few of these studies have examined the effect of morphine in models of more long-lasting inflammation and spontaneous pain-related behaviour, although such models are probably more relevant to postoperative pain in the clinical context.

The concept of pre-emptive analgesia is to prevent early sensitizing events in the nervous system and thereby obtain analgesic effects of morphine also after the plasma concentrations have decreased below the therapeutic levels. It would therefore, from a mechanistic point of view, be of value to follow the observed pre-emptive analgesic effect with the plasma concentration of morphine. To our knowledge this has not been done in previous clinical or preclinical studies.

PGE2 synthases in the endothelial lining of the barrier (Samad 2001), leading to an increased synthesis of PGE2 and activation of PG receptors in the brain.

Umut Heilborn

14

1.4 ANIMAL MODELS OF PAIN

In order for an animal model to have validity as representing any pain condition in humans, it must of course, mimic the human disease as closely as possible by one or several aspects. The main variables to be considered are the modality (temperature, mechanical, chemical or electrical), duration (acute, long lasting), location (exteroception, interoception, various organs) and pathophysiology (inflammation, neuropathy, ischemia) of the nociceptive stimulus. There is overwhelming evidence that the neurochemical as well as neuroanatomical pathways mediating different types of nociceptive input vary greatly between species (Sewards and Sewards, 2002). For instance, temperature, mechanosensation and interoception (sensation from inner organs such as the heart, joints and viscera) are all mediated through different neuroanatomical pathways, some of which have been demonstrated to exist in humans but not rodents (Craig, 2003). Therefore, data that are based on only one animal paradigm (for instance in drug development) must be interpreted with caution.

1.4.1 METHODS FOR INDUCING PAIN IN RODENTS

Most preclinical investigations of pain in animals measure the reaction of the animal to an acute, mildly painful mechanical or thermal stimulus, as in the tail flick or paw pressure tests (Le Bars et al., 2001). The validity of such animal models can be questioned both as to symptomatology and pathophysiology since, in contrast, the vast majority of clinical pain states originate from pathological conditions in the body resulting in spontaneous pain or pain induced by weight-loading (Negus et al., 2006).

Arthritis models in rodents

Models of inflammatory pain are commonly carried out by injecting an irritating or immunogenic agent subcutaneously or intraarticularly. This results in an inflammation which varies in duration and spreading. Injection of formalin is commonly used to causing a brief (1-2h), local inflammation in the skin resulting in pain-related behaviour and hyperalgesia (Tjølsen et al., 1992). However, the duration of pain in these models is probably too short to model most inflammatory conditions in humans. For investigations of long-lasting joint disease in rats and mice, immunogenic agents such as type II collagen or various antigens have been used to evoke an autoimmune reaction and subsequent polyarthritis (Wooley, 1991; Myers et al., 1997; Stills, 2005). These paradigms have validity by showing clinical and histological similarities to rheumatoid arthritis (RA) in humans. However, such models often lead to multi-organ involvement and severely affect the general state of health of the animals (Brand, 2005). This may render behavioural studies difficult, and in addition be questionable from an ethical perspective. Models of arthritis in which only one joint is affected (monoarthritis) have advantages from this point of view since they do not affect the general well being of the animals to the same extent (Coderre and Wall, 1988; Stein et al., 1988; Butler et al., 1992).

In rats, intraarticular injection of carrageenan is widely used to induce monoarthritis causing oedema and pain-related behaviour for 24 48 hours (Schött et al., 1994; Kissin et al., 2005). An alternative model of monoarthritis in rats with a longer duration (up to 6 weeks), is

Introduction

15

the adjuvant-induced arthritis (Butler et al., 1992) 3. Both carrageenan- and adjuvant-induced arthritis have been histologically verified to induce an inflammation in the joint that resembles human conditions of immunogenic arthritis (Sluka et al., 1994). The use of transgenic techniques in pain research has brought about the need for valid models of monoarthritis in mice. Carrageenan has mainly been used for intraplantar injections in mice, and its arthritic effect is less well documented. FCA injected subcutaneously has been reported to be a useful model of inflammation-induced nociceptive behaviour in mice (Larson et al., 1986; Chillingworth and Donaldson, 2003). However, since arthritis in man arises within the joint, intraarticular injection of adjuvant may be more appropriate to mimic human joint disease. To our knowledge, this technique has not previously been characterized in mice.

1.4.2 METHODS FOR ASSESSING PAIN IN RODENTS

Equally important as the choice of method for inducing nociception, is of course the choice of method for measuring the outcome. (for rev. see Blackburn-Munro, 2004). Since pain is defined as an unpleasant emotion evoked by nociceptive input, it has been questioned whether animals perceive pain at all. Indeed, there can be no doubt that many animal species possess the ability to detect potentially damaging stimuli such as excessive heat or mechanical pressure, however, this does not automatically mean that the same animals perceive pain (Flecknell and Waterman-Pearson, 2000). Since we lack the means to directly study the experienced pain in animals, indirect measures such as observation and interpretation of behavioural responses are being used. Experimental methods for assessing possible pain in animals can be classified into three categories based on the CNS-level required for the behaviour that is assessed (table 1). The first category is evoked reactions mediated by spinal reflexes in response to noxious or innoxious stimuli (principally mechanical, but also thermal and chemical). The stimulus intensity required for a specific response is assessed in classical models such as the hot-plate, tail-flick, and von Frey tests (Jensen et al., 2001). However, these tests may tell us relatively little about the ongoing nature of injury-induced pain. The second category is spontaneous behaviours indicative of pain. For example abnormal stance and gait, reduced mobility, licking, scratching, writhing or vocalisation can be observed as results of inflammatory, neuropathic or visceral disease (Calvino et al., 1987; Le Bars et al., 2001). Such spontaneous behaviours most probably have a higher relevance to clinical pain in humans, however, since most of these actions are mediated by brain stem regions, they lack the involvement of cortical regions which is by definition required for an experience to be defined

that is, painful. In contrast, the third category of tests involves assessment of learned, complex behaviours by which the animal can avoid or stop the noxious stimulation, the so-called operant paradigm (Colpaert et al., 2001; Vierck, 2006). Such behaviours, which require cortical involvement, not only indicate that the animal experiences nociception, but that the nociception induces a motivational drive for memory and learning, indicative of nociception-induced discomfort without affecting the nociceptive threshold itself (for rev. see Treede, 2006). In addition to the more directly pain-related behaviours and reflexes listed below,

3 Carrageenan is a polysaccharide produced from red seaweed. In addition to its usage in pain research, it is widely employed in the food industry as a thickening and emulsifying additive. Adjuvant (FCA) is a mixture of a non-metabolizable mineral oil, a surfactant and mycobacteriae (M. tuberculosis or M. butyricum) and is considered to be the most effective adjuvant for enhancing immunologic responses to antigens. It is therefore commonly used in vaccines.

Umut Heilborn

16

secondary signs of ongoing pain (such as sleep, weight, stress-hormones etc) can be observed. For example, in rats with adjuvant-induced polyarthritis, retardation of weight gain (Colpaert, 1987), abnormalities in sleep patterns, (Andersen and Tufik, 2000) and maladaptive changes in the hypothalamic pituitary adrenal (HPA) axis (Blackburn-Munro and Blackburn-Munro, 2003) have been shown to correlate tightly with inflammation and hyperalgesia.

Integration level in the CNS Response types Behavioural tests

Spinal reflexes (preserved after spinal cord transection)

Stretch, flexion or withdrawal responses

Tail flick, paw pressure Hargreaves test

Brain stem (preserved after decerebration)

Licking, biting, flinching, vocalising, struggling

Hot plate, vocalization, formalin tests

Cerebral (lost after decerebration)

Operant escape

Learned and motivated response that stops the noxious stimulation

Table 1. Nociceptive reactions categorised as mediated on spinal, brain stem or cerebral level (adapted from Vierck, 2006).

1.4.3 ETHICAL ASPECTS

The use of animals in experimental research, not least in pain research, inevitably raises a number of ethical issues. The experiments in the present thesis were carried out in agreement with the Swedish Animal experiments (DFS 2004:4 DLL55), as well as in accordance with the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (ETS No. 123). In brief, both these legislations are in line with the principles of the 3Rs : replace, refine, reduce (see Flecknell, 2002). The 3Rs imply that 1) alternative methods to animal experiments should be sought and when applicable, used; 2) the employed techniques should be continuously developed to minimise possible suffering, distress or lasting harm; 3) studies should be so well designed and carried out that the number of animals used is restricted to a minimum.

In the DFS 2004:4 DLL55, there are specific regulations regarding methods for induction of arthritis in rodents (§§ 13-16). These prescribe that inflammation must not be induced by intraplantar injection in rats or mice or by intramuscular injection in mice. The cage bedding and the placing of food and water must be adapted to the condition of the animals, which must be regularly supervised. Furthermore, the choice of strain and disease model, the spread and severity of the disease as well as the end-point of experiments should be considered and motivated. In the present thesis, we have complied with the above mentioned guidelines and we continuously follow the current development within laboratory animal research in order to meet the terms of the 3Rs.

Aims

17

2 AIMS

The specific aims of the study were:

I. To investigate a possible pre-emptive effect of morphine administered prior to

the induction of inflammatory pain in rats.

II. To develop a model for chronic inflammatory pain in the mouse that could be

useful for investigations of neurochemical mechanisms of pain and for the

development of analgesic drugs.

III. To investigate alterations in the release of CCK in the anterior cingulate cortex

during inflammation in rats.

IV. To investigate the effect of anti-inflammatory and analgesic drugs on the CCK

release in the anterior cingulate cortex of rats.

Umut Heilborn

18

3 METHODS AND METHODOLOGICAL REMARKS

For a more detailed description of the materials and methods used, please see the Materials and Methods section in each individual paper (I-IV).

3.1 LABORATORY ANIMALS

All experiments on mice and rats were done with permission from the local ethical committee for experiments on laboratory animals. After arrival to the animal department, one week of acclimatization to the environment was allowed before initiation of experiments. Thereafter the animals were handled once daily for a week in order to reduce the innate fear for humans in rodents and thus, minimize the stress imposed at the time of the experiments. The animals were housed in rooms with a temperature of 20-22 C and a relative humidity of 40-65 % on a 12:12 h light-dark cycle with lights on at 6.00 a.m.). They had free access to food and water.

In studies I, III and IV, male outbred Sprague-Dawley rats (B&K Universal, Sollentuna, Sweden) weighing 250 - 365 g at the time of experiments were used. The rats were group housed in macrolon type IV cages (595 mm x 380 mm x 200 mm) under standard conditions. In paper II, 8 - 10 weeks old female albino mice of inbred BALB/c and outbred NMRI strains, (B&K Universal AB, Sollentuna, Sweden) were used.

Comment

In paper I and III, repeated behavioural tests were performed on animals with arthritis. During these experiments, we randomised the treatment and the order by which the animal were taken out of their cages, since the selection order has been shown to stress the animals differentially (Brodin et al., 1994).

Since our aim in paper II was to develop a model for chronic inflammatory pain in mice, we planned to study the animals for up to 10 weeks after the injection of an immunogenic agent. During this time, the animals should preferably be group-housed since isolation is stressful and greatly influences the behaviour and physiology of mice (Van Loo et al., 2003; Zhu et al., 2006). However, group housed adult male mice can be severely aggressive to each other (Van Loo et al., 2003). We therefore used female mice which are not aggressive when group housed. BALB/c and NMRI mice were used since these strains have previously been used in studies of inflammation and antinociceptive effects (Wahlsten et al., 2003; Kalueff et al., 2007).

3.2 INDUCTION OF ARTHRITIS

In paper I, III and IV the rats were anaesthetised with 2% halothane and injected with 50 μl 0.7% (w/v) carrageenan lambda (Sigma-Aldrich, Stockholm, Sweden) dissolved in saline into the right tibio-tarsal joint with a 21-gauge needle as by Butler et al., (1992).

For induction of arthritis in mice in paper II, one of one of the following compounds was injected: 1 mg mycobacterium tuberculosis H37 Ra in a 1 ml mixture of paraffin oil and an emulsifying agent (Difco laboratories, Maryland, USA prod no 231131), (FCA-MT1) ; 1mg mycobacterium tuberculosis H37Ra, ATCC 25177, heat killed and dried in 1 ml of 0.85 ml

Methods

19

paraffin oil and 0.15 ml mannide monooleate (Sigma-Aldrich, prod nr 5881, MI, USA) (FCA-MT2) ; 0.5 mg mycobacterium butyricum in a 1 ml mixture of paraffin oil and an emulsifying agent (Difco laboratories prod no 263810) (FCA-MB) or a 2% dilution of lambda carrageenan (Difco laboratories) in saline. The animals were thereafter observed daily as long as there were visible signs of pain behaviour.

Comment

The most commonly used model of adjuvant-induced monoarthritis is a local injection of FCA into the tibiotarsal joint of the rat (Butler et al., 1992). There are also methods described for injecting FCA intra- and subdermally around the tibiotarsal joint of the rat (Donaldson et al., 1993; Grubb et al., 1991). Since the aim of the paper II was to develop a mouse model that originated from within the joint, we selected intraarticular injection as the route of administration of adjuvant. We first tried to make injections into the ankle joint, but this proved to be impractical due to the small size of the joint; we therefore proceeded by injecting FCA into the larger knee joint. Prior to the arthritis experiments, intraarticular test injections with 20 μl methylene blue were made in a separate group of mice in order to verify the injection technique. Under isoflurane anaesthesia, the animals were placed in a supine position, the knee was swabbed with 70% ethanol and a 29 gauge insulin needle (Becton Dickinson, UK) was inserted through the left patellar ligament into the knee joint with the knee in 90° flexion. The knee was thereafter extended so that the patella covered the injection site of the knee joint capsule. After injection, the animals were killed and the knee joint was dissected. The synovial fluid was tainted dark blue and there was a weak tint of blue in the surrounding tissue indicating intraarticular administration with minor periarticular leakage.

3.3 PHARMACOLOGICAL TESTING

3.3.1 STANCE SCORING

In paper I and III, the rats were placed in a transparent cage (30 cm x 20 cm x height 18 cm) and video recorded from below at a distance of 1 m during the first 5 min when the animals were most active. The stance of the rats was later scored according to a scale as follows: 0 = normal paw pressure, equal weight on both hind paws; 1 = slightly reduced paw pressure, paw is completely on the floor but the toes are not spread; 2 = moderately reduced paw pressure, foot curled with only some parts of the foot lightly touching the floor; 3 = severely reduced paw pressure, foot completely elevated from the floor. Intermediate scores (0.5, 1.5 and 2.5) were used for animals that displayed a behaviour in between the above described definitions.

In paper II, the mice were habituated to the experimental procedure for a week prior to testing. The mice were placed in individual chambers (125 mm x 100 mm, height 150 mm) with glass floors in a ventilated and temperature controlled (23.5-24.5°C) testing cabinet and recorded on videotape from below for 10 minutes.

Due to a gait and stance that is different from rats, the scoring of mice in paper II was not based on a 3-graded, but 2-graded scale as follows: 0 = No visible impairment of gait or stance. Foot firmly placed flat on surface with normal spread of toes. 1 = Moderate impairment of stance. The foot placed on the ground but with toes tightly contracted together. 2 = Severe impairment of gait and stance. Foot either entirely elevated from the ground or only the

Umut Heilborn

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lateral part of the foot very lightly touching the ground. Toes tightly pulled together. Since the stance in mice varied frequently during the observation period, the highest score maintained for at least 10 sec was therefore assigned as the final score. Intermediate scores (0.5 and 1.5) were used for occasional animals that displayed a behaviour in between the above described definitions. All naïve animals consistently obtained a score of 0.

Comment

The scoring technique described above was introduced by Coderre and Wall (1988) and has since then been widely used, especially in models of inflammatory pain. The rating scale was originally used for assessing pain in rats with urate-induced arthritis. It has later been slightly modified using intermediate scores (0.5, 1.5 and 2.5) for animals that displayed a behaviour in between the above described definitions in carrageenan-induced arthritis and further adjustments have, as mentioned, been made for the scoring of mice. A drawback of scoring pain-related behaviour is that it is influenced by the subjectivity of the scoring person. We tried to increase the objectivity of the method by letting two independent observers perform the scoring in a blinded manner. One fifth of the video sequences were scores were scored by both observers and the scores were later on compared in order to control for reproducibility. The scoring results of the two observers were identical.

In most behavioural models of inflammatory pain in animals, evoked responses to acute thermal, mechanical or electrical stimuli such as the hot-plate or tail-flick tests, are measured (see e.g. Mogil et al., 1999). In the present thesis, we have used measurement of spontaneous behaviour and impaired motor function since these parameters are considered to have greater relevance to clinical pain conditions. In addition, they may be less stressful for the animals (Coderre and Wall, 1988; Butler et al., 1992). Our own and other groups have shown that impaired stance or gait is strongly correlated to mechanical allodynia (Schött et al., 1994; Vrinten and Hamers, 2003). It is therefore likely that the antigen-induced arthritis used in paper II produced allodynia as reported for the similar model by Chillingworth and Donaldson (2003). The duration of allodynia and hyperalgesia can often be longer than that of spontaneous pain-related behaviour (Chillingworth and Donaldson, 2003; Nagakura et al., 2003). Thus, it is possible that if tested, we would have detected a provoked allodynia or hyperalgesia that remained after the spontaneous pain behaviour had subsided.

One drawback of the scoring technique is that the usage of a non-parametrical scale and a non-normal distribution of data, brings forth the need of more advanced tools for statistical analysis.

3.3.2 WEIGHT BEARING

In paper III, weight bearing was tested as previously described by Schött and collaborators (1994). In brief, the animals were placed in a test box, which measured the weight bearing of each hind leg. Two levers, functioning independently of each other, generate electrical signals recorded by a Grass polygraph. Calibration was performed so that a linear response was obtained in the range of 10 - 250 g. The lowest value used for calculation was 10 g and all registrations falling below this value were set to 10 g. The weight bearing ratio was expressed as the weight put on the left foot divided by the weight put on the right (inflamed) foot.

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Comment

Advantages of the weight bearing model compared to scoring of pain-related behaviour is objectivity and parametrical measuring and thereby easier statistics. However, this model is unfortunately not applicable on mice, since mice are too active. The method requires some training of the animals to stand still with each hind foot on each weight plate. Rats are able to learn this but not mice.

3.3.3 ANKLE OEDEMA

In paper I and III, we measured the ankle diameter for quantification of oedema as a parameter of inflammation. The anterior-posterior and medial-lateral aspects of the ankle joint were measured with an electronic calliper (Calliper PM 200, Sylvac SA, Switzerland) while the rat was held in a gentle grip. The mean of the two diameters obtained from these measurements was used to calculate the ankle circumference.

3.3.4 ADMINISTRATION OF DRUGS

In paper I and IV, morphine (injection solution 10 mg/ml; AstraZeneca, Södertälje, Sweden) was dissolved in saline (0.9% w/v NaCl), and administered subcutaneously (s.c.) in the back. Rats injected with morphine showed no apparent signs of sedation or respiratory depression. In paper IV, Diclofenac (injection solution 25 mg/ml; Voltaren®, Novartis, Täby, Sweden) was administered intramuscularly (i.m.; 0.15 ml) bilaterally into both quadriceps muscles. In paper II, diclofenac (containing, diclofenacsodium 25 mg, mannitole, sodiummetabisulphite 0,67 mg (antioxidant E 223), bensylalcohol, propylenglycol, sodiumhydroxide, pH 7,9, aqua; Voltaren®, Novartis, Täby, Sweden) was administrated to mice s.c. at doses of 3, 10 and 30 μmol/kg. Morphine (containing morphine hydrochloride 10 mg/ml, sodium chloride 7,5 mg/ml, sodiummetabisulphite 1 mg/ml, disodiumedetate 0,1 mg/ml, acetic acid to pH 3,0-3,5, sterile water; AstraZeneca, Södertälje, Sweden) was administrated s.c. at doses of 1, 3, 10 and 30 μmol/kg.

Comment

In paper IV morphine was administered to the rats during ongoing microdialysis. It has been shown that stress affects the effects of morphine (Valverde et al., 1997; Hawranko and Smith, 1999) and it cannot be excluded that the microdialysis procedure was stressful for the animals, thereby affecting the antinociceptive effects of morphine.

Diclofenac was administrated to rats intramuscularly since this is the recommended route of administration in humans. Since intramuscular injections are known to cause some degree of pain and muscle damage (Chellman et al., 1994), we could not exclude that the injection of diclofenac per se may affect the CCK-LI release. We therefore performed the injections under anaesthesia and we included a control group which received a subcutaneous injection of saline. We found that the intramuscular injection of saline or diclofenac alone had no effect on CCK-LI release in the ACC compared to the subcutaneous injection of saline in non-arthritic animals.

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3.4 MICRODIALYSIS

3.4.1 SURGICAL PROCEDURE

In paper III and IV microdialysis was carried out in freely moving rats. In brief, two days prior to the microdialysis experiments, a guide cannula (CMA 12, CMA/Microdialysis AB, Sweden) was implanted under halothane anaesthesia at the following stereotaxic coordinates: 0.7 mm caudally and 0.7 mm laterally to the Bregma (Paxinos and Watson,; 1998). The guide was lowered diagonally 3.3 mm from the skull surface (= 2.14 mm from the dura mater) at an angle of 60º from the horizontal plane aiming rostrally. The guide tip was thereby placed just above the left anterior cingulate cortex. The extension of the microdialysis membrane was + 0.95 +1.95 rostrocaudally to the Bregma and 1.86 3.59 mm ventrally to the dura mater. The guide was secured with dental cement and 2 mm anchor screws (Stockholm, Sweden) to the skull, and the skin was sutured.

In accordance with ethical guidelines for surgical procedures on animals, we provided postoperative analgesia, by intradermal injection of adrenaline + bupivacaine (bupivacaine

) around the wound at the termination of the operation. This combined preparation is commonly used in Swedish veterinary practice and is recommended for postoperative analgesia. The added adrenaline prolongs the duration of bupivacaine as well as reducing postoperative bleeding.

3.4.2 DIALYSIS

Following 2-3 days of postoperative recovery in individual cages, a microdialysis probe (CMA 12, CMA Microdialysis, Sweden) with a polycarbonate membrane (2 mm long, 0.5 mm diameter, molecular cut-off 20 000 Da), was inserted through the guide cannula. Microdialysis was performed in awake, freely moving rats and the probe was perfused with a Krebs solution (138 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 11 mM NaHCO3, 1 mM NaH2PO4) containing 0.2% bovine serum albumin, (Sigma-Aldrich, Stockholm, Sweden) 0.2% glucose and 0.03% of the peptidase inhibitor bacitracin (Sigma-Aldrich) at a rate of 3.5 μl/min. Following a wash-out period of 1 h after the insertion of the probe, samples of 105 μl were collected in 30 min fractions in Eppendorf vials. The dialysis probes were perfused for another 6.0 h (12 fractions) with normal Krebs solution. As a positive control of neuronal release (Vallebuona et al., 1993), the perfusate was shifted to a Krebs solution with elevated (100 mM) potassium concentration and correspondingly decreased sodium concentration (43 mM) 4 mimedium was then shifted back to normal Krebs solution. Animals which responded with an increase of less than 100% (3 rats out of initial 62) were excluded from the study. The microdialysis samples were stored in -20°C until quantitative immunoassay. After termination of the experiments, the animals were killed with an i.p. overdose of pentobarbital. The brains were dissected out and placed in a 25% sucrose-10% formalin solution. Later the brains were

for verification of probe placement .

Comment

Microdialysis is a perfusion technique used for measuring neurotransmitter release in vivo (for rev. see Ungerstedt, 1991). A thin semipermeable tube, perfused with a physiological

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solution, is stereotaxically implanted into the brain region of interest. Diffusion of neurotransmitters and other molecules occur as an effect of the concentration gradient between the two sides of a permeable membrane (Chen, 2006), in this case between the brain tissue and the solution in the dialysis probe. The diffusion can occur in both directions, allow administration of substances, e.g. drugs, into the brain tissue around the probe. Fick's law states that the diffusion rate is proportional to the concentration gradient and the net amount of molecules that are collected via the dialysis probe during perfusion is defined as recovery. The absolute recovery is the total number of molecules entering per time unit and the relative recovery is the relative concentration of a molecule in the perfusate compared to the concentration in the surrounding medium (Ungerstedt, 1991). A larger membrane area and a slower flow rate increase relative recovery (Lindefors et al., 1987). It is possible to determine the in vitro recovery of e.g. CCK for a specific microdialysis probe by comparing CCK-LI levels in the perfusate with the CCK-LI concentration in a test vial into which the probe is placed. This has been done in our group on the probe brands used in paper III and IV and the relative recovery for CCK-LI was found to be approx. 3% (see also Gustafsson et al., 1998). One must however, bear in mind that the in vivo the movement of molecules are more complex. Both the fraction of the extracellular space that is diffusible (the porosity), and the rate of transport through a tissue (the tortousity), is different in the brain compared to a liquid in a test vial. Furthermore, the concentration of neurotransmitters in the brain is affected by active processes such as transport over membranes, re-uptake and degradation.

Various methods can be used to control that the measured neurotransmitters during microdialysis are release synaptically. Examples of such methods are stimulation or inhibition of sodium channels, depolarisation by high-extracellular potassium or inhibition of calcium channels. Release during high-potassium stimulation is also considered to reflect the total

of transmitters in the synapses.

In paper III and IV, we used a perfusion solution with elevated (100 mM) potassium concentration and correspondingly decreased sodium concentration (43 mM) s a positive control of neuronal release (Vallebuona et al., 1993), during one collection fraction of 30 min. Animals who responded with an increase of less than 100% (3 rats out of initial 62) were excluded from the study. In paper III we also performed experiments where probes were perfused with a calcium-free, high-magnesium concentration (12 mM) medium. The potassium-induced CCK-LI release in the ACC was found to be calcium dependent (data not shown).

The main advantage of the microdialysis technique is that it permits continuous measurement of the extracellular concentration of neurotransmitters, allowing a temporal discrimination of physiological events and pharmacological effects in the intact brain of awake animals (for rev. see Stiller et al., 2003). A drawback of the technique is its invasive nature since implantation of the probe causes tissue trauma (Bungay et al., 2003; Chen, 2006). One of the technical difficulties of microdialysis in freely moving animals is the requirement of very long tubings, which may lead to a high flow resistance and consequent leakage of the probe.

3.5 RADIOIMMUNOASSAY

Briefly, assays were performed directly in the Eppendorf vials that were used for the sampling. Standards containing synthetic sulphated CCK octapeptide (CCK-8S, Peninsula Laboratories, USA) were diluted in Krebs medium of the same composition as used during the microdialysis. The dialysate and standard samples were pre-incubated for 24 h at 4°C with C-

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terminal-directed CCK/gastrin antiserum 2609/10 (from Prof. Jens Rehfeld, Rigshospitalet, Copenhagen, Denmark) (Rehfeld, 1978). After addition of 1000 cpm of {125I} gastrin I (Eurodiagnostica, Malmö, Sweden) to each vial, all samples were incubated at 4°C for another 72 h. Free radioligand was separated from antibody bound ligand by addition of 250 μl sheep anti-rabbit antibody coated sepharose suspension (Decanting suspension 3, Pharmacia Diagnostics, Uppsala, Sweden). After 30 min of incubation and centrifugation at 2600 x g for 10 min, the supernatant was discarded by aspiration. The radioactivity in the pellets containing the bound fraction was measured in a gamma-counter. counter. The CCK levels in the three sets of experiments described in section 4.5 below were analysed in separate assays, with a lower detection limit of 1.2 pM (=fmol/sample) in the two first sets and 2.4 pM in the third set.

Comment

The principle of RIA is based on the competitive binding of radiolabelled and endogenous (unlabelled) measurements of neuropeptides in microdialysis samples (for rev. see Lindefors et al., 1987) and is still the most sensitive method for detecting neuropeptides in very low (pM ranges) concentrations (for instance, see Rissler, 1995; Nyberg, 2004). A number of technical adjustments have been made to lower the detection limit of the assay. These improvements include the usage of small incubation volumes, comparatively small amounts of radioligand per incubation tube (1000 cpm) and sequential incubation of samples and radioligand with the antibody (Brodin et al., 1983). Further on, in order to reduce the loss of peptides in the samples, bovine serum albumin (BSA) is used for decreasing the adheration of peptides to the plastic material in the tubings and the peptidase inhibitor bacitracin is utilised for prevention of enzymatic degradation of the peptides. In the present thesis, we used a polyclonal antibody raised against gastrin which also binds with high affinity to CCK (Rehfeld, 1978) . CCK-antibodies with a high specificity (but lower sensitivity) are also available (for rev. see Rehfeld, 1998). However, since virtually no gastrin is present in the CNS of the rat, cross reactivity to gastrin was not a problem in the present studies. We therefore chose to use the gastrin/CCK-antibody with the highest affinity available.

3.6 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

3.6.1 CHARACTERISATION OF CCK-LI BY HPLC

In order to characterize the immunoreactive material in the above mentioned RIA experiments, high-performance chromatography (HPLC) was performed on a separate set of dialysates in paper III. In a group of rats, microdialysis with potassium stimulation was carried out in the ACC as described above. The collected samples were extracted using reversed-phase HPLC with a column containing hydrocarbon-bonded (C18) silica. The column was eluted with a linear gradient of acetonitrile, separating compounds in order of hydrophilicity and calibration with 0.1 pM synthetic CCK-8S was performed. The collected fractions were later re- -LI was performed as described above. The main fractions which were detected by RIA co-eluted with synthetic CCK-8S.

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3.6.2 QUANTIFICATION OF MORPHINE AND M3G BY HPLC

For quantification of plasma concentrations, morphine (3 mg/kg) was given subcutaneously to a separate group of rats (n = 40). After 0.25, 0.5, 1.0, 3.0, 6.0, 12.0, 24.0 and 48.0 h (n = 5 at each time point). The animals were decapitated and blood samples (3 - 4 ml) were collected in plastic tubes containing 10 IU/ml heparin. Trunk blood was also collected from five untreated rats. The tubes were centrifuged and the plasma was taken off and frozen at - 20 C until measurement of morphine and M3G was performed by high performance liquid chromatography (HPLC) with ultraviolet and electrochemical detection (Svensson et al., 1982; 1986). In brief, the method was based on solid-phase extraction with C18 cartridges, isocratic elution with a mobile phase containing 10 mM phosphate buffer (pH 2.1), 1 mM dodecyl sulphate and 26% acetonitrile using reversed phase chromatography. The lower detection limit of quantification for morphine was 0.001 nmol/l and for M3G 0.050 nmol/l.

Comment

HPLC is the method of choice for detection of morphine and its metabolites due to a higher specificity and more convenient procedure than methods such as RIA and gas-liquid chromatography (Milne et al., 1996). Svensson and collaborators (1982; 1986) improved the technique by combining electrochemical detection for morphine and ultraviolet detection for M3G. This procedure, which was used in paper I, has a high specificity and sensitivity with a lower detection limit for morphine (1 nmol/l) and M3G (50 nmol/l). Gas chromatography-mass spectrometry can be used for detection of even lower concentrations, e.g. several days after drug administration (Moeller and Kraemer, 2002).

3.7 CALCULATIONS OF RESULTS AND STATISTICAL ANALYSIS

3.7.1 BIOCHEMICAL DATA

When the concentration of CCK-LI in the microdialysates was lower than the detection limit of the RIA, the values were arbitrary set to the sensitivity of the assay (1.2 pM, 1.2 pM and 2.4 pM, respectively for the three separate assays)

In paper III we showed that the increase in CCK-LI release after carrageenan injection coincides with the time period when the rats show maximal pain behaviour, i.e. 3-7 hours after the induction of inflammation (Erel et al., 2004). Therefore, in paper III and IV, the mean basal release CCK-LI was defined as 3.0-7.0 h after carrageenan injection and probe insertion or probe insertion only. Stimulated release was in paper III, defined as the cumulative amount of CCK-LI exceeding basal level that was released during a 60 min period after the start of K+ - stimulation and was expressed in fmol (mean ± S.E.M.). Differences in the basal and stimulated release values between controls and carrageenan treated animals were analysed by unpaired non-parametric test (Mann-Whitney).

In the second set of experiments in paper IV, the mean CCK-LI release was calculated from the release during the 4 hours immediately after injection of morphine or vehicle. Since there was a variation in between groups in the pre-injection values, data were also normalised by calculating the mean release of CCK-LI during the 4 hours following injection as percent of the average release of CCK-LI during the 2 hours preceding the injection. Both normalised and non-normalised data were analysed statistically. The CCK-LI levels in paper IV were

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conformed to normality and differences in release values between groups were analysed with one-way ANOVA followed by Tukey's Multiple Comparison Test as post-test.

GraphPad Prism version 3.0 (GraphPad, San Diego, CA, USA) was used for statistical analysis in both paper III and IV.

3.7.2 BEHAVIOURAL DATA

In paper I and III, Friedman ANOVA followed by post-hoc test (respectively) were used for analysis of differences between observations at different time points compared to baseline values within the same group. Kruskall-Wallis test followed by the test for multiple comparisons of mean ranks for all groups, was used for analysis of differences between groups at each time point. The ankle dimension measurements were analysed by repeated measures one-way ANOVA. Differences were considered to be significant at p < 0.05.

Statistical analysis was performed in paper I with Statistica 6.0 (StatSoft, USA) and in paper III and IV with GraphPad Prism version 3.0 (GraphPad, San Diego, CA; U.S.A.). Data are presented as mean ± S.E.M.

In paper II, a statistical model was programmed using SAS® v8, (SAS Institute Inc., Cary, NC, USA) and Graph Pad Prism 2.01 (Graph Pad Prism Software Inc., San Diego, CA, USA). Data were presented as mean ± S.E.M. For analysis of differences in scores between mouse strains, time points, adjuvants, doses and drugs after adjuvant injection, chi-square tests of contrasts within a generalized linear model were used. The log odds ratios for the cumulative score probabilities were modelled as linear with one intercept per score category and coefficients depending on factor level, using a multinomial distribution for the scores. Animal was used as repeated measures factor, since the animals were followed over time generating dependent observations. Due to unbalances in the experimental designs and groups with zero variance, no interactions could be calculated. Instead all comparisons were carried out using only one factor besides animal and keeping all other factors fixed. For the time point comparisons some groups with zero variance were excluded, namely the FCA-MT2 and FCA-MB groups at time =10 days and all groups at time =12 and 16 days in the comparison between adjuvants, the BALB/c groups at 5 days in the comparisons with NMRI-mice and both groups at time=0 and the carrageenan group at time=48 hours in the comparison between carrageenan and FCA-MT1. In the experiment with drug treatments the saline and all diclofenac groups at time=0 hours and the saline group at all time points were excluded for the same reason.

Comment

The use of a subjective rating scale in paper I-III elicited the need of non-parametrical statistical methods. In all three studies, repeated measures were done comparing pain score in the same animal to pre-arthritis values. In paper III, comparisons were also made with non-arthritic controls. Furthermore in paper II, using repeated measures, we compared difference in pain score between two mouse strains and three arthritic agents. Thus, paper II contained the most complicated data in terms of statistical analysis since we used non-parametrical measures in a model with three variables.

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4 RESULTS AND DISCUSSION

4.1 INVESTIGATION OF A POSSIBLE PRE-EMPTIVE ANALGESIC EFFECT OF

MORPHINE IN RATS WITH MONOARTHRITIS (PAPER I)

The idea of pre-emptive analgesia is to block early sensitizing events locally at the site of inflammation or in the central nervous system and thereby obtain analgesic effects of morphine also after the plasma concentrations have decreased below the therapeutic interval. In paper I, we investigated and compared the antinociceptive effect of morphine given before or after the induction of monoarthritis in rats. We administrated morphine (3mg/kg, s.c.) either 30 min before (MO PRE group) or 2 h after (MO POST group) injecting carrageenan into the ankle joint of rats and we quantified the pain-related behaviour before and at 3, 5, 10, 24 and 48 h after the carrageenan injection. In order to examine whether the behavioural response to morphine was correlated to the plasma concentration we also followed the plasma kinetics of morphine following an acute injection of the same dose. In addition, the metabolite morphine-3-glucuronide (M3G) was measured since it has in some studies been shown to have a pharmacological, possibly antagonistic effect to morphine itself (Wittwer and Kern, 2006).

In the control group, which received carrageenan but no morphine, we observed a significant overall increase in pain score with a maximum at 5 h. As expected, when morphine was administrated 2 h after the induction of inflammation, the behavioural score was significantly reduced one hour later. There was still a tendency of reduction at 3 h after morphine administration. In the group which received morphine 0.5 h prior to inflammation, there was a small reduction in pain behaviour compared to controls at 3.5 h after morphine administration, but not at later time points. Our pharmacokinetic data following administration of 3 mg/kg of morphine in male Sprague-Dawley rats are in accordance with previous investigations (for rev. see Milne et al., 1996). The plasma half-life (T1/2) of morphine during 0 3 h was approximately 30 min and the highest concentration was measured at 15 min which is slightly earlier than previous studies showing a Tmax of 30-50 minutes after s.c. administration (Stain et al., 1995; van Crugten et al., 1997; Baker and Ratka, 2002). The concentration of morphine was lower than that of M3G at all measured time points, probably due to a rapid metabolism and the considerably larger volume of distribution (Vd) for morphine (Hasselström et al., 1996) than for M3G (Ekblom et al., 1993; Ouellet and Pollack, 1995). Our findings of a peak analgesic effect of morphine 1 h after administration and a tendency after 3 h is in accordance with others who have previously demonstrated a delay of approximately 30 min in the maximal effect compared to the Tmax in plasma (Melzacka et al., 1985; Stain et al., 1995; Hasselström et al., 1996; van Crugten et al., 1997). This concentration-effect delay is probably due to the required time for distribution of morphine from plasma to the CNS (Bouw 2000). Since M3G has no analgesic effect (Vaughan and Connor, 2003; Wittwer and Kern, 2006), it is not likely that it contributed to the antinociceptive effects of morphine observed in the present study.

Comparisons of the time course for the plasma concentration of morphine and the behavioural effects demonstrated that the only marked reduction in pain score compared to controls was in the MO POST group at 1.0 h after morphine administration when the morphine plasma concentration was relatively high (0.49 μM). In both the MO PRE and MO POST groups, there was a tendency for a reduction in behavioural score compared to controls

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at 3.0 h after morphine administration when the plasma concentration was lower than 0.03 μM. The slight reduction in pain-related behaviour when morphine was administrated before the induction of inflammation, was similar to the reduction in pain score demonstrated for morphine administrated after the induction of inflammation. Thus, the reduced stance score in the MO PRE group at this time point is probably not due to a pre-emptive effect. In other words, our data do not support a pre-emptive analgesic effect of morphine in this model of inflammatory pain.

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4.2 SPONTANEOUS NOCICEPTIVE BEHAVIOUR IN MICE WITH

MONOARTHRITIS (PAPER II)

The original objective of paper II was to develop a model of sustained inflammatory pain in mice in order to investigate the release of neurotransmitters during pain and pain-related behaviour, in genetically modified animals. Since such transgenic methods are most common in mice, our first aim was to develop a model of chronic inflammatory pain in mice. After initial tests of various techniques for inducing inflammation, intraarticular injection of Freund's complete adjuvant, was selected for further investigation. Using the stance scoring technique which quantifies pain-related behaviour, we characterized adjuvant- and carrageenan-induced monoarthritis, two inflammatory models which have previously been used in rats.

(FCA) from different manufacturers was compared in NMRI mice. The preparations contained 1 mg/ml mycobacterium tuberculosis H37Ra (FCA-MT1 and FCA-MT2) or 0.5 mg/ml mycobacterium butyricum (FCA-MB) in mineral oil. All of the three adjuvants caused a pronounced impairment of gait and stance at day 1-3 with a reduction at day 5. At day 7, the stance score was further reduced except in the FCA-MT1 group. Ten days after injection there was no significant pain-related behaviour in any of the treatment groups. There were no major differences in the responses to the three adjuvants. The slightly but significantly lower pain score in the FCA-MT-2 group compared to the other two adjuvants at day 1 may be due to the different strain and lower concentration of mycobacterium (Cannon et al., 1999).

Secondly, the effects of the adjuvants FCA-MT1 and FCA-MB were studied in two mouse strains frequently used studies of inflammation and antinociceptive effects (Wahlsten et al., 2003; Kalueff et al., 2007), inbred NMRI and outbred BALB/c mice. Both adjuvants produced high stance scores in both strains on day 1 after adjuvant administration. In NMRI mice, significant levels of increased stance score was observed for at least 7 days after injection of FCA while in BALB/c mice an altered behaviour was seen at 3, but not at 5 days. Thus, our experiments revealed that NMRI mice showed a more prolonged response than BALB/c mice to both FCA-MT1 and FCA-MB. Our findings are in agreement with previous investigations showing that various mouse strains exhibit considerable differences pain susceptibility (Elmer et al., 1998; Mogil, 1999).

As a third step, the responses to FCA-MT1 and carrageenan were compared in NMRI mice. Intraarticular injection of 2% carrageenan in NMRI mice caused an increase in stance score for less than 24 hours, with a maximal stance score of 1.0 at 3 h. The time course for the impaired stance score after carrageenan injection in mice was roughly in accordance with previous studies performed on rats (Tonussi and Ferreira, 1992; Schött et al., 1994) although the response appeared to be less pronounced in mice. The FCA-MT1 group, on the other hand, obtained an increased stance score for over 96 hours with a maximal score of 1.9. at 24 hours (figure 6). Thus, FCA seems more suitable than carrageenan for the induction of monoarthritis as a model of chronic inflammatory pain.

Due to the higher maximal stance score and longer duration of pain-related behaviour in NMRI mice injected with FCA-MT1 as demonstrated in part 1-3 of the study, we chose to use this combination of mouse strain and inflammatory agent for pharmacological characterization of the FCA model. Thus, as a fourth part of the study, two analgesic drugs with different mechanisms of action, morphine and diclofenac, were tested on NMRI mice at 3

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0 10 20 30 40 500.0

0.5

1.0

1.5

2.0 ***

Hours after injection

Stan

ce s

core

Figure 6. Time course of pain behaviour in NMRI mice injected with FCA-MT1 ( ) or carrageenan( ), n = 10 in each group. Data are presented as mean ± S.E.M. Comparisons at 48 were excluded due to zero variance in the carrageenan group.

days after injection with FCA- MT1. Morphine (s.c.) was found to reduce the stance score values at the doses 1, 3 and 30 μmol/kg, with the highest dose giving a maximal decrease of the stance score at 0.5 h after administration. At 3 h, the stance score was still significantly reduced in the 30 μmol/kg group compared to initial values and saline treated controls, while no significant reduction was seen at this time point with the other doses. Administration 10 and 30, but not 3 μmol/kg of diclofenac (s.c.), caused a significant decrease in stance score at 2 hours after administration. Following the highest dose, stance scores were still significantly reduced at 6 hours compared both to the values at 0 hours in the same group and to saline treated controls. In conclusion, both morphine and diclofenac were found to attenuate the FCA-MT1-induced behavioural effects with the highest doses giving the most pronounced effects. The time courses for both drugs are in accordance with previous behavioural studies performed on NMRI mice with morphine and diclofenac (Gupta et al., 1998; Kest et al., 2000; Vermeirsch and Meert, 2004).

Surprisingly, 10 μmol/kg of morphine showed a lower effect than both 1 and 3 μmol/kg at 0.5 and 1 h after administration. We have no obvious explanation for this finding, but others have previously demonstrated a bimodal effect of opioids, i.e. that lower doses may have excitatory effects on sensory systems while higher doses have inhibitory effects (Crain and Shen, 2000).

In order to provide a stable baseline for testing drug effects on pain related behaviour, an animal model of inflammatory pain should have a relatively long duration. In our study the stance score of NMRI mice was elevated for 7 days and returned to pre-injection values 10 after FCA injection. Chillingworth and Donaldson (2003) have reported an elevated pain score for 12-15 days using a different injection technique with double injections of FCA around the joint, which may account for the longer duration as compared to our study. However, our goal was to develop a model that closely resembles monoarthritis in man, with an inflammation that originates from the joint. We therefore chose to inject the adjuvant directly into the joint space. We found that intraarticular injection of FCA provides a robust model for pharmacological studies of inflammatory pain mice. However, we also found great inter-strain differences in the duration of pain related behaviour which could affect the outcome.

In accordance with our original project plan, we then proceeded by performing microdialysis in the anterior cingulate cortex of mice with FCA-induced arthritis and tried to detect changes in the release of cholecystokinin-LI measured by RIA. However, this proved to be very difficult. We were unable to obtain consistent results on CCK-LI release, probably due to the lower recovery of CCK using smaller microdialysis probes compared to in the rat. We therefore continued our microdialysis studies in the rat.

Results and Discussion

31

4.3 MONOARTHRITIS INCREASES CHOLECYSTOKININ RELEASE IN THE RAT

ANTERIOR CINGULATE CORTEX, AN EFFECT REVERSED BY NSAID BUT NOT

MORPHINE (PAPER III AND IV)

Based on the hypothesis that CCK in the ACC is involved in the modulation of pain and opioid effects, we investigated in paper III the effect of carrageenan-induced monoarthritis on the release of CCK-LI in the ACC. CCK-LI release was measured using microdialysis in freely moving male rats, and pain-related behaviour was studied with the stance scoring and weight bearing techniques.

The effect of carrageenan on the stance of the animals was confirmed by behavioural scoring and by measuring the weight-bearing ratio of the hind paws using a special registration device that has been developed in our group (Schött et al., 1994). In agreement with previous studies, (Schött et al., 1994; Kissin et al., 2005) we found that the stance score was increased for at least 24 h after carrageenan injection with a maximum at 5 h compared to controls at the corresponding time point. Similarly, the weight-bearing ratio in arthritic rats was significantly increased during 3 to 24 h with a peak at 5 h. The inflammation, which was quantified by increase in ankle dimension, was significantly increased during 3 to 48 h, reaching a maximum at 5 h compared to controls.

The extracellular concentrations of CCK-LI were measured in the ACC contralaterally to the carrageenan injected limb with start immediately after injection. The mean basal release of CCK-LI measured between 3 and 6 h after probe insertion, i.e. during a time period when the rats showed significantly increased pain behaviour, was significantly increased in animals with carrageenan-induced arthritis, compared to controls. Furthermore, potassium stimulation locally in the ACC produced a significantly higher evoked release of CCK-LI in arthritic rats compared to controls.

By means of reverse phase HPLC, the main component of the immunoreactive material in the ACC dialysates collected during potassium stimulation was found to co-elute with synthetic sulphated CCK-8 which is in agreement with previous observations of CCK-LI from microdialysis experiments in the prefrontal cortex (Maidment et al., 1991; You et al., 1994). The antibody used in the present assay cross-reacts to 33% with non-sulphated CCK-LI which likely arises from spontaneous desulphatation of CCK-8S (Rehfeld et al., 1985) and this is probably represented by one of the minor peaks in the chromatogram.

In order to strengthen the hypothesis that the increased CCK-LI release in the ACC during arthritis observed in paper III is involved in the modulation of pain, we next examined the effect of analgesic drugs. In paper IV, we used morphine and the non-selective cyclo-oxygenase-inhibitor diclofenac in doses within the ranges as have previously been shown to eliminate pain-related behaviour in rats with arthritis (Schött et al., 1994; Fernihough et al., 2004). Further on, we studied the effect of morphine by itself on the CCK-LI release in the ACC of non-arthritic rats, since morphine is known to induce CCK-LI release in other CNS-regions.

As observed in paper III, the mean release of CCK-LI was significantly increased in animals with carrageenan-induced arthritis compared to controls 3.0 to 7.0 h after injection of carrageenan, i.e. when maximal pain-related behaviour and oedema is observed. Administration of 10 mg/kg of morphine (s.c.) prior to carrageenan injection did not affect the mean release of CCK-LI 3.0 7.0 h after induction of arthritis compared to arthritic rats treated with saline. In contrast, treatment with 25 mg/kg of diclofenac (i.m.) counteracted the

Umut Heilborn

32

carrageenan-induced CCK-LI increase. In addition, the mean release of CCK-LI was significantly lower in arthritic rats treated with diclofenac compared to morphine treated animals (figure 7).

We could not find any effect of morphine by itself on the CCK-LI release in the ACC. Neither 5 nor 10 mg/kg of morphine s.c. affected the mean CCK-LI release in non-arthritic animals during the 4 hours after acute administration. The failure of morphine to affect the extracellular level of CCK-LI in the ACC in normal animals is in contrast to studies in other CNS regions such as the spinal cord (de Araujo Lucas et al., 1998; Gustafsson et al., 2001), the RVM (Xie et al., 2005), and the frontal cortex (Becker et al., 1999), indicating that the release of CCK is differentially regulated by opioids in various brain regions.

Since i.m. injections may in themselves cause both acute and chronic pain (Chellman et al., 1994), these injections were performed under halothane anaesthesia. When studying the effect of diclofenac (25 mg/kg i.m.) on the CCK-LI release in the ACC in normal animals, we used two control groups, which received saline injected intramuscularly or subcutaneously. In non-arthritic animals the overall mean CCK-LI release in the ACC during 3.0-7.0 h after probe insertion did not differ significantly between rats injected with diclofenac, saline i.m., or saline s.c. Thus, our results indicate that the effect of diclofenac on CCK-LI release in the arthritic rats is due to a true blockade of the arthritis-induced increase in CCK release.

In summary, we found that an increased release of CCK-LI in the ACC of rats with carrageenan-induced monoarthritis was counteracted by diclofenac but not by morphine, suggesting that prostaglandins may contribute to the increased CCK-LI release in the ACC during inflammation.

Figure 7. The release of CCK-LI in the ACC in control rats (n=6) and in rats with carrageenan-induced

arthritis (carr) receiving diclofenac (25 mg/kg, i.m.; n=6), morphine (10 mg/kg, s.c.; n=9) or saline (1 ml/kg, s.c.; n=8). Symbols are placed at the end of each 30 min collection period. Each data point represents the mean ± S.E.M. (A). The mean (± S.E.M.) release of CCK-LI during 3.0-7.0 hours after carrageenan injection in rats with arthritis receiving diclofenac (25 mg/kg, i.m.; n=6), morphine (10 mg/kg, s.c.; n=9) or saline (1 ml/kg, s.c.; n=8) and in control rats (n=6). ***p < 0.001 vs. control, +++p < 0.001 vs. animals with arthritis treated with saline, # # # p < 0.001 vs. animals with arthritis treated with morphine (B).

0 1 2 3 4 5 6 70.0

2.5

5.0

7.5

10.0

12.5controlcarr + salinecarr + morphinecarr + diclofenac

Time (h) after carrageenan

CC

K-L

I (pM

)

contr

ol

carr

+ sali

ne

carr

+ morp

hine

carr

+ diclo

fenac

0

1

2

3

4

5 *** ***

+++# # #

CC

K-L

I (pM

)

A B

General Discussion

33

5 GENERAL DISCUSSION

5.1 EVIDENCE OF PRE-EMPTIVE ANALGESIA

During the last decade there has been a change in anaesthetic practice that incorporates pre-emptive analgesia with opiates and other analgesics, although the evidence of the presumed beneficial effect for this practice in postoperative pain management has been very controversial (Bromley, 2006). Our main finding in paper I on carrageenan-induced arthritis, does not support a pre-emptive analgesic effect of morphine in this animal model of arthritic pain.

Our results are in agreement with a report by Sevostianova and collaborators (2003) who used the formalin test to study the antinociceptive effect of morphine in rats. They demonstrated that morphine administered after the early phase of the formalin reaction was more effective in suppressing the pain-related behaviour in the late phase compared to pre-emptive morphine treatment. Likewise Giamberardino et al. (2000), have reported that pre-administration of morphine or ketoprofen has no advantage in reducing behavioural indicators of visceral pain and referred hyperalgesia in rats.

Since in the present study, the plasma concentration of morphine was high at 0.5 h after s.c. administration, i.e. at the time point of carrageenan injection in the MO PRE group, one might have expected a prevention of the early neurochemical events leading to sensitisation and subsequent pain. However, previous studies have demonstrated a complicated interaction in the time course of the development of pain and inflammation and the time course of analgesic effect of morphine (Honoré et al., 1995; Sevostianova et al., 2003; Garlicki et al., 2006). For instance, Honoré et al. (1995) have shown that an increased expression of cFos in the spinal cord develops at 1.5, 2.0 and 2.5, but not at 1.0 h after formalin injection. The timing of pharmacological intervention for the prevention of such early molecular events preceding observable pain and oedema, is, in other words, probably crucial. Since there is a fast pharmacokinetic turn-over of morphine in the rat, it is possible that an injection at a different time point, for example at 0.5 hours after carrageenan, would have resulted in a different outcome. It is also a complicating factor that the inflammatory response in carrageenan-induced inflammation, although longer lasting than previously investigated models, has a relatively rapid spontaneous recovery. Consequently, it cannot be excluded that a presumed pre-emptive analgesic effect could be possible to demonstrate in an inflammatory model with a more stable and longer lasting inflammatory response.

One factor which seems important when studying the possible pre-emptive analgesic effect of morphine, is the route of administration. Intrathecal (Yamamoto and Yaksh, 1992; Abram and Yaksh, 1993) but not systemic (Abram and Olson, 1994; Sevostianova et al., 2003) morphine has been shown to prevent the late phase of the formalin response. A similar pre-emptive effect after intrathecal but not systemic morphine has been shown in a model of neuropathic pain (Luo and Wiesenfeld-Hallin, 1996). On the other hand, in models of surgical pain, a pre-emptive effect of morphine has been demonstrated after systemic (Abram and Olson, 1994; Sevostianova et al., 2003), but not intrathecal (Brennan, 1996) administration. Pre-emptive morphine administered at the site of inflammation, has been reported to reduce the c-fos expression (Locher-Claus et al., 2005) and vocalisation threshold (Perrot et al., 1999) during carrageenan-induced inflammation. Furthermore, locally pre-administered tramadol

Umut Heilborn

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prevents pain-related behaviour (Garlicki et al., 2006) in the same inflammatory model. In neuropathic pain models some studies have demonstrated a pre-emptive effect of morphine (Puke and Wiesenfeld-Hallin, 1993), while others have not (Munglani et al., 1995) and also in these models the analgesic effect depends on the route of administration (Luo and Wiesenfeld-Hallin, 1996).

Many previous studies on pre-emptive analgesia have suffered from inadequate study design (Bromley). For instance, in several human (Rockemann et al., 1996) and animal (Abram and Yaksh, 1993; Shavit et al., 2005) investigation of a possible pre-emptive opioid effect, various combinations with inhalation- or local anaesthetics or unselective drugs (Garlicki et al., 2006) have been used, thereby obliquing the specific role of the opioid used. Further on, fentanyl, a highly selective μ-receptor agonist, is widely used preoperatively due to its favourable cardiovascular effects, making it difficult to obtain valid controls. An additional problem is that many studies compare pre-surgical morphine with placebo rather than with post-surgical morphine (for rev. see Bromley, 2006).

In preclinical investigations pre-emptive analgesia, the choice of pain model and the methods for assessing pain, are of obvious significance for the outcome. Electrophysiological events (Chapman et al., 1994; Benrath et al., 2004) or reduction in c-Fos expression (Tölle et al., 1994; Honoré et al., 1995; Locher-Claus et al., 2005) have in several studies been quantified as an indication of pain. However, such parameters are not always correlated to pain-related behaviour in the pre-emptive paradigm (Gilron et al., 1999). Furthermore, such experiments are performed in anaethetised or decerebrated animals. In contrast, we have in the present study made direct observations of spontaneous pain-related behaviour. Moreover, we have used carrageenan-induced monoarthritis in order to induce a longer lasting inflammatory pain which (as discussed in 5.1) may be more relevant for the clinical setting .

In conclusion, in the present preclinical setting, our data do not support an advantageous effect of administration of morphine prior to the induction of arthritis compared to morphine treatment after the onset of inflammatory pain.

General Discussion

35

5.2 ANIMAL MODELS OF INFLAMMATORY PAIN

The aim of the first study was to develop a mouse model for pain during chronic inflammation. Thus, we characterized in female mice adjuvant-induced monoarthritis, an inflammatory model which is commonly used in rats. We found that intraarticular injection of FCA produces a robust and consistent increase in pain-related behaviour in both NMRI and BALB/c female mice. This is in agreement with a study in male mice showing that multiple, subdermal injections of FCA around the tibiotarsal joint produced hyperalgesia and allodynia (Chillingworth and Donaldson, 2003). We also validated our model pharmacologically using two different analgesic drugs with different mechanisms of action, and found both drugs to be efficient in decreasing the pain-related behaviour. Although no histological examination of the injected knee joint was performed in this study, other investigators have shown that intraarticular injections of FCA produce arthritis in the rat (Butler et al., 1992).

Our results also indicate that the choice of mouse strain is of importance for the results obtained. In paper II, NMRI mice were found to be significantly more susceptible to adjuvant-induced pain in terms of duration, than BALB/c mice. It has previously been shown that various mouse strains exhibit considerable differences in response to nociceptive stimulation (Elmer et al., 1998; Mogil et al., 1999) and morphine antinociception (Belknap et al., 1989; Elmer et al., 1998). Most of these studies have been performed with thermal or electrical pain stimuli to evoke acute pain (for review, see Mogil et al., 1999). Two previous studies have investigated the effect of genotype on nociception in the formalin (Mogil et al., 1998) and carrageenan (Mogil et al., 1999) tests. In these studies BALB/c mice have been shown to be of average or high susceptibility to inflammatory pain compared to other strains. However, it has also been shown that such strain differences in nociceptive sensitivity depend on the pain modality tested and that strains susceptible in one test can be resistant in another (Elmer et al., 1998).

In addition to the influence of strain in experimental pain models, the sex of the experimental animal may affect the outcome. Sex differences in pain perception have not been investigated in the present thesis. There is also evidence that oestrous cycle affects pain perception in rodents (Craft et al., 2004). Indeed, a number of studies have demonstrated differences due to sex sensitivity to pain and in the response to analgesic drugs in animals (Fillingim and Gear, 2004). Therefore, most animal studies of pain have been carried out in males. However, contradictory results have been published regarding how the oestrous phase affects nociceptive sensitivity and the results depend greatly on the behavioural pain assay used. It has also been shown that the intra-group variation due to oestrous cycle is smaller than the inter-strain difference (Mogil et al., 2006; Meziane et al., 2007), which is in accordance with our data that revealed major strain differences. In the present study, we did not control for the oestrous cycle. However, since the mouse oestrous cycle is very short, cyclic variations in pain perception probably has less impact on chronic studies with observations at several different time points and which lasts over the whole oestrous cycle such as the present one. In any case it is important to also study females in animal models of inflammatory pain. Clinical studies indicate that women are more afflicted with chronic pain than men (Wiesenfeld-Hallin, 2005) and that arthritic conditions such as RA are much more common in women (Cutolo, 2006).

Most preclinical studies of pain pharmacology in animals (for rev. see Mogil et al., 1999) have measured thermal or electrical pain stimuli to evoke acute pain) and not spontaneous pain behaviour. The methods used in the present thesis to assess the degree of pain, stance-

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scoring (paper I-III) and weight bearing (paper III), may be of greater relevance for the clinical situation. They have previously been validated pharmacologically (Coderre and Wall, 1988; Schött et al., 1994) and both paradigms are considered reliable although weight-bearing appear to give less variable results and to be more sensitive (Schött et al., 1994). Our own and other groups have shown that impaired stance or gait is also correlated to mechanical allodynia (Schött et al., 1994; Vrinten and Hamers, 2003). It is therefore likely that our model in paper II produced allodynia as reported for the similar model by Chillingworth and Donaldson (Chillingworth and Donaldson, 2003). However, since humans with chronic arthritis commonly have resting- and loading-pain, rather than mechanical allodynia or thermal hyperalgesia, the methods for assessing nociception in the present thesis may have a higher validity from a symptomatic point of view.

Translation of findings from animal studies to the clinical situation has met several problems, therefore, it is of importance to develop better behavioural models for pain research (Blackburn-Munro, 2004). One must question the validity, both in terms of neurobiological basis and symptomatic similarity, of the utilized animal model of pain to the clinical condition in humans. There are essential difficulties in interpreting pain in animals from both an ethological basis and a technical standpoint (Villanueva, 2000). In addition there are large species differences in pharmacokinetics and pharmacodynamics of analgesic drugs (Hill, 2000). The probability of misinterpreting the evidence obtained from experiments in species other than humans is always present because, obviously, animals cannot explain what they are feeling.

As discussed in the Introduction, pain consists not only of nociception, but also emotion. Thus, we do not know for certain if the effects of analgesics (paper I, II and IV) or changes in CCK release (paper III-IV), are truly related to pain. In order to investigate this, one should assess behaviours that are motivated, intentional, complex and learned. Place preference tasks such as the one used in this paper fulfil these criteria. They have proven to be sensitive to cortical lesions that do not affect spinal reflexes, especially in the ACC (Donahue et al., 2001; Gao et al., 2004; LaGraize et al., 2004). Thus, such models may provide the most useful tools for preclinical investigations of pain mechanisms and for the development of candidate analgesic drugs in the future.

General Discussion

37

5.3 THE POTENTIAL ROLE OF CCK IN PAIN MODULATION

In the present thesis, we have found that the CCK-LI release in the ACC was significantly increased in rats with carrageenan-induced arthritis compared to control animals under basal and potassium-stimulated conditions (paper III). The time interval after carrageenan injection when the increased CCK-LI release was observed coincides with the time course for the development of oedema of the limb and pain-related behaviour. The methods used to assess the degree of pain, stance-scoring and weight bearing, have previously been validated (Coderre and Wall, 1988; Schött et al., 1994). Both paradigms are considered reliable although weight-bearing appear to give less variable results and to be more sensitive (Schött et al., 1994).

Our group has previously shown that there is a bilateral increase in the evoked release of CCK-LI in the ACC in rats two weeks after axotomy, a model of phantom limb pain, although there was no detectable increase in basal release (Gustafsson et al., 2000). The finding that two entirely different pain models affect the release of CCK-LI in the ACC supports the hypothesis that CCK in this region is involved in pain processing. However, in contrast to the study by Gustafsson et al. (2000), also the basal CCK-LI release was increased in the present study. Such a dissimilarity suggests a differential role of CCK in the ACC in the modulation of different types of pain conditions. Interestingly, it has previously been shown that an electrolytic lesion of the ACC decreases inflammatory, but not neuropathic nociceptive behaviour in rats (Donahue et al., 2001). Furthermore, while in paper I and IV we have strong evidence that the rats experienced spontaneous pain during the periods when increased CCK-LI release was observed, it is unclear if animals experience pain 14 days after axotomy when the measurement of CCK-LI was performed. For instance, in the axotomy model, abnormal responses such as hyperalgesia and allodynia or abnormal behaviours such as autotomy it can be observed, while spontaneous pain has proved very difficult to demonstrate (Suzuki and Dickenson, 2006).

Human studies give evidence for involvement of the ACC in the emotional component of the pain experience (Casey, 1999; Verne et al., 2004; Bushnell and Apkarian, 2006). In rats, pain-induced anxiety-like behaviour can be abolished by lesions in the ACC (Johansen et al., 2001; Gao et al., 2004; LaGraize et al., 2004), Thus, we hypothesized that an increased CCK -release in this region could be involved in the mediation of emotional discomfort that is coupled to pain experience. Morphine, unlike diclofenac, is known to relieve the affective component of pain in humans (Zubieta et al., 2001) and such an effect has also been indicated in the rat (Oliveira and Barros, 2006).

Surprisingly, our results show that acute administration of diclofenac, but not morphine could reduce the arthritis-induced increase in CCK-LI release in the ACC (paper IV). Since morphine has previously been shown to effectively eliminate pain-related behaviour in the carrageenan model during the same time period and in the same dose, i.e. 10 mg/kg, as used here (Schött et al., 1994), it is likely that it produced analgesia also in our present experiments. However, it has been shown that chronic mild stress reduces morphine's rewarding (Valverde et al., 1997) and antinociceptive potency (Hawranko and Smith, 1999) and it cannot be excluded that the microdialysis procedure in the present study, including the preceding operation and single-cage housing, was stressful for the animals, thereby affecting the antinociceptive effects of morphine. Taken together, the present results showing no effect of morphine on the CCK release during acute pain argues against the hypothesis that CCK in the ACC is involved in the affective component of pain. However, the presently used animal

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model may not have been optimal to reveal an effect of morphine on CCK-LI release related to the affective component of pain. It has recently been reported that rats subjected to chronic stress in a social-defeat model, showed an increased susceptibility to acute pain. The increase in pain behaviour was accompanied by an elevated CCK-LI release in the frontal cortex which could be reversed by morphine (Andre et al., 2005). It is thus possible that the effect of morphine on CCK release in the ACC would have been different in a model of pain and stress with duration of several days, as described in the frontal cortex.

The finding that morphine by itself did not affect the release of CCK-LI in the ACC in control rats is in contrast to previous findings obtained in the spinal cord (de Araujo Lucas et al., 1998; Gustafsson et al., 2001) and RVM (Xie et al., 2005), where morphine administration has been shown to increase the release of CCK-LI. Becker and collaborators (Becker et al., 1999, 2000) have previously shown that systemic administration of morphine increases the release of CCK in the prefrontal cortex and have hypothesised that this mechanism is implicated in cortical pain-modulation. However, a more recent study from the same group found no effect of morphine in naïve control rats (Andre et al., 2005). Taken together, it appears that the release of CCK is differentially regulated by opioids in various brain regions and further studies are warranted to elucidate how opioids affect CCK release in different cortical areas. However, at present it is unclear whether CCK in the prefrontal and cingulate cortices acts to enhance or inhibit the pain experience. It is also quite possible that CCK in the cingulate and prefrontal cortex, respectively, is involved in different mechanisms of pain, as is shown in humans (Apkarian et al., 2005). In support for this assumption it may be mentioned that the CCK containing projections to these regions have different origin (Seroogy et al., 1985). Further on, at present it is unclear if CCK in the prefrontal and cingulate cortices act to enhance or inhibit the pain experience. In view of the notion that CCK may be involved in anxiety one can speculate that the observed increase of CCK-LI in the ACC during inflammation-induced pain, mediates the affective component of pain. However, CCK-LI release and tissue levels in CCK-LI in the prefrontal and frontal cortices have been shown to increase also during stress. Thus, it is possible that the enhanced CCK-LI transmission in the ACC observed in the present study during carrageenan-induced arthritis in rats may be related to stress-induced analgesia.

Interestingly, our results show that diclofenac abolished the increased release of CCK-LI in the ACC in arthritic rats. The dose of diclofenac used in the present study has previously been shown to alleviate pain-related behaviour in rat models of carrageenan-induced hind paw inflammation (Clarke et al., 1994) as well as osteoarthritis (Fernihough et al., 2004). The analgesic effect of NSAIDs is mediated by inhibition of prostaglandin (PG) synthesis through inhibition of cyclo-oxygenase (COX) enzymes. During peripheral inflammation, e.g. induced by carrageenan injection in animals, both COX-2 and PG levels are increased in CNS regions such as the spinal cord and the cerebral cortex (Samad et al., 2001; Schaible et al., 2002). Furthermore, binding sites for PGs have been demonstrated throughout the rat CNS including the cingulate cortex (Matsumura et al., 1992). There is increasing evidence that there are both peripheral and central mechanisms behind the analgesic effects of NSAIDs (for rev. see Burian and Geisslinger, 2005). For instance, intrathecally administrated NSAIDs which inhibit COX-2 in the CNS, have antihyperalgesic effects in models of peripheral inflammation (Dirig et al., 1998). Hence, the increase in CCK-LI release in the ACC in arthritic rats observed in the present study could be mediated by PGs, either directly in the ACC or indirectly through alterations in neuronal input to the ACC, an effect that was blocked by diclofenac, possibly through central inhibition of COX (as illustrated in figure 8).

General Discussion

39

In conclusion, while the findings with diclofenac are in line with a role for CCK in the ACC in the sensation of acute pain, the results obtained with morphine do not support such a role. Our observations suggest that the increased CCK-LI release in the ACC during peripheral inflammation may be mediated by an elevation in central prostaglandin levels, although this remains to be elucidated. Further studies on the interaction between CCK and prostaglandins in the brain may provide a future basis for the treatment of inflammatory pain in humans.

Figure 8. Illustration of a possible central mechanism for the increase cholecystokinin (CCK) release in the anteriorcingulate cortex (ACC) during peripheral inflammation,mediated by prostaglandin E2 (PGE2). CCK counteracts theopioid-mediated analgesia via the CCK2 receptor.

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Conclusions

41

6 CONLUSIONS

I. Our data on experimentally induced arthritis in rats, do not support a pre-emptive

analgesic effect of morphine compared to treatment after the onset of inflammation.

II. Adjuvant-induced monoarthritis, which has a limited effect on the general state of

health of the animals, constitutes a robust and reproducible model in mice. This model

could thus be a useful tool in patophysiological and pharmacological studies on

inflammatory pain. However, the choice of mouse strain is of importance for the

results obtained.

III. Both basal and evoked release of CCK-LI is increased in the ACC during peripheral

inflammation-induced pain. This effect was blocked by an NSAID, but not by morphine,

suggesting that this increase in CCK is mediated by an elevation in central

prostaglandin levels, although this remains to be elucidated. Further studies on the

interaction between CCK and prostaglandins in the brain may provide a future basis

for the treatment of inflammatory pain.

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7 SVENSK SAMMANFATTNING Smärta hör till de viktigaste funktionerna i nervsystemet. Syftet med smärta är att

förse oss med information om förekomsten av eller risken för skada. Ur detta perspektiv är smärta en nödvändig förutsättning för liv. Många människor är dock Drabbade av kronisk, handikappande smärta. Vanliga orsaker till sådan smärta är bl.a. ledsjukdomar som artros och reumatoid artrit. Den symptomatiska behandlingen av kronisk smärta är ännu otillräcklig. För att utveckla nya anti-inflammatoriska och smärtstillande läkemedel, är studier av de grundläggande mekanismerna bakom kronisk inflammatorisk smärta av mycket stor betydelse. I detta sammanhang behövs adekvata djurmodeller. I denna avhandling har vi i prekliniska djurmodeller studerat neurokemiska föränDringar i hjärnan vid långvarig inflammatorisk smärta. Vi har även utvecklat en djurmodell för studier av långvarig inflammatorisk smärta. Dessutom har vi använt oss av en djurmodell för att undersöka en farmakologisk strategi som hos människa har föreslagits kunna förebygga uppkomsten av kronisk smärta.

Kliniska studier har antytt att behandling med morfin före kirurgiska ingrepp skulle kunna leda till mindre smärta postoperativt. Vi studerade därför på råtta den analgetiska effekten av morfin administrerat före respektive efter induktion av ledinflammation (artrit). Det smärtrelaterade beteendet mättes och korrelerades till plasmakoncentrationen av morfin och dess metabolit, morfin-3-glukuronid. Morfin givet efter artrit-induktion hade som förväntat en hämmande effekt på det smärtrelaterade beteendet och en viss effekt kunde även ses av morfin givet före inflammation. Vi kunde däremot inte påvisa någon smärtförebyggande effekt av morfin givet före jämfört med morfin givet efter inflammation.

I den alltmer omfattande forskningen på så kallade transgena, d.v.s. genmodifierade djur, används huvudsakligen möss som försöksdjur. Vi ville därför utveckla en användbar modell för kronisk inflammatorisk smärta på mus. Vi framkallade inflammation i en led (artrit) och visade att denna modell svarar dosberoende på konventionell smärtlindrande och antiinflammatorisk behandling. Vi bedömer att metoden kommer att vara användbar vid prövning av den analgetiska effekten hos nya smärtlindrande och antiinflammatoriska kandidatsubstanser inom läkemedelsutveckling.

Cortex cinguli är ett område i hjärnan som hos människa är viktigt för bl.a. den känslorelaterade upplevelsen av smärta. Neuropeptiden cholecystokinin (CCK) tros spela en viktig roll vid signalering av smärta i centrala nervsystemet. Det har i andra regioner visats att både smärta och morfin kan frisätta CCK. Vi studerade frisättningen av CCK i cortex cinguli hos råtta i samband med artrit. Vi fann att CCK-frisättningen hos råtta är förhöjd vid artrit och att ökningen korrelerar väl tidsmässigt med förändring av det smärtrelaterade beteendet. Vi ville därefter studera om den artrit-orsakade CCK-ökningen i cortex cinguli kunde påverkas av morfin eller det antiinflammatoriska medlet diclofenac. Dessutom ville vi studera om morfin påverkar den basala frisättningen av CCK i cortex cinguli, eftersom det tidigare har visats vara fallet i andra regioner i nervsystemet. Våra data visade ingen påverkan av morfin varken på den artrit-orsakade eller normala frisättningen av CCK. Diclofenac däremot motverkade den ökade CCK-frisättningen i cortex cinguli. Detta skulle kunna tyda på att CCK-frisättningen påverkas av prostaglandiner i perifera eller centrala nervsystemet. Fortsatta studier av mekanismerna runt CCK skulle kunna utgöra en grund för utvecklandet av framtida smärtlindrande eller anti-inflammatoriska läkemedel.

Acknowledgements

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8 ACKNOWLEDGEMENTS Many people have contributed in different ways to this thesis and I wish to express my sincerest gratitude to all of them. In particular, I would like to thank

Professor Ernst Brodin, my supervisor, for introducing me to the fundamentals of Neuroscience and Pharmacology. For his extensive knowledge in pain research, his methodological expertise and careful scrutinisation of data. For encouraging my interests in teaching, emergency medicine and children, although they made the thesis take forever. For his generous and caring personality and for never seizing to believe in me, even when I did so myself.

Docent Lotta Arborelius, my co-supervisor, who joined in the later phase of my thesis work and provided the necessary constructive attitude for tying all the loose ends together into papers. For thorough analysis of results, meticulous correction of texts and for asking all the seemingly simple, but crucial questions. For always being a kind and considerate person.

My co-authors Professor Odd-Geir Berge for providing the opportunity to carry out the mouse experiments at AstraZeneca, for his genuine interest in behavioural pain research as well as scepticism to most methods used within this field, and for his kind personality. Docent Olof Beck for a valuable and interesting discussion on morphine kinetics. BSc Benjamin Rost for his brief but inspiring and productive visit as a guest student. For providing intelligent questions, inventive ideas and great breakfast pancakes. Dr Eva Schött, for teaching me to perform behavioural experiments, for helping me to overcome my big fear of small animals and for sharing good and bad times during the first years in the lab.

BSc Annika Olsson, For limiting all the damage I might have caused in the wet-lab by letting me label the tubes while she performed the RIAs included in this thesis.

Dr Marie Linder, AstraZeneca for valuable support with Statistical analysis.

BSc Anne Petrén, AstraZeneca for helpful assistance with behavioural experiments on mice.

Professor Leif Olgart for his broad knowledge in pain, inflammation and Pharmacology. For sharing my interest in teaching as well as in computer-aided illustrations. For his straight-forward advice and kind encouragements.

Dr Patricia Hedenquist, for her extensive competence within laboratory animal science. For prompt answers to any questions and helpful solutions to any problems. For being an excellent work mate making a day at work much less boring.

Docent Carl-Olav Stiller for introducing me to the group and teaching the microdialysis technique. For useful advice provided along with unbeatable German proverbs.

Present and former members of the Brodin group: Dr Henrik Gustafsson, Dr Abdullah Afrah, and Beata Bytner for good company and cooperation during the years.

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Mss Margareta Westling, Renée Andersson and Monica Pace-Sjöberg for invaluable help with educational, computational and administrative challenges, respectively, as well as for good humour and dashing style.

Present and former PhD-colleagues at the department, for being great colleagues and friends, on and off work, in particular Dr Sophie Erhardt, Dr Robert Frithiof, Sabina de Villiers, Dr Björn Schilström, Lotta Wiker and Dr Gunnar Schulte.

Mr Jan Gustavsson, Ms Eva-Britt Näslund, Mr Hans Svensson and Ms Marja Toumella-Lind for visible and invisible, appreciated service over the years.

Present and former heads of the department of Physiology and Pharmacology, Professors Stefan Ericsson, Bertil Fredholm and Peter Thorén for providing excellent facilities and a most stimulating environment for doctoral studies.

Drs Gülnur Müren, and Ender and also Nursel and and Gülay Ünüvar for early encouragement to scientific thinking as well as for being my stand-in relatives in Sweden.

Drs Linda Halldner Henriksson, Ulrika Ådén, Jill Thyboll, Ebba Bråkenhjielm and Hélène Schulte for private and professional friendship as well as superb dinners.

- Janet Holmén, Monica Marcus, Pernilla Videhult and Guilia Arslan for collegial, cultural and culinary company.

Drs Ann-Sofie Backman, Maria Elmberg, Ulrik Lindström, Ingrid Rydmark and Pia Tengnér, for sharing my years in medical school and, together with their families, being friends for life. Dr Steve Applequist and Henrik Holm for heroic although fruitless attempts to teach me how to use a racket and a car as well as for sharing thoughts on science, inventions and life.

Felicia Buijs and Karin Larsson, for always being there.

My in-law family for being my new family, special thanks to my mother-in-law Hedvig Svedlund for priceless baby-assistance during the thesis work.

My mother for giving me an early interest in reading and most of all, for infinite care and love.

Johan, supportive friend, enthusiastic colleague and loving husband, all in one! Thank you for making me finish this thesis, for making it possible and most of all, for making it worth it, because I will celebrate it with you for life!

Isabel and Emelie, my beautiful little flowers, for every smile!

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