Nociceptin/Orphanin FQ Is the Endogenous Ligand of the Orphan Opioid-Receptor-Like Receptor (ORL1)

From the Department of Laboratory Medicine, Division of Clinical Neurophysiology, Karolinska Institutet, Stockholm, Sweden

Neuropeptides and spinal antinociception: studies on galanin, nociceptin and endomorphin

by Stefan Grass

Stockholm 2003

About doing research and becoming an expert:

“An expert is one who knows more and more about less and less until he knows absolutely everything about nothing.”

(Murphy's Law of Super Speciality).

Abstract

Nociception and/or the sensation of pain is essential for the survival of the organism. Just as important are the antinociceptive mechanisms that reduce the nociceptive input and perceived pain. Without such mechanisms, it would be impossible to turn one’s focus away from even the most minor painful stimulus. The balance between excitatory and inhibitory functions in nociception is intricate and complex and some of the modulators involved may be important for future drug development for treating pain states. This work has tried to highlight some of the neuropeptides involved in the endogenous antinociceptive mechanism. Endomorphin-1 and endomorphin-2 are peptides that have high selectivity and affinity to the µ-opioid receptor, the receptor mediating the effect morphine. Intrathecal (i.t.) application of endomorphin-1 and –2 produced a dose-dependent short lasting depression of the spinal nociceptive flexor reflex in normal rats with comparable potency. The inhibitory effect of endomorphin-1 was significantly longer than that of endomorphin-2, suggesting a possible difference in enzymatic degradation. The reflex depressive effect of endomorphin-2 was maintained in rats after inflammation and in axotomized rats that did not exhibit neuropathic pain-like behavior (autotomy). However, in rats exhibiting autotomy behavior after nerve section, the effect of endomorphin-2 was significantly reduced. These results suggest that the presence of neuropathic pain decreases the antinociceptive potency of endomorphin-2, which is similar to what is observed with morphine. Nociceptin/orphanin FQ is the endogenous ligand of the orphan opioid-receptor-like receptor (ORL1). We now verified that the nociceptin analogue [Nphe1]nociceptin(1-13)NH2 is a selective antagonist of nociceptin in rat spinal cord. We have also systematically evaluated the effect of i.t. nociceptin on the flexor reflex in normal, inflamed and nerve injured rats. It was observed that nociceptin produced comparable depression of the flexor reflex in normal and inflamed rats and the effect of nociceptin was not reduced and if anything increased in rats with nerve injury. The results showed that nociceptin agonists may be useful analgesics in neuropathic pain. Galanin is a well documented inhibitory peptide in nociception. The work presented in this thesis provides further evidence for an inhibitory role of this peptide after inflammation. Moreover, we have developed a protocol to study flexor reflex in mice and studies on mice over-expressing galanin suggested that increased level of galanin is effective in reduced spinal hyperexcitability after repetitive C-fiber stimulation. However, we did not obtain evidence that galanin GAL-R1 receptor is tonically involved in inhibiting nociceptive input under normal condition in studies on mice lacking the GAL-R1 receptor.

Key words: endomorphin, flexor reflex, galanin, knock-out mice, neuropathic pain, nociceptin, orphanin FQ, transgenic mice

List of papers

The thesis is based on the following list of paper and will be referred in the text according to their roman numerals:

I. Xu IS., Grass S., Wiesenfeld-Hallin Z., Xu XJ. (1999). “Effects of intrathecal orphanin FQ on a flexor reflex in the rat after inflammation or peripheral nerve section.” European Journal of Pharmacology 370: 17-22.

II. Xu IS., Grass S., Calo G., Guerini R., Wiesenfeld-Hallin, Z., Xu XJ. (2002). “Intrathecal [Nphe1]nociceptin(1-13)NH2 selectively reduces the spinal inhibitory effect of nociceptin.” Life Sciences 70: 1151-1157.

III. Grass S., Wiesenfeld-Hallin Z., Xu XJ. (2000). “The effect of intrathecal endomorphin-2 on the flexor reflex in normal, inflamed and axotomized rats: reduced effect in rats with autotomy.” Neuroscience 98: 339-344.

IV. Grass S., Xu, IS., Wiesenfeld-Hallin Z., Xu XJ. (2002). “Comparison of the effect of intrathecal endomorphin-1 and endomorphin-2 on spinal cord excitability in rats.” Neuroscience Letters 324: 197-200.

V. Xu IS., Grass S., Xu XJ., Wiesenfeld-Hallin Z. (1998). “On the role of galanin in mediating spinal flexor reflex excitability in inflammation.” Neuroscience 85: 827-835.

VI. Grass S., Crawley JN., Xu XJ., Wiesenfeld-Hallin Z. (2003). “Reduced spinal cord sensitization to C-fiber stimulation in mice over-expressing galanin.” European J of Neuroscience (in press).

VII. Grass S., Jacoby AS., Ismaa TP., Crawley JN., Xu XJ., Wiesenfeld-Hallin Z. (2003) “Flexor reflex excitability in mice lacking galanin receptor GAL-R1.” Neuroscience Letters (in press).

Contents

Abbreviations 10

Introduction 11

Background...... 11 The concept of pain...... 11 Pain pathway...... 12 The nociceptive flexor reflex...... 13 Wind-up and central sensitization...... 13 Peptide transmission ...... 13 Peptides, nerve injury and inflammation ...... 14 Nociceptin/orphanin FQ ...... 15 Galanin...... 16 Galanin receptors ...... 16 Function of galanin during hyperexcitability...... 17 Endomorphin-1 and -2...... 18 Pharmacolgoy...... 18 Distribution ...... 18 Function...... 18 Animals studies of pain...... 19 Behavioral studies ...... 19 Physiological studies...... 19 Other methods ...... 19 Genetically modified mice ...... 20 Aims of the study 21

Materials and methods 22 Animals...... 22 Rats...... 22 Mice...... 22 Electrophysiology ...... 23 Rats...... 23 Mice...... 23 Wind-up ...... 24 Nerve injury ...... 24 Inflammation...... 25 Peptides...... 25 Statistics...... 25

8 Contents

Results 26 Study I...... 26 Summary ...... 26 Normal rats...... 26 Inflamed rats...... 26 Axotomized animals...... 26 Study II ...... 26 Study III ...... 27 Summary ...... 27 Normal rats...... 27 Inflamed rats...... 27 Axotomized rats ...... 27 Study IV...... 28 Study V ...... 28 Summary ...... 28 Baseline flexor reflex ...... 28 Wind-up and post-CS facilitation...... 28 Intrathecal galanin...... 29 Intrathecal M-35...... 29 Study VI...... 29 Study VII...... 30 Discussion 31 Nociceptin...... 31 Biphasic effect of nociceptin in the spinal cord...... 31 Effects of nociceptin in inflamed and nerve injured rats ...... 32 Nociceptin antagonist...... 32 Application of nociceptin-related compounds as analgesics ...... 33 Endomorphins...... 34 Effects of i.t. endomorphin-2 in normal, inflamed and nerve injured rats...... 34 Comparing endomorphin-1 and -2...... 35 Galanin...... 36 Role of galanin during inflammation ...... 36 Galanin over-expressing mice...... 37 GAL-R1 receptor knock-out mice ...... 37 Conclusions 39

Populärvetenskaplig sammanfattning 40

Acknowledgements 43

References 45

9 Abbreviations

Abbreviations cAMP Cyclic adenosine mono-phosphate ANOVA Analysis of variance CCK Cholecystokinin cDNA Complementary DNA CGRP Calcitonin gene-related peptide CNS Central nervous system CS Conditioning stimulus DBH Dopamine β-hydroxylase DRG Dorsal root ganglion EMG Electromyogram ES Embryonic stem cell GAL Galanin GAL-OE Galanin over-expressing IASP International association for the study of pain I.c.v. Intracerebroventricular I.t. Intrathecal LI Like-immunoreactivity M-35 Galanin antagonist, galanin (1-12)-pro-bradykinin-(2-9) NK-A and -B Neurokinin A and B NMDA N-metyl-D-aspartate NPY Neuropeptide Y PKC Protein kinase C PLC Phospholipase C ORL1 Opoid receptor-like (also known as OP4, the fourth opioid receptor) SP Substance P VIP Vasoactive intestinal peptide

10 Introduction

Introduction

Background The word “pain” originates from the Old French word “peine”, from the latin word “poena” which means “punishment”, “penalty”. In Greek and Roman mythology Poena was the goddess of punishment. Pain states, particularly those of chronic nature have become a major medical, social and economical problem in modern society. This can be attributed, to several factors, such as improved treatment of acute diseases, increased life expectancy of chronically ill patients, aging of the population and changes in the attitude of patients and doctors towards pain. Apart from leading to tremendous personal suffering for the individual pain patient, pain also has enormous societal costs. In a British study a questionnaire was sent to some 5000 patients. 50% of the respondents reported having chronic pain, defined as "pain or discomfort, that persisted continuously or intermittently for longer than 3 months" (Elliott et al. 1999). Similar numbers were obtained in a Swedish study in a rural area, where 55% of the respondents had perceived pain for 3 months and 49% for 6 months (Andersson 1994). Estimation of health care costs attributed to pain treatment and loss of work days are somewhat difficult due to the heterogeneity of diagnoses within the pain group. In UK, there may be 2.5 million people suffering from back pain only, at a societal cost of £6600 million to £12300 million for the year 1998 (Maniadakis and Gray 2000). A recent report from Sweden estimates that societal costs for back pain are three times higher than the total cost of all types of cancer (Jonsson 2000). The major reason for the huge economic burden on society resulting from chronic pain is loss of working days (Phillips 2001). Although being a major health problem, the resources allocated to pain research and treatment have been comparatively low. The scientific basis of pain physiology and pathology is however increasing through basic and clinical research, and there is a growing interest in dealing with pain associated problems in society. This introduction will give a brief pre- sentation of the basic knowledge in pain physiology, and then turn focus toward the spinal nociceptive mechanisms and endogenous pain-suppressing peptide systems, the primary subjects of this work.

The concept of pain We have all experienced pain, and it is easy to say whether we have pain or not. But how do we describe to concept of pain? In ancient times pain was thought to be related to demons, intrusion of objects into the body or magic fluids affecting us. Often it was associated with sin. During the Greek civilization great philosophers tried to explain the concept of pain,

11 Introduction

rather than just dismissing it as supernatural. Plato described pain as the opposition of pleasure, tied together “as having origin from the same head, while they are two” (Plato 360 B.C.). Aristotle introduced a concept of pain that in different forms was the fundament of teaching for nearly twenty centuries. In his work “De Anima” he identifies five senses: vision, hearing, taste, smell and touch, and describes pain as an increased sensitivity to touch (Aristotle 350 B.C.). The Roman physician Galen introduced some amazingly modern theories regarding the physiology of pain, primarily based on his anatomical studies. He concluded that the brain is the center of all sensation and that the nerves contain sensations. Perhaps the beginning of modern anatomy and physiology of pain was put forward by Leondardo Da Vinci. According to Da Vinci pain passes through the tubular, “perforated”, nerves to be transmitted to “sensorium commune”, located in the third ventricle. A modern definition of pain by the International Association for the Study of Pain (IASP) states that pain is “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (Merskey and Bogduk 1994).

Pain pathway In the skin, noxious stimuli are detected by nociceptors, that essentially are free nerve HQGLQJV 7KHUH DUHW ZR PDLQFDWHJRULHVRIQR FLFHSWRUVWKH$ /PHFKDQLFDODQGWKH& -polymodal. The C-polymodal nociceptor transfers its signal through slowly conducting, unmyelinated, C-fibers and gives rise to a delayed dull or burning pain (Ochoa and Torebjörk 1989). The C-polymodal nociceptors respond to noxious mechanical, thermal and chemical stimuli (Bessou and Perl 1969). 7KH$/PHFKDQLFDOQRFLFHSWRUVJLYHULVHWRVKDUSSULFking pain. They are best excited by mechanical stimuli that damage the skin and the signal is propagated in thin myelinated fibers with higher conduction velocity than the C-fibers. Their discharges are of higher frequencies and therefore deliver a more discriminative sensation to the CNS (Willis and Coggeshall 1991; Hansson 1997; Raja et al. 1999). After peripheral detection of the noxious input by the nociceptors, the signal is transferred through the primary afferents to the dorsal horn RIW KHV SLQDOF RUG 7KH$ /DQG &-fibers terminate predominantly in outer layers of the dorsal horn (Rexed’s laminae I and II) (Besson and Chaouch 1987). In the dorsal horn the primary afferent input is relayed to ascending projecting neurons, transporting the signal to supraspinal (e.g. primary sensory cortex), primarily via the spinothalamic tract (Besson and Chaouch 1987; Willis and Coggeshall 1991; Willis et al. 1995). The primary afferent also synapses to dorsal horn interneurons. Dorsal horn neurons that respond to peripheral stimulation are classified into three types: low threshold (LT) neurons, activated by innocuous (non-painful) stimuli, high threshold (HT) neurons responding exclusively to noxious stimuli, and wide dynamic range (WDR) neurons responding in a graded fashion to innocuous and noxious stimuli (Cervero et al. 1976).

12 Introduction

The nociceptive flexor reflex The interneurons that receive and modulate nociceptive signals are also involved in eliciting a withdrawal reflex (Doubell et al. 1999). Such reflexes occurring at the spinal cord level are important to protect the organism from extensive tissue damage. The flexor reflex induces withdrawal of a limb that is exposed to a noxious stimulus, and the reflex works through contraction of flexor muscles and relaxation of extensor muscles (Gordon 1991). The flexor reflex response to noxious stimuli has been extensively used in the studies of spinal nociceptive mechanisms both in man and animals (Schouenborg and Sjölund 1983; Endo et al. 1984; Wall and Woolf 1984; Cook and Woolf 1985). The strength of the withdrawal response is positively correlated to the response in the dorsal horn WDR neurons (Schouenborg and Sjölund 1983). In humans the magnitude of the flexor reflex response is also directly correlated with the degree of perceived pain (Willer 1977; Chan and Dallaire 1989). Thus the flexor reflex provides an easy way of measuring and quantifying the activity of dorsal horn WDR-neurons and level of pain. Studies on the flexor reflex have lead to important advances of our understanding of pain mechanisms, and the identification of activity-dependent increase in spinal cord excitability after activation of C-fibers (wind-up and central sensitization) (Wall and Woolf 1984).

Wind-up and central sensitization When C-fibers are repetitively stimulated, the WDR neurons gradually increase their response, a phenomenon known as “wind-up” (Mendell 1966). Such repetitive conditioning stimulation (CS) of C-fibers may also induce a state of spinal hyperexcitability following the wind-up. During this state the receptive fields of WDR neurons expand and these neurons increase their responsiveness and the flexor reflex is facilitated (Woolf 1996; Baranauskas and Nistri 1998). The duration of the hyperexcitability may last from minutes to hours, depending on the stimulus parameters, types of afferents activated and preparations used (Wall and Woolf 1984). Several mechanisms underlying the generation of wind-up have been proposed, including activation of NMDA-recptors, involvement of tachykinins and build up of Ca2+ in the presynaptic terminal (Woolf 1996; Herrero et al. 2000). The activity-dependent increase in spinal hyperexcitability after repetitive C-fiber stimulation is similar to that occurring after tissue injury and inflammation (Woolf 1996).

Peptide transmission During the 1970’s there was enormous intensity in peptide research. Many peptides were found to be expressed throughout important areas of the CNS and often co-exist with each other, and/or classical transmitters in the same neuron. Evidence for many peptides as neuro- transmitters and/or neuromodulators was presented. Peptidergic transmission was found to be different from that of classical transmitter in several aspects, including plasticity in expression

13 Introduction

under pathological conditions, release characteristics, involvement in more slow form of synaptic transmission and more of a modulatory role (Hökfelt 1991). Peptides have important roles in pain transmission and modulation. One of the best studied peptide is substance P (SP) in this regard. Almost 20 years after the discovery that substance P is present in the dorsal horn and dorsal roots (Lembeck 1953), the sequence of SP was found to be an 11 amino acid peptide (Chang et al. 1971). SP is expressed in a population of dorsal root ganglion (DRG) cells with small somata (Hökfelt et al. 1975), and has been shown to have a role in nociception, particularly in the activity-dependent increase in spinal cord excitability after intense activation of the C-fibers as well as under inflammatory conditions. In addition, a number of other peptides have also been found to be present in the DRG and dorsal horn, including CGRP (Wiesenfeld-Hallin et al. 1984) neurokinin A and B (Dalsgaard et al. 1985; Too and Maggio 1991), vasoactive intestinal peptide (VIP) (Gibson et al. 1981) and galanin (Ch'ng et al. 1985; Skofitsch and Jacobowitz 1985). Opioid peptides such as enkephalins and dynorphin are also found in the spinal cord (Hughes et al. 1975; Hökfelt et al. 1977).

Peptides, nerve injury and inflammation As described above, one of the characteristics of peptides is that they often exhibit plasticity in expression under pathological states, including in the somatosensory system. Thus, it was observed early on that the level of SP in the dorsal horn was markedly decreased after peripheral nerve transection (Jessell et al. 1979). The same is true for several other peptides, such as CGRP and somatostatin (Villar et al. 1991; Hökfelt et al. 1994). In contrast, the expression of VIP, galanin, neuropeptide Y (NPY) and cholecystokinin (CCK) was up- regulated after nerve injury (McGregor et al. 1984). Apart from acting as a trophic factor, VIP may also take over the role of SP as an excitatory mediator in nociception at the spinal level (Wiesenfeld-Hallin et al. 1990). CCK is another peptide involved in modulating nociceptive input. After nerve injury CCK and its receptor are up-regulated in the DRG (Verge et al. 1993; Zhang et al. 1993a). CCK has well established anti-opioid activity and may be important in determining sensitivity to opioid analgesia after nerve injury (Wiesenfeld-Hallin and Xu 2001). The up-regulation of peptides after nerve injury also involves inhibitory peptides. NPY and galanin are normally present in the superficial dorsal horn (Gibson et al. 1984; Merchenthaler et al. 1993), but not in DRG neurons in significant amounts. After nerve injury there is a large increase in the synthesis of NPY and galanin in DRG neurons (Hökfelt et al. 1987; Wakisaka et al. 1991; Zhang et al. 1993b). Just as VIP, NPY and galanin have been shown to have trophic effects. But more importantly, NPY and galanin may also serve to inhibit excessive nociceptive input after nerve injury (see below). During inflammation there is also a marked plasticity in different peptide systems in the dorsal horn and DRG. Many of the changes in these systems, however, differ from what is

14 Introduction

observed after nerve injury. Peptide expression in the DRG is often only slightly changed or unchanged, which is in contrast to the sometimes dramatic alterations in peptide expression seen after nerve injury. The changes in peptide and peptide receptor expression after inflammation usually occur in the dorsal horn (Hökfelt et al. 1997). A hypothesis proposed is that there are two separate pain-defense mechanisms: one in the dorsal horn reducing inflammatory pain, and one in the DRG neuron that fights neuropathic pain (Hökfelt et al. 1997). One of the noteworthy changes in peptide expression seen after inflammation is the marked increase in the expression of the endogenous opioids dynorphin and enkephalin in the dorsal horn (Weihe et al. 1994). Also the levels of excitatory tachykinins, such as NK-A and SP is increased during inflammation. Mice lacking the receptor for SP, NK-1 (neurokinin-1), have reduced inflammatory reaction after capsaicin as well as reduced spinal hyperexcitability (De Felipe et al. 1998). NPY as well as one of its receptor, Y1, is also up-regulated during inflammation (Ji et al. 1994). The functional importance of this is not entirely clear. Mice lacking the Y1-receptor exhibited hyperalgesia to acute cutaneous and visceral nociceptive input. Moreover, neuropathic pain was found to be increased in these mice (Naveilhan et al. 2001). Thus, NPY by acting on the Y1 receptor may play an inhibitory role during inflammation.

Nociceptin/orphanin FQ

The are three previously identified opioid receptors µ, δ and κ (Kitchen 1999). However, in 1994 in the process of cloning the classical opioid receptor a new receptor, with extensive homology with the other opioid receptors, was cloned from a cDNA library (Mollereau et al. 1994). The receptor was named ORL1 (opioid receptor like). Opioid peptides had very low affinity to this receptor and not until a year later, two groups independently published their finding of the identification of a peptide which binds with high affinity and selectivity to ORL1. The agonist, which appeared to be a naturally occurring peptide, was called nociceptin or orphanin FQ (Meunier et al. 1995; Reinscheid et al. 1995). The latter name, although used more frequently in recent literature, is somewhat misleading since the peptide causes primarily antinociception, rather than increase in pain as initially thought (see below). Nociceptin shares some sequence homology with opioid peptides, particularly dynorphin, but it does not interact with classical opioid receptors in a significant way. Both ORL1 and nociceptin are found throughout the CNS, in areas associated with neuroendocrine secretion, emotions, memory, anxiety, reward, feeding and pain (Taylor and Dickenson 1998; Neal et al. 1999a; Neal et al. 1999b; Harrison and Grandy 2000). Activation of the ORL1 receptor by nociceptin induces intracellular changes similar to those that occur after activation of other opioids, such as inhibition of adenylate cyclase, neuronal hyperpolarization via potassium currents and effects on calcium channels (Henderson and

15 Introduction

McKnight 1997; Yamamoto et al. 1999). As other opioid receptors the ORL1 receptor is G- protein coupled (Reinscheid et al. 1995). The initial studies on nociceptin showed that it induced hyperalgesia, after intracerebroventricular (i.c.v.) injection, while producing little effect in the spinal cord (Meunier et al. 1995; Reinscheid et al. 1995). Subsequent studies have, however, revealed a more complex picture both for supraspinal and spinal effects of nociceptin. It is generally believed today that nociceptin produces antinociceptive effects at the spinal level (Xu et al. 2000a). In most studies, opioid receptor antagonists at normal doses did not reverse the antinociceptive effect of nociceptin, suggesting an independence from classical opioid pathways. The physiological role of nociceptin is largely unknown, partly because of lack of selective receptor antagonists. Moreover, the effect and function of nociceptin in pathological pain states is also not clear, although some plasticity in the expression of nociceptin or its receptors has been noted after nerve injury or inflammation. Functional studies have shown that intrathecal nociceptin is antinociceptive during inflammation and after nerve injury (Yamamoto et al. 1997a, b; Hao et al. 1998).

Galanin Galanin is a peptide that originally was isolated from the porcine intestine (Tatemoto et al. 1983). It has 29 amino acids (30 in humans) and has been found in several species with considerable homology. The peptide has been found in many parts of the CNS and proposed to play a role in different physiological systems, such as feeding, endocrine modulation, cognition and nociception (Wiesenfeld-Hallin et al. 1992a; Bartfai et al. 1993; Merchenthaler et al. 1993; Crawley 1996). Since galanin is expressed in sensory neurons and the spinal cord dorsal horn, soon after its discovery it was hypothesized that it also may be involved in nociception. In behavioral models intrathecal administration of galanin induced moderate antinociceptive effects (Cridland and Henry 1988; Post et al. 1988; Wiesenfeld-Hallin et al. 1993), although hyperalgesia has also been reported (Kuraishi et al. 1991). Extensive studies using the nociceptive flexor reflex model in rats have revealed that galanin possesses a complex bi-phasic effect, with facilitation at low doses and inhibition at high doses (Wiesenfeld-Hallin et al. 1988, 1989a; Xu et al. 2000b).

Galanin receptors Three subtypes of galanin receptors have been cloned, GAL-R1, GAL-R2 and GAL-R3 (Habert-Ortoli et al. 1994; Smith et al. 1997; Wang et al. 1997)). They are all G-protein coupled, but activate different signal transduction systems. GAL-R1 and GAL-R3 produce inhibition by induction of hyperpolarization via Gi/o, and inhibit adenylate cyclase, which leads to decreased levels of c-AMP. GAL-R2 is excitatory and via Gq/11 stimulates phospholipase C (PLC), leading to increased levels of intracellular calcium and activation of protein kinase C (PKC) (Branchek et al. 2000; Liu and Hökfelt 2002). In functional studies, the inhibitory effect of galanin on C-fiber stimulation-induced spinal hyperexcitability was

16 Introduction

abolished in rats treated with GAL-R1 antisense probe, indicating that this receptor is important for the inhibitory effect of galanin in normal rats (Pooga et al. 1998). Recently, newly developed GAL-R1 and GAL-R2 receptor agonists were used in normal and rats with neuropathy. A combined GAL-R1 and GAL-R2 agonist reduced allodynia in rats with peripheral nerve injury, whereas a selective agonist of GAL-R2 receptor had no effect (Liu et al. 2001). This indicates again that the GAL-R1 receptor is important in mediating the inhibitory effect of galanin during neuropathy. The inhibitory role for GAL-R1 receptor under normal conditions and after nerve injury is also supported by recent studies on GAL-R1 knock-out mice. Thus, Hygge Blakeman et al. have reported that mice lacking the gene for GAL-R1 receptor had significant hyperalgesia to noxious heat stimulation and also exhibited increase neuropathic pain-like behavior after partial sciatic nerve injury (Hygge Blakeman et al. 2003).

Function of galanin during hyperexcitability As mentioned above, galanin exhibits considerable degree of plasticity in response to nerve injury. The expression of galanin in DRG neurons is markedly up-regulated after peripheral nerve injury (Hökfelt et al. 1987; Villar et al. 1989). The functional significance of this was demonstrated by Wiesenfeld-Hallin and co-workers, who found that intrathecal galanin produced stronger inhibitory effect after nerve injury (Wiesenfeld-Hallin et al. 1989b). Furthermore, injection of M-35, a galanin antagonist, induced a more profound increase in spinal cord hyperexcitability in rats with sectioned sciatic nerves than normal rats (Wiesenfeld-Hallin et al. 1992b). Thus, galanin may serve to inhibit increased nociceptive input after nerve injury. During other states of spinal hyperexcitability galanin also seem to play a role. After repetitive stimulation of C-fibers, a spinal hyperexcitability (wind-up) is seen. Galanin appears to be very effective in blocking this C-fiber-induced hyperexcitability (Wiesenfeld- Hallin et al. 1989a; Xu et al. 1990; Xu et al. 1991). After inflammation the situation seems to be more complex. Galanin expression in the DRG neurons is down-regulated after inflammation, whereas the expression in the superficial dorsal horn is up-regulated (Ji et al. 1995). GAL-R1 receptors in the DRG are transiently down-regulated and GAL-R2 receptors up-regulated (Xu et al. 1996; Shi et al. 1997). The function role of galanin under condition of inflammation is unclear.

17 Introduction

Endomorphin-1 and -2

Pharmacolgoy Endomorphin-1 and -2 are two recently discovered endogenous peptides, that are agonists at the µ-opioid receptor (Zadina et al. 1997; Goldberg et al. 1998). They both have high affinity and specificity for the µ-receptor, with affinity comparable to other known µ-opioid receptor agonists, such as DAMGO and morphine (Goldberg et al. 1998), but often with higher 35 selectivity. Binding of endomorphins to the µ-receptor stimulates [ 6@*736 ELQGLQJ LQ membrane preparation, indicating that the effect of endomorphins are linked to G-protein activation (Narita et al. 1998). There is some debate as whether the endomorphins are partial or full agonists as regarding the activation of G-protein or second messenger systems. Some of the differences observed in vitro pharmacological studies, can probably be explained by differences in tissue preparations, G-protein and µ-receptor presence in the preparations (Horvath 2000). Cellular changes induced by endomorphin binding include inhibition of calcium-currents (Mima et al. 1997), induction of outward potassium currents that lead to pronounced hyperpolarization (Hayar and Guyenet 1998) and inhibition of adenylate cyclase activity (Spetea et al. 1998; Monory et al. 2000). Endomorphin-1 and -2 activation of the receptor also triggers receptor internalization, something that is not seen with morphine (Burford et al. 1998; McConalogue et al. 1999). There is also evidence that endomorphin-1 and -2 activate different G-proteins than morphine (Burford et al. 1998).

Distribution Endomorphins are distributed throughout the CNS in areas involved in pain processing, reward, stress and autonomic functions (Martin-Schild et al. 1999). In the spinal cord dorsal horn endomorphin-like immunoreactivity (LI) is detected in the superficial laminae, co- localized with substance P and CGRP. Dorsal rhizotomy or capsaicin treatment abolished endomorphin-2-LI, indicating that endomorphin is present in the primary sensory afferents (Martin-Schild et al. 1998; Pierce et al. 1998; Martin-Schild et al. 1999). There are some differences in distribution, with a higher level of endomorphin-1 in the brain, whereas endomorphin-2 seems to be more abundant in the spinal cord (Martin-Schild et al. 1999).

Function Consistent with their high affinity for the µ-receptor, the endomorphins exhibit strong, but short-lasting, antinociceptive effects in behavioral studies (Stone et al. 1997; Zadina et al. 1997). In an electrophysiological study endomorphin-1 and -2 dose-dependently inhibited all components of electrical evoked C-fiber responses of spinal neurons (Chapman et al. 1997). The difference in their distribution has lead to some discussion regarding potential functional differences between endomorphin-1 and -2. Some authors have reported that endomorphin-1 and -2 induce antinociception with similar efficacy and potency (Stone et al. 1997; Horvath et al. 1999; Sakurada et al. 1999; Soignier et al. 2000). Others, however,

18 Introduction

suggests that endomorphin-1 is more efficacious and potent, both after intrathecal and intracerebroventricular administration (Goldberg et al. 1998; Przewlocka et al. 1999; Tseng et al. 2000). In the physiological study mentioned above, endomorphin-1 was able to block the $DQG&-fiber response of dorsal horn neurons, whereas endomorphin-2 only affected the C- fiber activity (Chapman et al. 1997). Only few studies have been published examining the effect of endomorphins during pathological states. In a neuropathic pain model endomorphin -1 and -2 displayed significant antinociceptive effects, and morphine was found to be ineffective (Przewlocka et al. 1999). During inflammatory states endomorphins reduce peripheral edema and inhibit inflammatory response (Jin et al. 1999; Khalil et al. 1999).

Animals studies of pain

Behavioral studies Behavioral testing of acute pain usually involves application of a noxious stimulus (thermal, mechanical, electrical, chemical etc.) and recording of the elicited responses. The response is often measured in threshold or in magnitude. These tests are frequently used in normal animals to examine the efficacy of analgesics. However, it has becoming increasingly clear in recent years that many clinical pain conditions involve mechanisms that are distinctly different from acute pain. Therefore, animal models of tonic or chronic pain have been developed such as after inflammation or nerve injury (Wall et al. 1979; Bennett and Xie 1988; Seltzer et al. 1990; Kim and Chung 1992; Gazelius et al. 1996). In particular, a large number of animal models of neuropathic pain are available to address different aspects of mechanisms and treatments of neuropathic pain.

Physiological studies A different approach of studying pain is to directly measure the activation in pain-related structures in the CNS by noxious stimulation. These may include, among others, recordings from peripheral nerve, spinal roots, dorsal horn neurons and neurons in the brain as well as study on various nociceptive reflexes. These studies can be performed in vivo or in vitro. The advantage with the electrophysiological methods is that it can be better controlled and quantified and are not subjected to some variability affecting behavioral measurement.

Other methods There are other techniques that are also useful in studies of pain mechanisms. Anatomical studies may reveal important neural circuits, expression patterns of important pain-related transmitter as well as structural plasticity after injury. In recent years, the ability to monitor chemical changes in vivo by different methods, such as microdialysis, have opened up possibilities of measuring transmitter release in response to noxious stimulation. Also the rapid advances in functional imaging techniques, such as fMRI and PET, have made it possible to study pain mechanisms, particularly those involving higher integration of pain processing.

19 Introduction

Genetically modified mice The remarkable progress in molecular biology has resulted in the possibility to produce new exciting potential tools of studying pain physiology and pharmacology. Thus, it is now possible to add, duplicate or remove genes from genome of animals. Mice are most commonly used mammals because of their short life span which makes breeding less time consuming. There are several types of genetically modified mice. The most commonly used are knock-out and transgenic mice. In knock-out mice a gene has been removed or made inactive. This is done by introducing a mutation in a critical part of the gene of interest. Factors are inserted that make the gene only translated when resistant to gancyklovir and neomycin. The resultant gene construct is then inserted into embryonic stem cells (ES) and some of the cells will incorporate the gene construct via homologous recombination into their genome. Only the cells which have the construct properly inserted will survive in the presence of gancyklovir and neomycin. These cells are then injected into mouse blastocysts that are inserted into pseudopregnant female mice. The offspring of this mouse will be a mix (chimera) of the original blastocyst cells and the genetically modified cells. Only mice where the integration of the gene construct is in the germ line can be used for the future breeding to create homozygous offspring. In contrast, transgenic mice have a duplicate of an existing gene or an entirely new gene added to the genome by attaching the gene to a suitable promoter. This promoter-gene construct is then injected into a fertilized mouse oozyte, which is then introduced into a pseudopregnant female mouse. Hopefully, the gene construct is integrated into the genome before cell division, and thus all of the cells in the newborn mouse will contain the gene. If the integration is correct this founder mouse will be bred to create a line of mice with the transgene. As with knock-out mice only animals were the construct gene is integrated in the germ cells will transfer the gene to next generation.

20 Aims of the study

Aims of the study

The general aim of this thesis is to study spinal nociceptive mechanisms during normal and pathological conditions. The focus will be on the role of endogenous antinociceptive peptides, nociceptin, galanin and endomorphins. In particular the aims were to:

1. Further characterize the action of the opioid-like peptide nociceptin, its antinociceptive effects in normal rats, after inflammation and nerve injury. Also, examine the potential activity of a nociceptin analogue as ORL1 receptor antagonist in the spinal cord.

2. Investigate the role endmorphin-1 and -2 as antinociceptive peptides. The effect of endomorphin-2 during different pathological state and a comparison between the two endomorphins will be studied.

3. Study the role of endogenously released, as well as exogenously applied, galanin on spinal flexor reflex excitability during inflammation.

4. Adapt and develop a flexor reflex model for mice in order to study genetically modified mice.

5. Study the role of galanin in modulation of flexor reflex excitability in galanin over- expressing (GAL-OE) mice and mice lacking the GAL-R1 receptor.

21 Materials and methods

Materials and methods

Animals All animals were housed in standard laboratory conditions, with constant room temperature and 12 h light/dark cycle. Food and water was accessible ad libitum. The experiments were carried out according to the ethical guidelines of the International Association for the Study of Pain (IASP), and all experimental protocols were approved by the local ethic committee.

Rats In the electrophysiological experiments on rats, female Sprague-Dawley rats (B&K Sollentuna, Stockholm) weighing 200-300 g were used.

Mice GAL OE A GalOE construct was produced by linking a section of the human dopamine β- hydroxylase promoter (hDBH) to the mouse galanin gene. This construct was then injected into fertilized mouse eggs (SJL x C57BL6) creating four founders. Three founder lines were established and one of the lines (1923) was backcrossed into the C57BL6/J strain for at least six generations (see (Mazarati et al. 2000) for details). This generated the GAL OE mouse strain used in the present study. Both male and female mice weighing 25-35 g, were used in the study. The animals were obtained from Jackson Laboratories (Maine, USA) where the animals were bred and genotyped. Both homozygote and heterozygote over-expressors were used. GAL-R1 knock-out The GAL-R1 knock-out mice were produced by injection of embryonic stem cells from 129Sv, carrying a mutant allele, into C57BL/6J blastocysts. Mating of the resultant chimeric mice with C57BL/6J mice, produced heterozygous (GAL-R1 +/-) mice. This heterozygous progeny was then backcrossed to C57BL/6J to generate heterozygous mice for mating to produce GAL-R1 knock-outs (GAL-R1 -/-) and wild type (GAL-R1 +/+) littermates for control (see (Jacoby et al. 2002) for detailed description). The animals weighed 25-35 g and were all bred and genotyped at Jackson Laboratories (Maine, USA).

22 Materials and methods

Electrophysiology

Rats On the day of experiment, the rat was briefly anaesthetized with an intraperitoneal (i.p.)  injection of methohexital (Brietal , Lilly, Indianapolis, USA). A tracheal cannula was inserted and the animal was artificially ventilated with room air. It was then decerebrated by aspiration of the forebrain and midbrain. The thoracic spinal cord was exposed by a laminectomy and transected at level Th 8-9. After a small incision in the dura mater, a catheter (PE 10) was inserted i.t. distal to the transection, with its the tip aimed at spinal segment L4-L5. Between spinalization and start of experiment, the animal was allowed to recover for at least 1 hour. A single electrical shock (10 mA, 0.5 ms) at 1/min was applied to the sural innervation area of the hindpaw or the exposed sural nerve (after nerve injury or inflammation, see below). The elicited nociceptive flexor reflex was recorded as electromyographic (EMG) activity through a pair of stainless steel needle electrodes from the ipsilateral biceps femoris/semitendinosus (hamstring) muscles. The EMG was amplified, filtered (Neurolog, 0.1 Hz, 5 KHz bandwidth) and fed into a spike trigger. The signal was integrated over two separate channels, a 1 s (continuous recording) and a 2 s (triggered by the stimulus eliciting the reflex) integration and recorded on a chart recorder. The heart rate was monitored and rectal temperature was maintained around 37ºC with a thermostatic heating pad. A stable baseline of responses was established for at least 20-30 minutes before application of conditioning stimulation (CS) or i.t. drug application. The CS consisted of a train of 20 electric shocks at 1 Hz, identical in intensity to those applied to elicit the reflex 1/min. The change in reflex magnitude to each stimulus in the CS train (wind-up), as well as following termination of the train (“central sensitization”), was quantified (see below). Drugs were injected with a micro-syringe, usually in a 10 µl volume and flushed with 10 µl of saline. The change in reflex magnitude integrated over 2 s was expressed as percent increase or decrease compared with baseline value. The proper location of the i.t. catheter position was controlled at the start of the experiment by i.t. injection of 10 ng SP, which elicited a marked increase in flexor reflex response usually lasting for about 5 minutes if the tip of the catheter is on the lumbar enlargement. Small adjustment of the catheter location was made if SP did not produce a typical effect.

Mice The mouse was anaesthetized with urethane (ethylcarbamate, Merck, Germany) at 1.5 g/kg body weight, but was not decerebrated or spinalized. When stable anesthesia was achieved, the biceps femoris/semitendinosus was exposed and a pair of stainless steel electrodes inserted. Similarly to rats, the sural nerve innervation area was stimulated electrically 1/min, but the CS train consisted of 10 stimuli at 1 Hz instead of 20 as in rats, since it was observed that maximal wind-up reached a plateau after the 4th stimulation and continued stimulation

23 Materials and methods

above 10 stimulations caused a decrease in response magnitude in some animals, something not observed in rats. The same procedures for recording as in rats were used. For i.t. injection, a catheter was implanted 3-4 days prior to the acute experiment. A needle was inserted between vertebra at the level of cauda equina and a stretched PE10 catheter with a guided wire inside punctured the dura. Upon penetration of the dura, which was accompanied by typical tail twitch, the wire was withdrawn from the catheter. The catheter was then carefully implanted rostrally, aiming its tip at the lumbar cord enlargement. After every experiment the catheter position was verified visually by laminectomy, and proper function was assessed either by injection of dye or saline.

Wind-up In some of the experiments (study V for rats and VI and VII for mice) a conditioning stimulation (CS), consisting of 20 stimuli (rats) and 10 stimuli (mice) at 1 Hz, was delivered at same intensity as that eliciting the baseline flexor reflex. This stimulation is supramaximal for C-fiber activation. During the CS, the magnitude of the flexor reflex gradually increased (wind-up). After termination of the CS there was a period of increased flexor reflex response (post CS facilitation), reflecting spinal hyperexcitability (Herrero et al. 2000). The calculation of the wind-up during the CS was done according to the formula (Gjerstad et al. 1996):

Σ(wS)/ pS x nr of stimuli.

Where Σ(wS) is the total sum of all flexor reflex values during the CS, and pS is the pre stimulation baseline (calculated as the mean of the 10 stimulation values preceding the CS). The pre stimulation value (pS x nr of stimuli) thus represents the theoretical sum of what the reflex would be without the CS. The results were then presented as percent increase in reflex magnitude during wind-up. The post-CS facilitation was simply expressed as percent increase compared with pre-CS baseline.

Nerve injury In some of the studies (I and III) the sciatic nerve was sectioned 10-14 days prior to electrophysiology. This was done by tight ligation, followed by transection distally to the ligation, of the three branches of the sciatic nerve (tibial, peroneal and sural) at mid-thigh level. The sural nerve was cut as distally as possible, to allow the stump to be placed on a silver hook electrode and stimulate it electrically to elicit the flexor reflex. Some of the nerve sectioned rats developed autotomy behavior showing as scratching and biting of denervated toenail, toes and hindpaw. If a rat displayed such a behavior, it was quickly used for the electrophysiological experiment.

24 Materials and methods

Inflammation

,QVWXGLHV,,,,DQG9LQIODPPDWLRQZDVLQGXFHGE\VXEFXWDQHRXVLQMHFWLRQRI-carrageenan (Sigma) into the plantar skin of one of the hindpaw of the rat. Signs of inflammation, such as redness, swelling and impaired function, were observed. The peak of the inflammatory reaction was seen after 24 h, and gradually decreased, to be virtually absent after 5-6 days. To limit the influence of any factor in the peripheral tissue, the flexor reflex in theses animals was evoked in the same way as in the axotomized animals, by stimulating the nerve directly (see above).

Peptides Most of the peptides used in the studies were purchased from commercial laboratories (see 1 individual papers for further information). [Nphe ]nociceptin(1-13)NH2 was synthesized at Department of Experimental and Clinical Medicine, section of Pharmacology, University of Ferrara, Italy and M-35 synthesized at Department of Neurochemistry and Neurotoxicology, University of Stockholm.

Statistics All statistics were calculated with StatView on an Apple Macintosh computer. Results are expressed as mean ± SEM. Data were analyzed with a variety of tests, including ANOVA followed by a post-hoc test such as Fischer PLSD in studies I, III, IV, V, VI and VII. In some of the experiments unpaired t-test was used, study II, IV, and VI. Regression analysis was used in study I, III and IV. In two of the studies, III and V, Mann-Whitney U-test was used, as 2 ZHOOD V: LOFR[RQ VLJQHG UDQN WHVW LQ VWXG\ 9 7KH $ -test was also used in study I. Significance level was set to p<0.05

25 Results

Results

Study I

Summary The effect of intrathecal nociceptin on the flexor reflex in normal, inflamed and axotomized animals was tested. Four doses of nociceptin 10 ng, 100 ng 1 µg and 10 µg were administered. All groups displayed a dose-dependent depression of the flexor reflex after an initial facilitation (Fig. 1, study I). Inflamed animals exhibited significantly greater facilitation and weaker depression compared with both normal and axotomized rats (Figs. 2-4).

Normal rats 10 and 100 ng i.t. nociceptin briefly facilitated the flexor reflex in about half of the experiments. The facilitation lasted for 1-2 min. After the 100 ng dose there was a depression of the flexor reflex in half of the experiments, whereas no depression was seen after 10 ng (Fig. 1). At the two highest doses nociceptin rarely facilitated the reflex, but instead induced a strong depression (Fig. 1). Regression analysis indicated that the depression was dose- dependent (Fig. 4).

Inflamed rats The electrophysiological experiments were carried out 1-3 days after inflammation. Flexor reflex discharges had a longer duration compared with normal animals. After 10 ng and 100 ng nociceptin, there was also an initial facilitation, that at 100 ng was followed by a depression (Fig. 1). 100 ng and 1 µg frequently induced a depression of the flexor reflex. The initial facilitation was significantly stronger in the inflamed animals than in normal rats (Fig. 2), and the subsequent dose-dependent depression was significantly weaker and shorter- lasting than normal and axotomized rats (Fig. 3).

Axotomized animals The experiments were conducted 10-16 days after axotomy. As in normal animals, lower doses of nociceptin usually induced a short-lasting facilitation of the flexor reflex (Figs. 1-2) whereas higher doses produced a dose-dependent depression of the reflex (Figs. 3-4). The depression was significantly stronger compared with inflamed animals, but no difference was seen compared with normals (Figs. 3-4).

Study II The study was conducted to examine the effect of a proposed novel nociceptin receptor 1 1 antagonist, [Nphe ]nociceptin(1-13)NH2, on the flexor reflex in rats. I.t. [Nphe ]nociceptin(1-

26 Results

13)NH2 dose- dependently facilitated the flexor reflex (Fig. 1). At the two lowest doses, 5.5 nmol and 16.5 nmol, the duration of facilitation was brief, but at the highest dose, 38.5 nmol, the facilitation lasted for 5-20 minutes (Fig. 1, study II). When pre-treating with 16.5 nmol 1 [Nphe ]nociceptin(1-13)NH2 before i.t. injection of nociceptin, the initial facilitatory effect of nociceptin normally observed was significantly increased (Figs. 2 and 3). Moreover, the depressive effect of nociceptin after the initial facilitation was almost completely blocked by 1 pretreatment with [Nphe ]nociceptin(1-13)NH2 (Figs. 2 and 4). In contrast, the facilitatory and inhibitory effects of i.t. endomorphin-2 were not affected by pre-treatment with 1 [Nphe ]nociceptin(1-13)NH2 (Figs. 3 and 4), indicating that the drug did not interact with the µ-opioid receptor.

Study III

Summary Endomorphin-2 was administered i.t. to normal, inflamed and axotomized rats and the flexor reflex was examined as previously described. The axotomized rats were subdivided into rats with autotomy (self mutilating behavior) and rats without autotomy. In most experiments, i.t. endomorphin-2 induced an initial brief facilitation of the reflex that in all experiments was followed by an inhibition (Table 1, study III). The inhibition was dose-dependent and nearly 100% at the highest dose, but short-lasting. There was no difference in the magnitude or duration of depression between normal, inflamed and axotomized rats without autotomy. Axotomized rats with autotomy had significantly weaker and shorter lasting depression of the flexor reflex.

Normal rats I.t. endomorhpin-2 at 40 ng, 400 ng and 4 µg induced a potent, but short-lasting inhibition of the flexor reflex (Fig. 1, study III). At 40 ng the depression of the reflex was about 20% and lasted for 5 minutes. 400 ng of endomorphin-2 induced a depression around 50-60% lasting for 10 minutes. After 4 µg the depression was approximately 95% that lasted for 25 minutes. ANOVA and regression analysis showed that the depression was dose-dependent (Figs. 2-3).

Inflamed rats Signs of inflammation were clearly observed 24-48 hours after subcutaneous injection of carrageenan into the hindpaw. Although not measured quantitatively, the reflex in the inflamed rats appears to be stronger and the after-discharge was more prolonged than in normal rats. Similar to normal rats, endomorphin-2 induced a dose-dependent depression of the flexor reflex that did not differ significantly from that seen in normal rats (Figs. 2-3).

Axotomized rats A minimum of 10 days was allowed for the animals to recover after sciatic nerve section. Animals were regularly inspected for signs of autotomy, such as scratching and biting of toe

27 Results

nails, toes or hindpaw. Autotomy was seen in 5 of the 11 rats at days 12-19 after axotomy. If displaying such behavior, the animals were immediately used for electrophysiological experiments. In axotomized rats without autotomy, the reflex depressive effect of endomorphin-2 was not significantly different from normal or inflamed animals (Figs. 2-3). In contrast, in axotomized rats showing the autotomy behavior, endomorphin-2 induced virtually no depression at the two lowest doses (Figs. 2-3). At 4 µg the depression was around 20% and lasted only 5 minutes, which was significantly lower than in other groups of rats (Figs. 2-3).

Study IV This study was conducted to compare the effect of endomorphin-1 and endomorphin-2 on the flexor reflex in rats. The initial facilitation of the reflex was similar between endomorphin-1 and –2 (Fig. 1, study IV). Also, i.t. endomorphin-1 and endomorphin-2 at 4 ng to 4 µg induced a strong depression of the flexor reflex dose-dependently (Figs. 2-3). There was no difference in magnitude of reflex depression between the two endomorphins (Fig. 2). The duration of depression, however, was significantly shorter after endomorphin-2 (Fig. 2).

Study V

Summary I.t. galanin and galanin antagonist M-35 were injected i.t. to normal rats and after carrageenan-induced inflammation. The effect on wind-up and the subsequent facilitation of the flexor reflex after C-fiber CS was assessed. Inflamed animals had significantly reduced wind-up and post-CS reflex facilitation compared with normal controls. This difference was reversed by i.t. M-35. On the other hand, the effect of galanin was also reduced in the inflamed rats.

Baseline flexor reflex As described above, at one to three days after carrageenan-induced inflammation, the magnitude of the integrated flexor reflex was significantly increased compared to normals (Table 1, study V). Moreover, the duration of EMG activity was also significantly prolonged (Table 1). At four to eight days after the injection of carrageenan when the signs of inflammation were diminishing, the magnitude and duration of the reflex become normalized (Table 1).

Wind-up and post-CS facilitation A C-fiber CS produced wind-up of reflex magnitude during the CS in normal rats (Table 2, Fig. 1, study V). In inflamed rats, the extent of wind-up was significantly reduced and in some rats, we observed a decrease in reflex excitability during the CS, “wind-down” (Table 2, Fig. 1). Such reduction in wind-up was recovered in rats 4-8 days after carrageenan injection

28 Results

(Table 2, Fig. 1). Similar to wind-up, the post-CS facilitation was also significantly reduced in inflamed rats compared to normal or recovered rats (Fig. 2).

Intrathecal galanin I.t. galanin at 100 ng, 1 µg and 10 µg dose-dependently blocked the reflex facilitation induced by CS in normal animals while producing no effect on wind-up (Fig. 4). In inflamed rats, the effect of i.t. galanin on reflex facilitation was significantly reduced (Fig. 4) and in recovered rats galanin’s effect was abolished (Fig. 4).

Intrathecal M-35 The galanin antagonist M-35 produced a moderate facilitation of the flexor reflex in normal and inflamed rats (Fig. 5). This facilitation was significantly reduced in recovered rats (Fig. 5). Moreover, the duration of facilitation in inflamed and recovered rats was significantly shorter. Wind-up was not influenced by M-35 in normal and recovered rats. In inflamed rats, the reduced wind-up was completely reversed by i.t. M-35 (Fig. 6). The reflex facilitation was potentiated by M-35 in normal and inflamed rats, whereas in recovered rats this potentiation was not present.

Study VI In this study a mouse strain over-expressing galanin gene was used to study spinal cord excitability. The flexor reflex model, previously used extensively in rats, was successfully adapted to mice. Electric stimulation of the sural nerve innervation area of the hindpaw in anaesthetized mice, elicited a brisk contraction of the hamstring flexor reflex in a similar manor as seen in rats. It was possible to obtain a stable reflex response for baseline measurement. A CS of 10 stimuli at 1 Hz induced wind-up and the subsequent reflex facilitation lasted for about 5 minutes which is similar to rats. Also, a new method of chronically implanting i.t. catheter in mice was developed, enabling the spinal effects of drugs on flexor reflex excitability to be studied. The flexor reflex response in mice over-expressing galanin (GAL-OE) was similar to that seen in wild-type (WT) mice. During the CS there was a gradual increase in flexor reflex response, with a plateau after the 5th stimulation. The total flexor reflex increase during wind- up was approximately 300% and no difference between GAL-OE and WT mice was observed (Fig. 1, study VI). After delivery of the CS, there was a period of reflex hyperexcitability for on average 3 minutes in GAL-OE mice and 6 minutes in WT (not significantly different). The reflex increase was about 160% in WT mice, whereas GAL-OE had significantly lower faciliation, only around 20% (Fig. 2). In some experiments, the galanin antagonist M-35 was administered i.t. shortly before the CS was delivered (Fig. 3). Whereas no change in post-CS facilitation was seen in WT mice after M-35, in the GAL-OE mice, the facilitation (in these

29 Results

mice that was rather modest) was increased dramatically by M-35 to the same levels as in WT (Fig. 3).

Study VII A newly developed mouse strain lacking the galanin receptor GAL-R1 was studied using the mouse model of the flexor reflex. Baseline flexor reflex values did not differ between GAL- R1 knock-out, wild-type or heterozygote mice. A conditioning stimulation (CS) was applied (10 stim, 1 Hz), that gradually increased the flexor reflex (wind-up). The total increase during CS was about 400% with no differences observed between the three groups. The subsequent facilitation was around 150% in all three groups, no significant difference observed. After intrathecal injection of galanin at 500 ng and 5 µg no significant effect on the total reflex value during the CS was seen. The post-CS facilitation, however, was dose- dependently blocked in wild-type and knock-out mice, no significant difference observed between the two groups. In heterozygote mice the blocking effect of galanin was stronger, but only significant compared to knock-out mice.

30 Dicsussion

Discussion

Nociceptin

Biphasic effect of nociceptin in the spinal cord After an initial brief facilitation, intrathecal nociceptin dose-dependently depressed the nociceptive flexor reflex in rats. Facilitation was usually seen after administration of low doses. An initial phase of facilitation after intrathecal delivery of inhibitory compounds is often seen, and the mechanisms behind this are still unclear. One possible explanation may be that inhibitory interneurons are inhibited, resulting in disinhibition. It is possible that the inhibitory neurons are more sensitive and are inhibited at lower doses. For example, it has been shown that at very low doses, administration of i.t. nociceptin produce allodynia and hyperalgesia that is alleviated by glycine (Hara et al. 1997). This suggests that the facilitatory effect of nociceptin may be due to inhibition of inhibitory glycinergic interneurons. There is also evidence that nociceptin-induced facilitation is caused by the release of substance P (Inoue et al. 1999). This has previously been observed with morphine, where low doses of morphine facilitates the nociceptive flexor reflex, probably through the release of SP and other tachykinins (Wiesenfeld-Hallin et al. 1991). Finally, it is known that ORL1 receptors may be present in different splice variants and theses may account for the facilitatory and inhibitory effects respectively (Wang et al. 1994; Xie et al. 1999). I.t. nociceptin produced dose-dependent inhibition of the flexor reflex in spinalized rats. This is in agreement with extensive literature demonstrating a spinal antinociceptive effect of this peptide. Since nociceptin-induced spinal antinociception is usually not naloxone reversible, it appears not to involve classical opioid receptors. Several lines of evidence suggest that the inhibitory effect of nociceptin in the spinal cord may involve both pre- and post-synaptic mechanisms. Thus, it has been shown that the mRNA for ORL1 receptors is expressed in rat dorsal root ganglia (DRG) (Neal et al. 1999b) and in vitro studies showed that nociceptin reduced N-type and non-N, non-L type calcium current in disassociated rat DRG cells with strongest effect observed for the small cells (Abdulla and Smith 1997, 1998). Liebel et al. reported that nociceptin inhibited excitatory postsynaptic potentials (EPSPs) in superficial dorsal horn neurons to electrical stimulation of dorsal roots, but not to the application of glutamate (Liebel et al. 1997). Nociceptin also reduced the frequency, but not the magnitude, of spontaneous miniature EPSPs of superficial dorsal horn neurons (Liebel et al. 1997). These findings indicated a presynaptic site of action for nociceptin by reducing the release of excitatory transmitters from afferent terminals. On the other hand, several studies have shown that nociceptin reduces the responses of dorsal horn neurons to excitatory amino acids and substance P (Wang et al. 1996; Shu et al. 1998). Lai et al. also reported that nociceptin at high concentration hyperpolarized some dorsal horn neurons (Lai et al. 1997).

31 Dicsussion

These, together with the fact that some dorsal horn neurons also express ORL1 receptors (Neal et al. 1999a), suggest that nociceptin may also have a postsynaptic inhibitory action.

Effects of nociceptin in inflamed and nerve injured rats It has become increasing clear in recent years that the endogenous opioid systems undergoes significant changes during different pathological states, such as nerve injury or inflammation (Dickenson 1994a; Dickenson 1994b; Stanfa and Dickenson 1995). Such changes may underlie differential opioid sensitivity in different painful states. Clinically, it has long been suggested that neuropathic and idiopathic types of pain responded poorly to opioids (Arner and Meyerson 1988). Although the efficacy of opioids in treating neuropathic pain is still a matter of debate, there is extensive experimental and clinical evidence that the potency of opioids in alleviating neuropathic pain is reduced in comparison to nociceptive pain. On the other hand, the antinociceptive effect of opioids may be increased during inflammation (Hökfelt et al. 1997). The reduction in opioid responsiveness in neuropathic pain may be explained by up- regulation of anti-opioid systems and plasticity in the opioid systems, with down-regulation of µ-receptors after nerve injury (Wiesenfeld-Hallin and Xu 1996; Hökfelt et al. 1997). Since nociceptin produces antinociception through mechanisms independent of activation of classical opioid receptors, it is possible that nociceptin will not exhibit changes in effect after inflammation or nerve injury in the same way as morphine. Results presented in this thesis showed that this is indeed the case. The potency of nociceptin to depress the flexor reflex is slightly higher after nerve injury, whereas after inflammation it is somewhat reduced. Recently, a study has been published demonstrating increase in expression of ORL1-receptors after nerve injury (Briscini et al. 2002). This may well be an explanation for the increased, or at least not reduced, effectiveness of nociceptin after nerve injury observed in our study. It is also possible that the endogenous anti-opioid system, involving for example CCK, may be inactive against nociceptin. Overall, the data would suggest a possible clinical application for nociceptin in treating neuropathic pain.

Nociceptin antagonist Much of the previous research on the physiology and pharmacology on the nociceptin systems, have been hampered by the lack of a selective and high affinity antagonist for the ORL1-receptor. The earlier developed antagonists have suffered from low potency and selectivity and in some cases being in fact agonists (Xu et al. 1998). In the work presented in this thesis, we evaluated a novel nociceptin analogue 1 [Nphe ]nociceptin(1-13)NH2 as an ORL1 receptor antagonist in rat spinal cord. This compound did not depress the nociceptive flexor reflex, indicating lack of agonist property. The initial facilitation seen after intrathecal nociceptin was significantly increased by 1 [Nphe ]nociceptin(1-13)NH2, whereas the inhibitory effect of intrathecal nociceptin was 1 blocked. An explanation for the increase in facilitation seen after [Nphe ]nociceptin(1-13)NH2 may be derived from the bi-phasic effect of nociceptin (discussed above). As seen earlier, low

32 Dicsussion

doses of nociceptin facilitate the flexor reflex and therefore it is possible that the competitive 1 antagonist [Nphe ]nociceptin(1-13)NH2 shifts the dose-response curve to the right, making facilitation the predominant effect. It is also possible that the bi-phasic effect of nociceptin is exerted by the different splice variants of the ORL1-receptor and the antagonism of 1 [Nphe ]nociceptin(1-13)NH2 only is effective at either one of the receptor variants. 1 The potency of antagonism by [Nphe ]nociceptin(1-13)NH2 was rather weak, at least 30- 1 fold higher dose of [Nphe ]nociceptin(1-13)NH2 was required to block the effect of nociceptin. This has been predicted in earlier in vitro experiments (Calo et al. 2000). Nevertheless, this compound appears to reduce the inhibitory effect of nociceptin selectively 1 since the effect of endomorphin-2 is not affected. Thus, [Nphe ]nociceptin(1-13)NH2 may be a useful tool in the future studies of the physiological function of nociceptin.

Application of nociceptin-related compounds as analgesics Several factors suggest that agonists of the ORL1 receptor may have potential use as novel analgesics. Not only did spinal administration of nociceptin produce potent antinociception in rodents, but this effect is also independent of the activation of classical opioid receptors. Thus, nociceptin-mediated antinociception represents novel mechanisms. Further, in contrast to morphine, the antinociceptive potency of nociceptin is not reduced after nerve injury and nociceptin may be particularly effective in treating mechanical allodynia, a hallmark of human neuropathic pain syndrome. I.c.v administration of nociceptin did not induce place preference or aversion (Devine et al. 1996), but rather is anxiolytic (Jenck et al. 1997). Nociceptin may therefore lack abuse potential and also has a favorable CNS side effect profile LQFRPSDULVRQWR-opioid agonists. Finally, we have shown that there is no cross tolerance between the antinociceptive effect of i.t. morphine and nociceptin (Hao et al. 1997). Drugs acting on the ORL1 receptor may therefore be useful in chronic pain patients tolerant to opioids analgesics. On a final note, it needs to be emphasized that the evidence that i.t. nociceptin is antinociceptive and potentially analgesic has only been obtained in rodent models of acute and chronic pain. It is unclear at the moment whether such findings can be generalized to other species, including primates. Intravenous administration of nociceptin decreases blood pressure and heart rate (Champion et al. 1997; Giuliani et al. 1997), which may present a problem for systemic application. Moreover, as the supraspinal effect of nociceptin is largely pro-nociceptive (Henderson and McKnight 1997; Darland et al. 1998; Yamamoto et al. 1999) the overall effect of systemic ORL1 receptor agonists on nociception cannot be determined without the development of brain penetrating non-peptide agents.

33 Dicsussion

Endomorphins

Effects of i.t. endomorphin-2 in normal, inflamed and nerve injured rats In normal rats, i.t. endomorphin-2 induced a substantial, but relatively short-lasting, dose- dependent depression of the flexor reflex. The effect was naloxone-reversible. This supports earlier behavioral and physiological evidence showing that endomorphin-2 produces antinociception (Chapman et al. 1997; Zadina et al. 1997) and would agree with nature of this peptide being a µ-receptor agonist. Although the depressive effect was nearly total after the highest dose of endomorphin-2, the average duration of depression was only around 30 minutes, which is substantially shorter than morphine. The endomorphins are short tetra- peptides and may be vulnerable to rapid degradation by peptidases, such as dipeptidyl peptidase IV (Peter et al. 1999; Shane et al. 1999). Another possible explanation for the short- lasting effect of endomorphin-2 is that the µ-receptor is internalized after agonist binding, something that is not seen with morphine (Burford et al. 1998; McConalogue et al. 1999). The initial brief facilitation seen after intrathecal endomorphin-2 usually occurred at lower doses, and was followed by depression at higher doses. This phenomenon is often seen after administration of inhibitory compounds, and some of the potential reasons behind this have been discussed in previous paragraphs. Most likely, the facilitation seen after endomorphin-2 shares common mechanisms with morphine, involving the release of excitatory peptides (Wiesenfeld-Hallin et al. 1991). A further possibilityLVWKDWORZGRVH- opioids have been found to activate excitatory G-proteins (Crain and Shen 1998), an effect that is overshadowed by inhibition at high doses, which activates inhibitory G-proteins. After inflammation the effect of endomorphin-2 was similar to that of normal rats. There are a few previous studies examining the effect of endomorphins during inflammation. In a recent study it was shown that endomorphin-2 is predominantly expressed in the dorsal horn of the spinal cord, and the expression is unchanged during inflammation (Mousa et al. 2002). The expression of µ-opioid receptors, however, was significantly up-regulated in lumbar segments of the spinal cord ipsilateral to inflammation (Mousa et al. 2002). This may explain why endomorphin-2 still is fully effective during inflammation, when peripheral nociceptive input is increased markedly. In peripheral tissue endomorphin-expression is found in immune cells (Jessop et al. 2000), and the expression of both endomorphin-1 and -2 are up-regulated in these cells in response to inflammation (Mousa et al. 2002). Thus, endomorphins may be acting both at the periphery and in the spinal cord to reduce the increased nociceptive activity during inflammatory states. As discussed above, after nerve injury the effect of morphine may be reduced (Arner and Meyerson 1988; Ossipov et al. 1995). In rats with sectioned sciatic nerves the effect of endomorphin-2 was equally good as in normal rats, but only in the subgroup of animals without autotomy behavior. In the group with autotomy behavior the inhibitory effect of endomorphin-2 was markedly reduced. Similar differences have previously been reported for morphine, where autotomizing animals have increased threshold for the antinociceptive effect

34 Dicsussion

of intrathecal morphine (Xu and Wiesenfeld-Hallin 1991). Autotomy occurs after transection of a nerve and there is evidence that autotomy represents a sign of ongoing neuropathic pain (Wall et al. 1979; Coderre et al. 1986). Thus, it is possible that autotomizing rats have reduced response to the antinociceptive effects of endomorphin-2, and other µ-opioid agonist, due to higher basal levels of nociceptive input. There might also be differences in µ-opioid expression and differences in activity of anti-opioid systems such as CCK. It has been shown that the µ-opioid receptor is down-regulated after nerve injury (Zhang et al. 1998), but no specific analysis of regulation of receptor or agonists in autotomizing vs non-autotomizing animals have yet been performed. In the clinic it has been observed that some patients respond poorly to opioids (Arner and Meyerson 1988; Hanks and Forbes 1997). It is thus possible that some genetic difference in opioid pharmacology and physiology account for the differential opioid responses seen in autotomizing vs non-autotomizing animals. Studies comparing autotomizing and non-autotomizing animals using genetic mapping may be needed to localize possible differences in opioid, and pain-related genes. This may be of importance clinically, both to find ways of identifying potential poor opioid-responding patients to be able to individualize dosage and treatment, as well as finding possible future therapeutical alternatives for this group of individuals.

Comparing endomorphin-1 and -2 Intrathecal endomorphin-1 and -2 induced a short-lasting, dose-dependent and strong depression of the nociceptive flexor reflex. Both endomorphins initially facilitated the reflex, especially at lower doses. The effect of endomorphins was naloxone-reversible. There were no differences in efficacy or potency between endomorphin-1 and -2, agreeing with some, but not all, earlier published data (Stone et al. 1997; Horvath et al. 1999; Sakurada et al. 1999; Soignier et al. 2000). At the highest dose the depression of the reflex was nearly total for both endomorphins. However, the duration of depression was significantly shorter after intrathecal endomorphin-2 than endomorphin-1. This may explain why endomorphin-1 seem to have stronger efficacy and potency in some of the behavioral studies, since measurements usually are made 10-15 minutes after administration. The reasons for the difference in duration for the two endomorphins are unclear, but there are several possibilities. First, there may be differences in receptor binding or G-protein activation between the endomorphins. However, since the potency and efficacy is equal for two endomorphins, the most probable reason for the difference in duration is differences in peptide metabolism. It has been shown that different peptidases, such as carboxypeptidase Y, aminopeptidase M and dipeptidyl peptidase IV, may be involved in degrading the peptides. Carboxypeptidas Y degraded endomorphin-1 in about 20 minutes, whereas endomorphin-2 was no longer present after 5 minutes, and proteinase A metabolized endomorphin-1 in 8 hours and endomorphin-2 in 6 hours (Peter et al. 1999). Thus, endomorphin-2 seems more sensitive to rapid degradation.

35 Dicsussion

Galanin

Role of galanin during inflammation After plantar injection of carrageenan, the magnitude and duration of the nociceptive flexor reflex was increased compared to normal rats and rats that had recovered from inflammation (4-8 days after carrageenan injection). This probably reflects spinal hyperexcitability during inflammation (Xu et al. 1995). Despite the increase in baseline reflex magnitude during inflammation, the wind-up during repetitive C-fiber stimulation as well as post-CS reflex facilitation was significantly reduced. In a previous study, Stanfa et al. also reported that carrageenan-induced inflammation was associated with reduction in wind-up in some dorsal horn neurons, particular those with high degree of initial response (Stanfa et al. 1992). It is not likely that the reduced wind-up and reflex facilitation in inflamed rats reflects saturation due to higher baseline reflex level since the galanin receptor antagonist M-35 is able to normalize the reduced wind-up and reflex facilitation. On the other hand, the results with M-35 in inflamed rats suggest there exists an endogenous galaninergic control on spinal hyperexcitability during inflammation. This would fit with the finding that inflammation induced up-regulation of galanin synthesis in dorsal horn neurons. Moreover, increased basal release of galanin in the dorsal horn has also been reported after inflammation to the joint (Hope et al. 1994). In contrast to the role of endogenous galanin, it appears that exogenous galanin became significantly less effective in blocking the reflex facilitation in inflamed rats. An explanation for this observation may be that galanin GAL-R1 receptors in the dorsal root ganglia are reduced after inflammation (Xu et al. 1996), whereas GAL-R2 excitatory receptors are up- regulated (Shi et al. 1997). Such reduction of the number of inhibitory galanin receptors coupled with an increased in the activity of endogenous galanin may render the exogenous galanin less effective. Between four to eight days after carrageenan when the signs of inflammation were reduced the baseline flexor reflex, as well as the wind-up and post-CS facilitation was normalized. This indicates that the spinal hyperexcitability during inflammations is a reversible process and that the inflammatory process in the periphery is needed to maintain the hyperexcitability. Interestingly, i.t. galanin did not block the facilitation of the reflex after C-fiber CS in recovered rats, as it did in normal rats. Moreover, i.t. M-35 also did not potentiate the effect of C-fiber CS. Thus, it appears that the C-fiber CS was no longer influenced by either exogenous or endogenous galanin. Again, this may be related to the reduction in inhibitory GAL-R1 receptors in DRG after inflammation. The reduction is maximal at three days and partially recovered at five days. However, this time course may not correlate precisely with functional changes since newly synthesized receptors need to be transported to the afferent terminals from the cell body. Form this perspective, it would be interesting to hypothesize that the effect of exogenous galanin will eventually recover at a later date after inflammation.

36 Dicsussion

Galanin over-expressing mice GAL-OE mice displayed a baseline flexor reflex that was not different from wild-type littermates. Wind-up during repetitive C-fiber stimulation was not altered in GAL-OE, but the subsequent facilitation of the flexor reflex was significantly reduced. This agrees with previous studies in rats, where i.t. galanin did not influence wind-up during the CS, but blocked the reflex facilitation (Wiesenfeld-Hallin et al. 1989a; Xu et al. 1990; Xu et al. 1991; Wiesenfeld-Hallin et al. 1992a; Xu et al. 2000b). The ability of galanin to reduce C-fiber CS- induced facilitation may be due to its ability to reduce the excitatory effect of SP and CGRP (Xu et al. 1990) which are important for activity-dependent increase in spinal excitability. After i.t. administration of the galanin antagonist M-35, the reduced facilitation after wind-up in the GAL-OE mice was reversed, and the facilitation increased to similar levels as seen in wild-type mice. Immunohistochemical studies have shown that this strain of GAL-OE mice had increased levels of galanin in the DRG and the dorsal horn (Hökfelt, personal communication). This indicates that the reduced facilitation in the GAL-OE mice is primarily a galanin-mediated effect through inhibitory control exerted by galanineric neurons in the DRG or dorsal horn. Previous behavioral studies in GAL-OE mice also showed that these mice have increased nociceptive threshold to heat stimulation and reduced development of neuropathic pain-like behavior after partial nerve injury (Hygge Blakeman et al. 2001; Hygge Blakeman et al. 2002). After nerve injury the expression of galanin in DRG neurons is markedly up-regulated (Hökfelt et al. 1987; Villar et al. 1989). Thus, galanin over-expressing mice may be a useful model to study endogenous galanin physiology and pharmacology during this state of elevated galanin levels. Moreover, the studies on galanin over-expressing mice have shown that it is possible to adapt and develop a nociceptive flexor reflex model for mice. This allows for future studies in genetically modified mice.

GAL-R1 receptor knock-out mice There is an increasing body of evidence supporting an inhibitory role for galanin acting on the

GAL-R1 receptor. The coupling of GAL-R1 to inhibitory Gi/Go protein leads to decreased neuronal activity and/or neurotransmitter release (Branchek et al. 2000). Spinal administration of antisense probe directed against the GAL-R1 receptor gene reduces the inhibitory effect of intrathecal galanin on spinal cord hyperexcitability (Pooga et al. 1998; Rezaei et al. 2001). Moreover, the antiallodynic effect of galanin in nerve injured rats has also been suggested to be mediated by GAL-R1 receptor (Liu et al. 2001). Behavioral studies on GAL-R1 null mutant mice, although supporting an inhibitory role of this receptor under normal conditions and after nerve injury, showed that the difference between the wild-type and knock-out mice is quite modest (Hygge Blakeman et al. 2003). In the present study, we observed however that GAL-R1 null mutant mice did not differ from wild-type mice in term of pattern of baseline flexor reflex, wind-up during C-fiber CS, and post CS reflex facilitation. Thus, under normal conditions and during the spinal hyperexcitability induced by repetitive C-fiber stimulation, the GAL-R1 receptor subtype may have only limited physiological importance. Alternatively,

37 Dicsussion

plasticity may have occurred in that other subtypes of galanin receptors have taken over the role played by the GAL-R1 receptor normally in the knock-out mice (see below). In wild-type mice, i.t. galanin significantly reduced reflex facilitation induced by C-fiber CS. This is in agreement with earlier studies showing a similar effect by galanin in rats, an effect that has been shown to be mediated by the GAL-R1 receptor. However, galanin produced similar inhibitory effects in GAL-R1 receptor knock-out mice. This would support the plasticity theory since other galanin receptor subtypes are indeed capable of mediating the inhibitory effect of galanin. It would be interesting to test mice with mutations in other galanin receptor subtypes, and to evaluate double and triple knock-outs with deletions in two or three galanin receptor subtypes, to further define the physiological role of endogenous galanin and its receptors in nociception.

38 Conclusions

Conclusions

Endogenous antinociceptive peptides are important in reducing nociceptive input, especially during states of spinal hyperexcitability, such as after inflammation, nerve injury or activity dependent increase of spinal responsiveness. From the specific studies on the peptides included in this work, the following conclusions can be drawn:

1. Nociceptin dose-dependently reduces nociceptive input, during normal conditions as well as after nerve injury and inflammation. The antinociceptive effect of nociceptin during inflammation seems slightly decreased, whereas the effect after nerve injury is increased.

1 2. [Nphe ]nociceptin(1-13)NH2 acts as a ORL1 receptor antagonist, with weak potency. The compound does not display any agonist properties.

3. Endomorphin-2 induces a short-lasting but strong depression of spinal excitability during normal conditions, as well as during inflammation and after nerve injury. The effect of endomorphin-2 is reduced in rats with signs of on-going neuropathic pain.

4. There is no difference between the potency of endomorphin-1 and endomrophin-2. The duration of effect is shorter for endomorphin-2. This may indicate differences in metabolism.

5. Endogenous galanin acts as a antinociceptive brake during inflammation. Spinal hyperexcitability seen during inflammation is a reversible and plastic process, involving changes in the expression of galanin and galanin receptors.

6. We have adapted and developed a flexor reflex model for mice in order to make it possible to study genetically modified mice.

7. Galanin over-expressing mice have reduced spinal hyperexcitability after repetitive C- fiber stimulation (wind-up). These mice may be used as a model of states of high galanin expression.

8. The flexor reflex, wind-up and following spinal hyperexcitability in galanin GAL-R1 receptor null mutant mice have is similar to wild-type littermates. I.t. galanin is able to reduce spinal hyperexcitability in GAL-R1 knockouts.

39 Populärvetenskaplig sammanfattning

Populärvetenskaplig sammanfattning

Kronisk smärta är ett stort och växande problem. Det drabbar dels den enskilde patienten, men också samhället, med ökade kostnader för behandling, sjukfrånvaro och rehabilitering. En nylig rapport från SBU (statens beredning för medicinsk utvärdering), slår fast att kostnaderna för endast ryggsmärtor är tre gånger så höga som för all cancer. Kroppen har flera sinnrika inbygga system för att minska omfattningen av smärta och i detta arbete studeras några av dessa system. Genom att förstå kroppens egen smärtlindring kan förhoppningsvis nya läkemedel tas fram, med bättre behandlingsresultat och mindre biverkningar. Ryggmärgen är den första omkopplingsstationen för de smärtnerver som förmedlar sin signal till hjärnan. Det sjuder av aktivitet i ryggmärgen: nerver kopplar till varandra i komplexa mönster, signaler kommer in från de olika hud- och kroppsnerverna och signalsubstanser ser till att nerverna kan kommunicera med varandra. I detta invecklade system finns det ämnen som både ökar och minskar intensiteten i smärtsignalerna. Under sjukliga förhållanden som leder till kronisk smärta, t ex inflammationer, nervskador, cancer, ökar produktionen av smärtlindrande substanser som ett försvar mot för mycket smärta. Några av de smärtlindrande ämnen som är av betydelse och studerats i detta arbete är: endomorphin- 1, endomorphin-2, nociceptin och galanin.

Opioider Endomorphin-1 och -2 och nociceptin är kroppsegna opioider. Opioider är ämnen som binder till opioidreceptorer (mottagarmolekyler för opioider) och därigenom utövar en stark smärtlindrande effekt. Morfin, som är ett av de mest använda starka smärtlindrande läkemedlet i kliniskt bruk, binder till samma opioidreceptor som endomorphin-1 och -2, den s k µ-receptorn. Förutom µ-receptorn finns det flera andra opioidrecepoter såsom /RFKGHQ nyupptäckta ORL1. Nociceptin binder till ORL1. Endomoprhin-1, -2 och nociceptin har alla visats ha smärtlindrande egenskaper, både under normala förhållanden, men också under olika sjukdomstillstånd med smärta. Ett mysterium som gäckat smärtforskare under lång tid är varför morfin, som normalt har en mycket stark smärtlindrande effekt, efter nervskada minskar eller förlorar sin förmåga att åstadkomma samma effektiva smärtlindring som före skadan. Genom våra studier på råttor visade det sig intressant nog att nociceptin tycks bevara, och kanske tom öka sin förmåga att lindra smärta efter nervskada. Det indikerar att de vägar och mekanismer i kroppen nociceptin använder sig av för att ge upphov till smärtlindring, skiljer sig åt från morfinets. Därför kanske nociceptin kan utgöra ett komplement till morfin i en framtida behandling av smärttillstånd efter nervskada.

40 Populärvetenskaplig sammanfattning

µ-receptorn anses vara opioidernas viktigaste förmedlare av smärtlindrande signaler. Både endomorphin-1 och -2 binder mycket starkt till denna receptor, faktiskt starkare och mera specifikt än morfin. Endomorphin-1 och -2 finns kroppseget i stora delar av vårt nerv- system och tycks alltså vara vårt ”eget morfin”. I de experiment vi gjort har endomorphinerna mycket starkt smärtlindrande egenskaper i normala råttor och i råttor med inflammation, men även i en undergrupp av nervskadade råttor. Råttorna med nervskada delades upp i två grupper: de som hade ett beteende då de bet och kliade sig på den del av kroppen som blivit av med sin nervförsörjning, och de som betedde sig normalt. Endomorphin-2 hade ingen effekt i den förstnämnda gruppen, men fullgod effekt i de djur som betedde sig normalt. Det inger misstanke om att genetiska skillnader kan ligga bakom den förlorade känsligheten för opioidernas smärtlindrande effekter efter nervskada. Om dessa skillnader kan studeras närmare, kanske mysteriet med morfinets förlorade förmåga efter nervskada kan komma närmare en lösning.

Galanin Galanin är en unik molekyl som inte tillhör någon annan ”molekylfamilj”. Den har hittats i flera olika arter, däribland människa, och skiljer sig mycket lite åt i struktur mellan arterna. Efter nervskador ökar produktionen av galanin enormt i smärtnerverna och frisätts i rygg- märgen. Galanin tros ha en nervskyddande effekt, men har även visats ha smärtlindrande egenskaper, särskilt efter nervskador. I våra studier har vi hittat bevis på att galanin också är effektiv som smärtlindring under inflammatoriska tillstånd. Mekanismerna för detta verkar dock mer komplicerade än efter nervskada. Galaninproduktionen vid inflammation ökar nämligen inte lika dramatiskt som efter nervskada, utan minskar närmast i nerven, men ökar i stället i ryggmärgen. Genom att tillföra en galanin-antagonist, som blockerar galaninets effekt vid dess receptor, kunde vi se att råttorna fick ökad aktivitet i smärtnerverna efter inflammation. Det talar för att kroppseget galanin i ryggmärgen bromsar smärtnervernas signaler innan de kopplar om till hjärnan.

Genetiskt modifierade möss Numera finns det möjlighet ändra arvsmassan hos djur och därigenom lägga till eller ta bort gener. Djuret föds alltså med en gen för mycket, en mångfaldigad gen eller en eller flera gener för lite. Det öppnar nya möjligheter att studera en specifik gens betydelse. Vi har studerat möss med överproduktion av galanin. Dessa möss överuttrycker genen för galanin och har alltså sedan födseln konstant höga nivåer av galanin i ryggmärgen och smärtnerverna. Detta tillstånd liknar situationen efter nervskada, då ju produktionen av galanin ökar kraftigt. De galanin-överutryckande mössen har påtagligt reducerad retbarhet i ryggmärgen. Retbarheten studeras genom att stimulera nerverna i hög frekvens hos en sövd mus. Man observerar då att nervimpulserna inte bibehåller samma styrka trots konstant stimulering, utan gradvis ökar sin signalering. Det kallas ”wind-up”, dvs smärtsignalen trissas upp i styrka, och

41 Populärvetenskaplig sammanfattning

är ett fenomen som delvis kan ligga bakom uppkomsten av den ökade retbarhet som ses i ryggmärgen vid kroniska smärttillstånd. Galanin i dessa möss kan alltså blockera ”wind-up” och därmed minska retbarheten och upptrissningen i ryggmärgen. Det är ytterligare ett bevis på galaninets smärtlindrande effekt. Den andra gruppen av genetiskt modifierade möss vi studerat, är s k ”kock-out” möss. I dessa möss har man tagit bort en gen, redan innan musägget börjar växa till i livmodern. De möss vi undersökt saknar genen för en mottagarmolekyl för galanin, galanin GAL-R1 receptorn. Galanin binder till flera receptorer, vissa av dem verkar förmedla smärta, medan andra leder till minskad smärta. Systemet är alltså mycket komplext. Galanin GAL-R1 knock- out-mössen visade sig inte skilja sig nämnvärt från normala möss. ”Wind-up” var densamma, även galaninets förmåga att blockera ryggmärgens retbarhet var bibehållen. Kanske GAL-R1 receptorn inte har någon större betydelse vid denna experimentella form av upptrissning av ryggmärgen. I flera tidigare försök har GAL-R1-receptorn visats förmedla smärtlindrande signaler. Slutsatsen av den studien är att galaninsystemet är mycket komplext och i och med att flera receptorer är inblandade är det svårt att bena ut vilken receptor som förmedlar vilken signal. Det kommer krävas betydligt fler studier för att komma närmare en lösning på den gåtan. Vi har alltså med våra studier som ligger till grund för den här avhandlingen undersökt ett flertal kroppsegna smärtlindrande substanser. Många av dem verkar i oerhört komplicerade miljöer, där det kan vara svårt att förstå hur kroppen gör för att bibehålla balansen mellan smärta och smärtlindring. Med dessa studier har vi endast gläntat på dörren för de hemligheter som ligger förborgade i ryggmärgens invecklade värld.

42 Acknowledgements

Acknowledgements

My research career already started during the physiology course of my basic medical studies. I did a small 2-week project at the Division of Clinical Neurophysiology, Huddinge hospital, and was offered to come for the summer. So I came that summer. And the next summer, and the next… And suddenly I found myself writing this thesis. This work had not been possible without the contribution of many people. I particularly would like to express my gratitude to:

My supervisor, Professor Zsuzsanna Wiesenfeld-Hallin, for asking me to come that first summer, and letting me “stay all the way”. Your great generosity and willingness to share your profound scientific knowledge is truly remarkable. Also, your balance between “hardcore research” and pleasure is a perfect combination that creates an inspiring environment. No subject has been too small or too big to discuss during our lunches at “Personalmatsalen” in Huddinge. I will always remember my years in your lab as a happy time. Lastly, I want to thank you for persuading me to stay during my period of doubt…

Docent Xiao-Jun Xu, my co-supervisor, for your enormous amount of time and effort spent in helping me with every aspect of the work I have done during the years. You have always been ready to listen, comment and discuss and your scientific skills and way of thinking has served as a model for me. I appreciate our friendship and the many discussions we have had about science and almost all other subjects imaginable. I also would like to thank you for your excellent guiding of your Beijing, making our trip to China a fantastic experience (despite trouble in the World).

Dr Jing-Xia Hao, for helping me with all the practical matters in the lab (and elsewhere). Your always friendly, happy and curious personality made the sometimes tedious work in the lab to a great time. I enjoy all our talks, about people, cultural differences and so on, and the thought of your friendly laughter instantly makes me in a good mood.

Present and past members of the group: Dr Wei-Ping Wu, for all the help in the lab and bringing me Chinese tea. Dr Aleksandra Bulka, Francois Kouya, Dr Margareta von Heijne, Dr Isabella Shi Xu, Helle Erichsen, Dr Wei Yu, Dr Guilherme Lucas, Dr Aida Plesan, Eva Brimark and Monika Mäkinen for creating a pleasant working environment. Dr Karin Hygge Blakeman for fun times and practical superiority. Dr Pawel Alster for sharing room and friendship, rest in peace.

My co-workers: Prof Jacqueline N. Crawley, Prof Tiina P. Ismaa for helpful comments with the manuscripts and sharing your mice. Dr Arie S. Jacoby for fruitful collaboration.

43 Acknowledgements

Everyone working at the clinical section of the Department of Clinical Neurophysiology for creating a friendly atmosphere and for nice “brännboll” games. Dr Thomas Andersson for musical cooperation (Brahms clarinet sonata). Yvonne Borin for keeping things organized. Doc Anders Persson for rewarding discussion during my examination. Doc Rolf Hallin for nice skating tours, lunch talks and good sense of humor. Anders Jonsson for help with technical details and for sharing interest in gadgets.

Fellow students and former students, sharing the hardships of graduate studies, especially: Mats Ekstrand for high flying moments and Dr Jon A. Tsai for giving good advice and friendship.

To all my friends, who helped shaping me to the one I am.

My parents, Eva and Martin, who have raised me to become critically thinking and independent, for their love, care and support. My brother Christian for many fun moments and being the best of brothers.

Maria, the love of my life. For always believing in me, lifting me up, sharing all the wonderful moments in life as well as being a support during hard times. Utan dig skulle jag bara överleva- inte leva.

44 References

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