Chapter 6 Neurogenic mechanisms and neuropeptides in chronic pain

G. Carli and M. Zimmermann (Eds.) Progress in Brain Research. Vol. I10 0 1996 Elsevier Science B.V. All righa reserved.

CHAPTER 6

Neurogenic mechanisms and neuropeptides in chronic pain A. Dray Sandoz Institute for Medical Research, 5 Gower Place, London WCIE 5BN. VK

Introduction: neuropeptide containing afferent neurons All tissues are innervated by fine afferent fibres but the properties and physiological function of these fibres may differ depending on whether they are somatic or visceral afferents. A large subgroup of primary afferent fibres are nociceptive but this is less clear in visceral systems under normal physiological conditions. However a significant portion of afferent fibres become responsive once sensitised by irritants or inflammatory mediators (Habler et al., 1990; Schmelz et al., 1994). Most afferents also contain one or more peptides but the pattern of peptide coexistence and regulation is not well understood. Neuropeptide-mediated cell signalling is complex, and it is not yet clear what the entire range of functions is. Changes in neuropeptide synthesis and release are related to the pain symptoms which follow chronic inflammation and neuropathic injuries. These specific features of neuropeptide containing afferent neurones are discussed in this article but readers are also directed to several recent publications (Holzer, 1988; Willis and Coggeshall, 1991; Scott, 1992; Levine et al., 1993; Rang et al., 1994) that have described some the properties of sensory neurones in considerably greater detail.

Neuropeptides and the orchestration of inflammation Various products of tissue damage and inflammation stimulate afferent fibres to induce pain, hyperalgesia and to release neuropeptides which pro-

duce a variety of effects in the periphery. Some of the mediators of inflammation whose actions are best understood are summarised in Fig. 1. These include peptidic growth factors and cytokines released from target tissues and immune cells as well as neuropeptides released from primary afferents (substance P) and from sympathetic neurones (neuropeptide Y). The most comprehensively investigated of the sensory neuropeptides are substance P, neurokininA (NK-A) and calcitonin gene-related peptide (CGRP) which play a critical role in the responses elicited by sensory nerves. These are important for orchestrating a number of events that occur in inflammation (Dray, 1994) (Fig. 2). For example substance P causes vasodilatation in part via the release of NO from vascular endothelium. In addition contraction of endothelial cells in venules allows the extravasation of plasma, immune cells, and other active substances (bradykinin, ATP, 5HT, histamine) which thus gain access to the site of tissue injury. However vasodilatation and extravasation do not always occur together when fine afferent nerves are stimulated (Janig and Lisney, 1989) supporting the functional heterogeneity of afferent fibres and the release of different peptides. Indeed CGRP produces vasodilatation of arterioles with little direct effect on vascular permeability. The increases blood flow into venules results in synergistic actions with substance P in causing plasma extravasation (Brain and Williams, 1985; Gamse and Saria, 1985; Green et al., 1992). Mast cell degranulation, by substance P, also releases other inflammatory mediators including histamine and 5HT as well as proteolytic enzymes which

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target tissues

Spinal cord

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Fig. 1. Interactions between afferent nerve terminals and other cells and structures in peripheral tissues. The mediators responsible for most of the interactions are mainly peptides. Thus growth factors and cytokines are released from target tissues and immune cells while neuropeptides such as substance P, and neuropeptide -Y are released from peripheral afferent fibres and sympathetic fibres respectively.

catalyse the production of kinins. The latter may also evoke pain and induce a number of proinflammatory effects (Dray and Perkins, 1994; Rang et al., 1994). Substance P, NK-A and CGRP, when injected intradermally, are also capable of causing hyperalgesia (Nakamura-Craig and Gill, 1991).Presently, it is not clear whether this can be attributed to direct stimulation of sensory fibres or to a number of indirect mechanisms involving sympathetic nerve fibres or the vasculature. It is useful to remember that the effects of neurokinins may be complemented or modified by the concomitant release of other sensory neuropeptides. For example, galanin, somatostatin and neuropeptide Y may have inhibitory actions on afferent excitability and thereby reduce substance P release from sensory fibres during neurogenic inflammation (Gazelius et al., 1981; Green et al., 1992). This mechanism may be important in some pathological situations, e.g. rheumatoid arthritis, in which substance P plays a critic role (Levine et al., 1993) and where the expression of sensory neuropeptides may be altered by inflammatory processes. Lymphatic tissues are innervated by sensory fibres containing substance P and CGRP (Weihe et al., 1991) and it is likely that these neuropeptides regulate lymphoid tissues. Indeed the immune re-

sponse (secretion of antigen-specific antibody) to antigen challenge is reduced following pretreatment of rats with capsaicin, to selectively damage sensory fibres and deplete neuropeptides. The reduced antibody response can be restored by exogenously administered neurokinins (Helme et al., 1987; Eglezos et al.. 1991). Stimulation and recruitment of inflammatory cells is another important role of substance P in inflammation (Payan et al., 1983). For example, stimulation of cytokine production from monocytes (Lotz et al., 1988) leads to the expression and activation of adhesion molecules necessary for the attraction and movement of leukocytes along the vascular endothelium. Effects such as these offer an explanation for the observed close proximity of peptide-containing fibres to immune cells (Weihe et al., 1991). In addition, the persistence of macrophages in neuroma tissue following nerve cuts or ligations suggest that interactions between nerves and immune cells may be important for chronic pain symptoms as well as neuronal regeneration and remodelling (Friesen et al., 1993). The release of neurokinins has also been suggested to be involved directly in receptor mediated regenerative processes particularly in the control of NGF release, the regrowth of connective tissue and the revascularization that follows an injury (Nilsson et al., 1985; White et al., 1987; Ziche et al., 1990; Fan et al., 1993). Ultimately, the effects of neurokinins are medi-

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Fig. 2. Neuropeptides orchestrate peripheral inflammation and alter spinal excitability.

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ated through the activation of one or other of the specific neurokinin (NK) receptors, NKl, NK2 or NK3 to which substance P, NKA and NKB respectively are the preferred naturally occumng ligands (Maggi et al., 1993). So far, actions mediated by NKl and NK2 receptors have been implicated in the effects of neurokinins outlined above. This characterisation has been facilitated by the availability of several selective NK 1 (CP99994, RP67580, SR140333) and NK2 receptor antagonists (MEN 10376, FK888, SR48968) (Maggi et al., 1992). Little work has been done on the role of NK3 receptors, due to the present lack of specific pharmacological antagonists. However increased expression of NK3 receptors in the spinal cord has been demonstrated only after peripheral inflammation (McCarson and Krause, 1994). Interestingly the mismatch between neuropeptide release and synaptic receptors in the spinal cord suggests that effects via non-synaptic elements may also be of importance during inflammation (Liu et al., 1994; Valtschanoff et al., 1995). Indeed, NK receptor activation may induce the release of neuroactive substances (ref) including glutamate from glial cells made reactive by inflammatory processes. A specific pathophysiological role for neuropeptide-containing afferents, in the trigeminovascular system, has been suggested in the etiology of migraine (Moskowitz and Macfarlane, 1993). Thus activation of perivascular trigeminal neurones leads to release of a number of peptides (substance P, CGRP) producing vasodilatation and extravasation in the dura mater. Experimentally, the extravasation induced by capsaicin or trigeminal nerve stimulation can be suppressed by a number of drugs (sumatriptan, ergot alkaloids) whose activity correlates with their potency against clinical migraine. Vasoconstriction or effects on afferent excitability have been suggested to explain the mechanism of action of these drugs (Humphrey and Feniuk, 1991; Moskowitz and McFarlane, 1993). Recently, a role for neurokinins in plasma extravasation in the dura has been further supported by the fact that NK1 receptor antagonists (Moussaoui et al., 1993; Shepheard et al., 1993) potently reduce extravasation in experimental models.

Changes in peptidergic fibre functions imposed by tissue injury and inflammation During inflammation the events described above are likely to be amplified and the contribution of peptidergic afferent fibres increased. This is partly because of increased neural activity and peptide release; but more importantly because increased peptide synthesis and nerve sprouting occur as a result of increased trophin or growth factor activity (Noguchi et al., 1988; Donnerer et al., 1994). During inflammation or nerve injury the neuropeptide content of sensory nerves may be initially reduced (Gillardon et al., 1991) due to nerve activation and exhaustion of releasable stores of peptide. Following this, a number of changes occur due to the effects of NGF and other neurotrophins which are secreted in greater amounts (Donnerer et al., 1994) by a variety of cell types (fibroblasts, keratinocytes, Schwann cells) upon stimulation by inflammatory mediators such as the cytokines interleukin 1-p (ILl-p) and tumor necrosis factor -a (TNF-a). Increased NGF has been measured in several inflammatory conditions including pleurisy, rheumatoid arthritis as well as in blister fluid and in the skin following experimental inflammation (Woolf et al., 1994). NGF binds with a specific tyrosine kinase receptor (trk A) on small sensory neurones and is transported to the cell body where it stimulates increased mRNA production coding for neurokinin precursor peptides. This involves increasing gene transcription by stimulating transcription activators (e.g. Oct-2) or gene promoters (e.g. calgcat) (Watson et al., 1995). Tracer experiments suggest that increased amounts of neuropeptide may be secreted or leak from the terminals of afferent fibres both in the spinal cord (Valtschanoff et al., 1992, 1995) as well as in the periphery. NGF-induced sprouting of sensory fibres may further amplify these events (Diamond et al., 1992). The effects of NGF or other neurotrophins such as ciliary neurotrophic factor (CNTF) or neurotrophin-3 (NT3) are not exerted uniformly on sensory neurones, since their corresponding trk receptors trkA, trkB and trkC are heterogeneously distributed on different populations of neurones. However, it is likely that some

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neurotrophins affect sensory neurones by interaction which do not involve the known trk receptors (McMahon et al., 1994). NGF also produces a number of other indirect effects on afferent fibres via the release of proinflammatory mediators such as histamine and prostanoid derivatives such as leukotriene C4 (LTC,) from mast cells and leukocytes. As a result of these effects, NGF induces a prolonged increase in afferent fibre sensitivity and synaptic efficacy resulting in hyperalgesia particularly to mechanical stimuli. Consistent with this are studies showing that inflammation, or the injection of NGF increase tissue sensitivity and responsiveness to noxious stimuli. Treatment with anti-NGF antibodies reduce these effects (Lewin and Mendell, 1993; Woolf et al., 1994) and also prevent the increased substance P and CGRP mRNA expressed in sensory neurones (Woolf et al., 1994). In addition there may be secondary contributions to NGF induced hyperalgesia through opioid and kinin mechanisms (Apfel et al., 1993; Rueff and Mendell, 1994).

Changes in neuropeptide nerve function induced by nerve lesions Peripheral nerve lesions or nerve ligations, which remove or prevent the transport of the target tissue sources of NGF and possibly other neurotrophins, reduce neurokinin and CGRP synthesis in small sensory nerves. Deficits in substance P and CGRP are also seen in small afferent fibres following experimental diabetes induced by streptozotocin. This is partly due to the lack of NGF in skin and muscle as well as other metabolic and neurotrophic insufficiencies affecting fine afferent fibres. The peptide deficit can be corrected following insulin treatment (Fernyhough et al., 1994). The hyperalgesia associated with peripheral nerve injuries produced by nerve ligation can also be reduced by NGF infusions (Thomas et al., 1993) possibly due to an increase in the survival of damaged afferents. On the other hand NGF has been shown to be an important mediator of the hyperalgesia caused by inflammatory stimuli (Woolf et al., 1994) and treatment of normal animals with NGF induces

hyperalgesia to thermal and mechanical stimuli (Lewin and Mendell, 1993). Several mechanisms may account for this including overexpression and release of peptidergic mediators of nociception as mentioned earlier as well as the proliferation of sensory and sympathetic nerve fibres. While nerve lesions reduce the expression of substance P and CGRP, similar nerve injuries increase the expression of other peptides such as galanin, neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and their receptors which are not normally detectable in sensory neurones (Villar et al., 1991; Wakisaka et al., 1992; Nahin et al., 1994). This may be explained in part by the absence of target-derived inhibitory factors which normally suppress the expression of some neuropeptides. It is also conceivable that injured tissues induce hitherto unidentified trophins which are able to alter the phenotype of specific sensory neurones. Large myelinated neurones undergo a number of spectacular changes; they begin to express neurokinins (Marchand et al., 1994), they become abnormally excitable and discharge spontaneously, and they may sprout abnormally to innervate areas of the spinal cord which normally transmit specific pain signals (Woolf and Doubell, 1994). These changes are likely to contribute to post injury pain and allodynia following stimulation of low threshold A-fibres, but no particular correlation has been established so far between peptide release and A-fibre induced pain. Although it is not clear what function the newly expressed peptides serve; neurokinins are likely to be facilitatory, while other neuropeptides induce a net inhibition in the spinal cord to compensate for the increased excitability seen after peripheral inflammation or injury. For example, NPY which is normally present in sympathetic fibres, may be released both from the sprouting sympathetic fibres which innervate sensory neurones after peripheral nerve injury (McLachlan et al., 1993) and from large DRG neurones which express NPY after injury (Wakisuka et al., 1992; Itogawa et al., 1993). Inhibition may occur through NPY receptors which are upregulated on small DRG neurones (Mantyh et al., 1994; Zhang et al., 1994) and

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NPY may cause antinociceptive by blocking calcium conductance and transmitter release (Colmers and Bleakman, 1994). On the other hand NPY has also been associated with producing hyperalgesia following neuropathy but this appears to be via activation of Y2 receptors located on sympathetic fibres (Tracey et al., 1995). Interestingly, while peripheral nerve transection produces a decreased synthesis of neurokinins and CGRP, dorsal rhizotomy induces an increase in the synthesis of these peptides in sensory neurones. This would suggest that neuronal phenotype may also be regulated by centrally derived neurotrophins. So far however the identity of these substances has not been determined (Villar et al., 1991; Inaishi et al., 1992).

Sensory neurones and growth regulators Although much attention has been focused on neurotrophins and their role in sensory neurone regulation, the function of these cells may also be dramatically influenced by other cellular growth regulators. For example acidic fibroblast growth factor (aFGF) has been identified in sensory neurones (Elde et al., 1991) from which it may be released to affect nearby cells. Furthermore depletion of acidic FGF upon nerve injury may play a role in the pathophysiology of neuropathic pain as treatment with acidic FGF accelerates the regeneration and functional recovery of sensory fibres after nerve injury (Laird et al., 1995). The release of acidic FGF from sensory nerve terminals may also affect the growth of nearby tissues or act as a growth regulator for sensory neurones themselves, as acidic FGF receptors have been identified on sensory cells. In addition transforming growth factor a (TGFa) which is synthesised by skin keratinocytes, also facilitates sensory nerve regeneration as it enhances the survival of dorsal root ganglion cells grown in culture (Chalazonitis et al., 1992) and regulates the production of target tissue derived NGF (Buchman et al., 1994). Presently it is not known whether growth regulators exert specific effects on sensory neurone excitability but they have been shown to regulate early gene expression (c-Fos, c-Jun) (Gold et al., 1993; Lo and

Cruz, 1995) and thus are capable of producing phenotypic changes in sensory neurones. Whether this directly affects the expression of sensory neuropeptides is not known at present. Since constant regulatory interactions are likely to occur between sensory neurones and surrounding tissues, changes in sensory neurone function may develop because of some dysfunction in the neurochemistry of target tissue following injury. It is conceivable that such abnormalities also contribute to chronic pain.

Sympathetic neurons, neuropeptides and afferent fibres Sympathetic nerves are important in the generation of certain types of chronic pain, but the reasons for this are poorly understood (see review by McMahon, 1991). Interestingly sympathetic neurones, which contain catecholamine transmitters, may also normally make neuropeptides (e.g. NPY) but in addition are able to express other peptides when stimulated by inflammatory products such as cytokines. Curiously NGF is required for the survival of sympathetic neurones but does not normally cause the expression of neurokinins in these cells. However, another cellular regulator, leukaemia inhibitory factor (LIF) also generated during inflammation has recently been shown to induce the expression of substance P (Jonakait, 1994). In addition, a number of interactions between sympathetic and afferent neurones have been described. Thus, neurokinins, released from activated sensory neurones, may stimulate post ganglionic sympathetic fibres to alter vascular calibre, induce changes in local blood flow, and indirectly affect plasma extravasation. In keeping with this, sympathectomy reduces the plasma extravasation induced by either noxious stimulation or by the administration of inflammatory mediators. However, in the knee joint, the sympathetic transmitters noradrenaline and NPY reduced plasma extravasation (Levine et al., 1993); probably due to an inhibition of calcium permeability necessary for neuropeptide release (Colmers and Bleakman, 1994). Direct interactions of sympathetic nerves or sympathetic transmitters with afferent fibres have not been easy to demonstrate (McMahon, 1991;

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Treede et al., 1992) except after peripheral nerve damage or inflammation. Thus, afferent fibres can be sensitized during inflammation to induce hyperalgesia, partly by the release of prostanoids from sympathetic fibres (Levine et al., 1993). In addition, sympathetic nerve stimulation, or the direct administration of noradrenaline, excited some C-fibre afferents after a partially injury to a sensory nerve trunk (Sato and Perl, 1991) or after sciatic nerve transection (Devor et al., 1994). These effects were attenuated by phentolamine and block of a- adrenergic receptors which are presumed to be expressed on C-fibres (Sato and Perl, 1992) as well as on large A fibre afferents (Devor et al., 1994). Clearly, further characterization of the specific adrenoceptors expressed on afferent fibres is necessary.

Sensory neuropeptides in the spinal cord To date there is little evidence that neurokinins directly alter the excitability of the central terminals from which they are released. However, direct depolarization of DRG neurones by substance P has been described (Dray and Pinnock, 1994) and NKl receptors on primary afferents have been postulated (Malcangio and Bowery, 1994). These observations may explain why spinal administration of substance P and the depolarization of primary afferent nerve terminals enhanced plasma protein extravasation in the skin; though stimulation of other supraspinal pathways or preganglionic sympathetic neurones may also have contributed to this (Kerouac et al., 1987). In addition, the inhibition of afferent nerve excitability that was observed following substance P administration into the spinal dorsal horn may have been due to depolarization block of afferent nerve terminals (Davies and Dray, 1980). It remains an intriguing possibility that presynaptic actions of NKs may be important for regulating peripheral nerve excitability as well as the excitability of afferent terminals within the spinal cord. Other neuropeptides (e.g. somatostatin, opioid peptides) can inhibit afferent terminal excitability and thereby control transmitter release in the spinal cord. As in peripheral terminals, these substances act upon receptors,

often coupled with G-protein which regulate ionic conductances and thereby calcium coupled transmitter release (Levine et al., 1993; Rang et al., 1994). Most small (capsaicin-sensitive) primary sensory neurones terminate in the superficial dorsal horn but some fibres also project to the contralatera1 dorsal horn and a significant portion of these contain substance P (Ogawa et al., 1985). It is therefore very likely that ipsilateral stimulation of fine afferents induces contralateral excitability changes in the spinal cord, especially in inflammatory conditions where their signaling capacity can be increased by nerve sprouting and by increased peptide synthesis and release. Indeed, following the establishment of an inflammatory injury, the release of neuropeptides (substance P and neurokinin A) which is normally too small to measure, can be readily detected in the spinal dorsal horn but can also be detected at some distance from the site of release (Schaible et al., 1990). These data suggest that neurokinins are normally efficiently removed following neurosecretion and they remain restricted to the dorsal horn. However, during inflammation their sphere of activity may be significantly extended so that they can participate more extensively in increasing spinal excitability. In the spinal cord, post-synaptic receptors for neurokinins have been extensively characterised. Neurokinins play an essential role, together with glutamate which is also released from fine afferent nerve terminals, in enhancing the excitability of dorsal horn neurones (Urban et al., 1994). Normally neurokinins released by acute stimulation of nociceptors has little affect on the excitability of dorsal horn cells and any excitation is little affected by NK receptor antagonists. Repetitive stimulation of C-fibres and peripheral inflammation, which induces a prolonged activation of nociceptors, increases the appearance of NK1 receptors (McCarson and Krause, 1994) and enhances the sensitivity of spinal neurones to NKl antagonists (Thompson et al., 1994; Urban et al., 1994) (Fig. 3). Indeed, during inflammation, spinal excitability is increased by specific NKlMMDA receptor interactions on wide dynamic range, dorsal

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Fig. 3. This summarises the effects of the NMDA receptor antagonist D-APS and the changes in effectiveness of neurokinin receptor antagonists on spinal excitability in nalve rats and in animals that had received a peripheral injury with ultraviolet irradiation which induced hyperalgesia of the skin. D-APS (20pM) reduced the VRP in both n a h e and UV treated animals whereas RP67580 and CP-96345 (NKI antagonists) and MEN10376 (NK2 antagonists) only reduced responses following the induction of hyperalgesia. (modified from Thompson et al., 1994).

horn cells and by the release of glutamate and several inflammatory mediators from glial cells by NK1 receptor activation (Urban et al., 1994). In keeping with this, the NKl antagonists RP67580 and CP96345 are more efficacious in producing analgesia in conditions of chronic rather than acute pain (Birch et al., 1992). Several reports indicate that different modalities of noxious stimulation determine the type of peptide released in the spinal cord (Wiesenfeld-Hallin, 1986). For example, somatostatin release occurred only during heat or chemical stimulation whereas substance P was released by mechanical stimulation (Kuraishi et al., 1985). Such data further support the possible functional heterogeneity of nociceptive afferents but so far similar studies have not been performed during chronic pain to indicate what the pattern of neuropeptide release might be. This is an important issue which requires further investigation. Summary and perspectives

Fine afferent fibres contain a multiplicity of neu-

ropeptides whose functions, for the most part, are unknown. This discussion has focused on the neurogenic effects of the neurokinins which are localised in many afferents and which have been most extensively studies. The effects of substance P, exemplify the diversity of actions which have been described for sensory neuropeptides. In the spinal dorsal horn, substance P induces changes in excitability of afferent nerve terminals, spinal neurones and glial cells; events associated with the propagation of nociceptive signals and the modulation of acute and chronic pain responses. Following peripheral inflammation, neuropeptide synthesis and released are increased and there some indications that release may also occur without afferent nerve stimulation though the significance of this is unclear (Valtschanoff et al., 1992). On the other hand, reduced synthesis and release of neurokinins occur after peripheral nerve injury when target-tissue derived trophic factors are removed. In addition, there is evidence for the expression of several neuropeptides in small and large sensory neurones which are not normally found there. The significance of this for chronic pain or for the processes of regeneration after injury are unknown. In the periphery, a multiplicity of effects produced by the release of neuropeptides suggests that they orchestrate several components of inflammation. Indeed, sensory neuropeptides provide an important chemical interface for neuroimmune regulation. Neuropeptides also appear to have an emerging role in the regeneration and remodelling of peripheral nerves and tissues after injury, but further studies are necessary here. It is clear that all features of afferent nerve function can be tempered by their chemical environment and involves dynamic interactions between afferent (and sympathetic) fibres and surrounding tissue. This affects neural excitability, but perhaps more significantly over the long term there are changes in the peptide composition, concentration and release. The plastic features of afferents make difficulties for precise neurochemical and functional classification of different groups of fibres. However, these changes are likely to play an important role in producing the diversity of symptoms observed in chronic pain.

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