Visceral pain

British Journal of Anaesthesia 1995; 75: 132–144

Visceral pain S. B. MCMAHON, N. DMITRIEVA AND M. KOLTZENBURG Persistent pain of visceral origin is a much greater clinical problem than that from skin, but the overwhelming focus of experimental work on pain mechanisms relates to cutaneous sensibilities. Until relatively recently, it was often tacitly assumed that the ideas derived from cutaneous studies could be transferred wholesale to the visceral domain. However, there are several reasons to believe that the neural mechanisms involved in pain and hyperalgesia, and indeed our conceptual framework of these mechanisms, do not apply equally to superficial and to deep structures. First, the skin encounters mainly external perturbations and to these the organism can mount a defence behaviour. This consideration led Sherrington in his pioneering studies on pain to conclude that “pain seems the psychical adjunct to protective reflexes” [97]. Yet these external threats impinge much less on the body's viscera, which are more commonly exposed to the ravages of disease. In many cases, such as passage of a ureteric calculus or spasm of the gut, the protective nature of pain is less obvious, as it is not clearly associated with any effective countermeasures or “protective reflexes”. A second problem concerns the concept of nociceptive afferents. It was Sherrington again who postulated the existence of a specific set of receptors that would only be activated by tissue threatening or damaging stimuli and which would evoke flexion reflexes. Several decades after this hypothesis was formulated, direct experimental support was forthcoming for the existence of large numbers of such receptors in the skin. However, it is difficult to transfer the concept of a noxious stimulus to viscera since some major forms of life-threatening tissue destruction, such as perforation of hollow visceral organs or neoplastic processes, are frequently not painful and, conversely, some experimental stimuli which are painful (such as distension of the hollow viscera) are not always tissue damaging. A final reason to suggest that the mechanisms for cutaneous and visceral pain are different is that the nature of pain from these two tissues exhibits marked differences.

Clinical and experimental features of superficial and visceral pain Table 1 lists some of the key differences in the features of visceral and somatic pain. (Br. J. Anaesth. 1995; 75: 132–144) Key words Pain, visceral. Pain, somatic. Pain, psychological variables.

EFFECTIVE STIMULI

In the normal healthy person, a variety of intense stimuli applied to skin readily produce pain. These include mechanical and thermal stimuli that might be considered noxious (i.e. tissue damaging), as well as non-damaging events such as electrical stimuli and some chemical irritants. When the intensities of cutaneous stimuli are raised above threshold, pain does not usually radiate but, conversely, frequently becomes more focal, as can be appreciated if a pencil point is increasingly pushed against the skin. Deep somatic tissues, such as joint and muscle, are similarly sensitive to direct tissues-threatening stimuli. For instance, strong mechanical pressure on a muscle or distortion of a joint beyond its working range causes pain. Irritant chemicals, directly injected into muscles or joints, can also induce pain (see Mense [72]). The sensibility of some visceral tissue differs profoundly. Some structures, such as the lung, liver and the parenchymatous part of the kidney, appear not to give rise to pain at all, even after their gross destruction by malignant growth. The early surgeons, using only local anaesthesia of the body wall, were surprised to find that a wide variety of traumatizing stimuli, including crushing, cutting, tearing and burning, often failed to cause any sensation at all when applied to healthy visceral tissue [15, 63, 75]. (There are a few exceptions, for example the mesenteries are said by most authors to be sensitive to tension or clamping.) Viscera are, however, sensitive to other forms of stimulation, the best recognized of which is distension of the hollow muscular-walled organs. The gastrointestinal tract from oesophagus to rectum, the urinary tract from kidney pelvis to bladder, and the gall bladder, all yield pain upon distension [11a, 13, 19, 20, 29, 37, 53, 75b, 83, 87a, 91]. The severity of this distension-induced pain is often modest, at least in experimental studies on healthy subjects. Such pain arises with a short latency (measured in seconds), suggesting that indirect effects (e.g. ischaemia) are not to blame. Active contractions of smooth muscle around, for instance, an obstruction may exacerbate pain and result in pain that comes in waves, illustrated by the pain of labour. STEPHEN B. MCMAHON, BSC, PHD, AND NATALIA DMITRIEVA, BSC, MSC, Department of Physiology, St Thomas’s Hospital Medical School (UMDS), Lambeth Palace Road, London SE1 7EH. MARTIN KOLTZENBURG, BSC, PHD, MD, Department of Neurology, University of Würzburg, Josef-Schneider-Strasse 11, D-97080 Würzburg, Germany. Correspondence to S. B. M.

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Table 1 A comparison of the features of somatic and visceral pain (See text for full details)

Effective stimuli Summation Localization Primary hyperalgesia Secondary hyperalgesia

Visceral

Cutaneous

Direct trauma ineffective Distension and ischaemia effective Yes?? Local—diffuse Often referred Yes Yes, at site of referral

Direct trauma effective No Local—precise Not referred Yes Yes, around site of damage

Another effective stimulus for visceral pain is ischaemia, the best recognized example being that of ischaemic heart disease. It is likely, however, that ischaemia of other visceral tissues produces pain [52, 84]. With coronary occlusion there is the possibility of secondary, mechanical effects (for instance the spasm of arteries [78]) but it is frequently assumed that an important component of the stimulus is an accumulation of pain producing chemicals in the ischaemic tissue [60, 77]. A number of well recognized algogenic chemicals do produce pain when applied to human visceral tissues. The best studied is the naturally occurring agent bradykinin. This substance produces pain when infused into the abdominal cavity of healthy volunteers [53], although it is less clear if it is normally algogenic in the heart [77, 79]. Other irritants can produce pain in some viscera such as urinary bladder [34, 59, 76]. A final, and clinically important, circumstance where visceral pain may be triggered is in inflammatory states [34, 112]. In the urinary and alimentary tracts, inflammation is common and can be painful. In cystitis, for example, the sensations during bladder emptying often become unpleasant and painful [76, 80].

LOCALIZATION

SUMMATION

REFERRED PAIN

For noxious cutaneous stimuli, increasing the area over which a stimulus acts causes a modest increase in perceived pain but, importantly, the threshold for pain is not markedly reduced [85]. In the visceral domain one of the problems in applying the concept of “nociception” is that, for the hollow viscera, the distending pressures associated with pain are not tissue-damaging (e.g. approximately 35 mm Hg in the case of bladder distension). Estimates of the threshold pressures producing pain in a particular viscus often vary considerably. One reason is that the area of tissue stimulated may be a crucial determinant of threshold. Unlike skin, spatial summation may drastically reduce the effective threshold for pain. This viewpoint was already known to clinicians at the beginning of the century [28]. Comparisons of different studies in humans and animals suggest that spatial summation can appreciably lower the threshold for visceral pain [52, 81]. The existence of appreciable spatial summation of visceral inputs may explain the failure of localized mechanical stimuli, even frankly damaging ones, to produce pain.

In contrast with pain deriving from stimulation of skin, much visceral pain is localized to distant structures, a phenomenon known as referred pain. The area of referral is generally segmental and superficial, that is to muscle, skin or both, innervated by the same spinal nerves as the affected viscus. A classic example is the pain that develops shortly after myocardial infarction. While the initial pain in these cases may be felt as deep within the chest, with time (usually measured in minutes) it is often felt in parietal structures. Here it is still not well localized, but most often perceived as diffuse within the anterior chest and left arm. In some patients, with the passage of time, the referred pain becomes yet more superficial to involve cutaneous structures [86]. An important feature of referred pain is that the site of referral may additionally show hyperalgesia [28a, 34, 86]. This is true for both the pain referred to muscle and to skin. Such tenderness develops slowly, taking many minutes or even hours to become manifest and, equally, persisting for prolonged periods, measured in hours.

Pain of cutaneous origin is usually focal (i.e., with well defined boundaries), and well localized. Even if tactile cues are removed (by block of large diameter afferent fibres), a noxious stimulus to the skin of the hand can be localized to within 10–20 mm [44a, 52]. For visceral pain, two distinct types of localization have been noted, both distinct from that seen in skin. In some cases, visceral pain may be referred to distant structure, as described in more detail below. In other cases the pain is perceived as being deep within the body. This type of visceral pain, often called “true” visceral pain (or by early authors “splanchnic pain”, [92]), is usually perceived as arising in the midline, and perceived as anterior or posterior. One example is the initial sensation perceived after myocardial infarction [86]. Another is the early pain of appendicitis, initially felt in the midline. “True” visceral pain is usually extensive rather than focal (i.e. perceived over an area much larger than that of the stimulus) and with diffuse boundaries. It is frequently associated with a sense of nausea and illbeing. Autonomic and motor reflexes associated with deep pains are often extreme and prolonged.

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Figure 1 Summary diagram illustrating the various theoretical mechanisms of referred pain. See text for details. (From Morrison [75a].)

British Journal of Anaesthesia MacKenzie was convinced that the viscera were wholly insensitive and therefore that visceral afferent activity never of itself gave rise to pain. He proposed instead that this activity was capable of creating an “irritable focus” within the spinal cord, so that other, segmentally appropriate, somatic inputs could now produce abnormal and, of course, referred, pain sensations [63]. His theory did not find general acceptance, in part because it implicitly denied the existence of “true” visceral palm. However, the theory offers an explanation for referred hyperalgesia and, perhaps, the delay of referred sensations (allowing for the generation of an “irritable focus”). The concept of an irritable focus has more recently been resurrected with another label—central sensitization, which appears to be of major importance in hyperalgesia from somatic and visceral structures. A final view of referred pain is illustrated in figure ID, and suggests that interactions at supraspinal levels lead to the phenomenon [104]. However, the electrophysiological data we have do not suggest the existence of separate pathways from spinal cord to brain, an inherent assumption of this theory.

Mechanisms of referred pain A number of explanations have been offered for the existence of referred pain (fig. 1) The first case shown originated from Sinclair and colleagues [98]. The suggestion is that primary sensory neurones have widely bifurcating axons and innervate both somatic and visceral targets, thus obscuring the source of afferent activity, and explaining the segmental nature of referred sensations. This suggestion has repeatedly surfaced since that time. While there is some supporting evidence [11, 82, 102, 103], some of these findings have been challenged on technical grounds [22], and others have failed to find such axons, at least in any appreciable numbers. The only positive data for sensory neurones with receptive fields in two tissues comes from a study by Mense and colleagues [73] who reported a few single sensory neurones with both skin and muscle fields innervating the tail of the cat. Also, the hypthesis does not explain the time delay in the evolution of referred pain, or the referred hyperalgesia that frequently develops, since antidromic activity (that might invade the distant branch) does not appear capable of inducing a sensitization of peripheral terminals [74a, 88]. Another suggested mechanism for referred pain, and one for which there is considerable experimental evidence, is that visceral and somatic primary sensory neurones converge onto common spinal neurones (fig. 1B). This is the convergence-projection theory, suggested as such by Ruch [93], but derived from earlier ideas of Sturge [101] and Ross [92]. This theory proposes that the activity in ascending spinal pathways is misconstrued as originating from somatic structures and thus offers a ready explanation for the segmental nature of referred pain, but does not address explicitly the issue of referred hyperalgesia. A variation on the theme of convergence-projection (fig. 1C) derives from the ideas of MacKenzie [63] and is the convergence-facilitation theory.

PRIMARY HYPERALGESIA

In the wake of strong stimuli, the sensitivity of skin changes markedly. Previously innocuous stimuli become capable of evoking pain, and noxious stimuli produce more pain than in normal tissue. This phenomenon is called primary hyperalgesia. There is a wealth of experimental evidence that this hyperalgesia arises in part from a sensitization of primary sensory nociceptors. Surrounding the area of damage, skin also sometimes becomes more sensitive, a process called secondary hyperalgesia. The widespread area of secondary hyperalgesia that is sometimes seen, argues strongly that the underlying cause must lie not in the properties of primary afferents but within the central nervous system, and indeed there is considerable experimental support for such a view. The opportunities to observe such changes in sensitivity of visceral structures are much more limited, and it is not surprising that we have only meagre information on this point. None the less, there are a number of anecdotal reports that visceral structures may become hyperalgesic, particularly in inflammatory states. Kinsella [43] reported that direct mechanical stimulation of the inflamed, but not the healthy, appendix caused pain. Other reports exist for the ureter, kidney, bladder, ovary, stomach and oesophagus [34, 37, 40, 64, 94, 80, 112]. One example comes from the much-studied patient Tom, who had a gastric fistula. The gut mueosa of Tom was normally insensitive to pinching, but the same stimuli produced pain when the mucosa was inflamed. Quantitative studies of the increased sensitivity of inflamed viscera are few, but some data exist for patients with irritable bowel syndrome and non-cardiac chest pain [62]. The altered sensibility of visceral tissue in pathological conditions such as inflammation may indicate the emergence of new neurophysiological processes, a view for which there is growing experimental evidence.

Visceral pain Comment It is clear that the nature of pain from cutaneous and visceral tissues differs in a number of important respects. We do not know why this is so, although there are some general reasons one could propose: First, quantitative differences in the density of innervation (visceral structures probably have an innervation density of less than 1 % of that found in skin) may have a major impact in the precision of perceived sensations. It is also true that the tissues exhibit very different physical properties, such as their degree of viscoelasticity, and these may greatly affect the encoding properties of sensory fibres. Alternatively, the observed differences in sensory capacities may have arisen secondarily to the evolution of other systems. For instance, the skin is endowed with a numerically large and highly specialized system of fast conducting afferent fibres which, with great precision, relay information about tactile stimuli. This afferent system is associated with a large and detailed cortical representation. It may therefore be that the high degree of localization seen for cutaneous pain derives incidently from the existence of the accurate cortical map of the body surface. Finally, visceral and somatic systems may be fundamentally different for good teleological reasons—for instance that the tissues are normally exposed to very different types of stimuli and participate in different behavioural repertoires. In the remainder of this review we will consider experimental data relating to peripheral and central neuronal processing of visceral information. We will focus on one particular visceral organ—the urinary bladder—since it has been reasonably well studied, shows all the features of visceral pain described above, and we have an animal model of a persistent pain state associated with this structure.

The urinary bladder as a model for visceral pain Most of the time, the bladder has a storage function, slowly distending as urine forms. Bladder pressures usually vary between 0 and 15 mm Hg in this phase. Intermittently, micturition contractions empty the bladder. During normal micturition, intravesical pressure typically reaches about 25 mm Hg. Humans can perceive a variety of innocuous sensations associated with normal motility (for review see Jänig and Koltzenburg [41]). Contractions of the urinary bladder against an acute obstruction lead to increased pressures and are often painful. An estimate of the psychological pain threshold in healthy humans is therefore about 30–40 mm Hg. The urinary bladder receives a dual afferent innervation, with one set running with the sympathetic nerves (the hypogastric and lumbar splanchnic) and projecting to the thoracolumbar spinal cord, and another set running with the parasympathetic nerves (the pelvic nerve) and projecting to sacral segments. Despite the claims of many textbooks, there is much evidence demonstrating that all bladder sensations, including the pain of acute over-distension or cystitis, can be signalled by

135 pelvic nerve afferents [12, 20, 21, 30, 35, 80, 90, 114]; for a detailed review see Jänig and Koltzenburg [42]. The bladder also displays a very limited, and probably non-physiological, thermal sensitivity. Intravesical injections of chemical irritants can clearly evoke pain [34, 59, 76]. This chemosensitivity is likely to be of minor importance in normal bladder, but it may become important during tissue damage and inflammation. There are clear changes in bladder function and sensations occurring with inflammation. Bladder capacity is severely compromised and urgency and exaggerated frequency of micturition are hallmarks of cystitis. Moreover, the sensations associated with bladder emptying become painful. The pain of cystitis has a burning and disagreeable quality which is different from the vague and dull sensations evoked by acute over-distension of the normal bladder. There are also anecdotal clinical reports that the mucosa of the bladder displays increased mechanical sensitivity such that light pressure which is normally not perceived becomes painful [76, 80]. Thus, the urinary bladder might be considered an ideal representative example of visceral tissues in general, exhibiting as it does nearly all features of visceral pain, under both normal and pathophysiological conditions. The changes seen with cystitis suggest that new neural mechanisms of pain signalling emerge as a result of bladder inflammation.

Sensory mechanisms of the normal urinary bladder The physiological properties of primary afferents have been studied in detail by recording single afferent neurones in sacral dorsal roots or peripheral visceral nerves in cats and rats [9, 10, 24, 26, 27, 32, 33, 110]. All studies agree that pelvic afferents have very little or no resting activity when the bladder is empty. Increases of the intraluminal pressure excites mainly myelinated fibres in the cat, but both thin myelinated and unmyelinated fibres in the rat. The threshold for excitation of many fibres corresponds to the pressure values when humans start to appreciate a sensation of bladder fullness and virtually all units respond in the pressure range that is reached during non-painful micturition. A typical record of the response properties of this type of afferent is shown in figure 2. In both cat and rat the units display monotonically increasing stimulus response functions covering the entire spectrum of the magnitude and temporal profile of the biologically relevant changes of intraluminal pressure. In the cat, there is a striking dichotomy between myelinated and unmyelinated fibres innervating the pelvic organs. Out of several hundred fibres that were shown (with an unbiased electrical search procedure) to project into the pelvic nerve and therefore to innervate pelvic organs, less than 3 % responded to changes of intravesical pressure up to 75 mm Hg. These few mechanosensitive C-fibres also behaved very differently from the myelinated visceral fibres in that they had significantly higher thresholds, between 30 and 50 mm Hg. The unmyelinated fibres showed a slowly adapting dis-

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British Journal of Anaesthesia

Figure 2 Example of a typical myelinated mechanosensitive afferent unit of the pelvic nerve responding to filling and distension of the urinary bladder of the cat. (A) The activation of the unit (upper trace) during a spontaneous contraction of the bladder (intravesical pressure shown in lower trace). (B) Neural activity and corresponding. intravesical pressure evoked by slow filling (2 ml min91) of the bladder and isotonic distension. Arrow indicates emptying of the bladder. (C) Stimulus-response function of this unit for filling and distension. This relationship is typical for the homogeneous population of myelinated mechanosensitive afferents. (!) filling; (–––) distension.

charge and could only encode intravesical pressure changes that are at the top end or even beyond the biologically relevant range of intravesical pressure. Pelvic afferents in the rat showed a similar, but quantitatively less marked, distinction [24]. Afferent units recorded in cats and projecting in the hypogastric or lumbar sympathetic nerve exhibited very similar properties to the thin myelinated pelvic afferents. That is, they usually had thresholds in the innocuous range and show a graded coding of intravesical pressure extending into the noxious range. Some of the units displayed ongoing activity even in the absence of tissue perturbation. Thus, it seems likely that primary afferents innervating the normal urinary bladder are largely a homogeneous population of afferents responding in a graded fashion to increases of intravesical pressure throughout the biologically relevant range. Therefore, unlike somatic tissues there are only few fibres that might be called specific nociceptors. Given their small percentage and their stimulus response function, it is questionable whether these fibres contribute significantly to pains of acute over-distension. Together, these observations suggest that these visceral afferents encode information according to an “intensity” rather than “specificity” theory. This concept represents an important departure from normal textbook dogma, for instance implying that the pain threshold might be variable and set by the CNS and not determined principally by the recruitment of a particular high threshold population of afferents. The responses of spinal cord neurones to activation of bladder afferents have been examined in a number of animal species and under a number of differing conditions [17, 66, 70, 71, 74]. As might be expected, neurones responding to urinary bladder inputs are concentrated in the sacral and upper

lumbar segments of the spinal cord. In these segments, most neurones responsive to bladder distension have thresholds clearly in the innocuous range and threshold distribution of spinal neurones are like those of mechanosensitive primary afferent units [70, 71]. Also like the primary afferents, they usually encode levels of distension throughout the physiological and into the supra-physiological, presumably noxious, range. In cat and rat, there are also neurones which are activated by synchronous afferent volleys generated by electrical stimulation of the pelvic nerve, but which do not respond to distensions or contractions of the bladder [66, 70, 71]. Some of these cells might have peripheral receptive fields in other visceral structures, but it is likely that some simply receive only weak connections from bladder afferents (which, of course, discharge asynchronously during natural stimuli and are thus not capable of bringing the cell to threshold). The urinary bladder does not have a dedicated, private, spinal transmission. Rather, bladder information is represented in a number of well recognized somatosensory pathways such as the spinothalamic tract, spinomesencephalic tract, etc. [107]. This statement is also true on a single, neurone basis, for practically all cells studied to, date which had a visceral receptive field also had a somatic one; that is, somatovisceral convergence is the norm. The somatic receptive fields are sometimes exclusively low threshold and sometimes contain high threshold components. An interesting question, to which we do not presently have an answer, is how the central nervous system selectively extracts information about individual peripheral targets, given this extreme convergence onto spinal neurones. In other respects the existence of somatovisceral convergence has some explanatory power: it is very probably the substrate for referred pain, as discussed above. It

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Figure 3 Schematic summary of the changes seen in peripheral and central sensory neurones following experimental inflammation of the urinary bladder. In the periphery, inflammation increases the slope of the stimulus-response functions of the mechanosensitive afferents, and additionally leads to the recruitment of previously mechanically insensitive afferents. In the spinal cord, the stimulus-response functions of dorsal horn cells also show excitability increases, both to bladder and convergent somatic ones. See text for fuller details. (–––) normal bladder; (– – –) inflamed bladder.

should be realized, however, that convergence could underlie other phenomena, such as the integration of somatic and visceral information in reflex function. Figure 3 shows schematically the encoding properties of peripheral and spinal neurones representing the urinary bladder.

Changes in the inflamed urinary bladder—mechanisms of persistent pain To investigate the neural mechanisms involved in persistent visceral pain states such as cystitis we have used an animal model of bladder inflammation, generated by exposing the mucosal surface of the bladder to irritant chemicals. A number of agents including turpentine oil, mustard oil, croton oil or xylene have been used [2, 44, 58, 67]. When used in dilute concentrations there is little tissue destruction [32, 44]. With these treatments, there is an initial neurogenic extravasation resulting in vasodilatation and plasma extravasation to which primary sensory neurones contribute [44, 57, 67], followed by a genuine inflammatory response, with infiltration of the bladder by polymorphonuclear leucocytes, which can persist for days. Animals show a number of sensory and reflex changes that are similar to changes occurring in cystitis in humans [44, 67]. These include increases in bladder motility and increased frequency of micturition. These reflex changes are of a neurogenic origin as they disappear after denervation. Animals also display a behaviour consistent with a persistent urinary tract irritation, such as excessively grooming or licking the ventral abdomen and periurethral area. Pelvic afferent neurones have been studied at the onset of an acute experimental inflammation [31, 32, 33]. Myelinated mechanosensitive afferents were vigorously excited by intraluminal application of irritants such as mustard oil with a short delay of typically less than 5 min. Afferents displayed on-

going activity when the chemical irritant was removed from the bladder. Ten min after mustard oil application, the mean frequency of these fibres was 2.1 impulses per second. Such discharge frequencies would be elicited by an intravesical pressure of 10–20 mm Hg in the normal non-inflamed bladder, suggesting that the persistent firing could contribute to the frequency and urgency of cystitis. In addition, some mechanosensitive units displayed sensitization and responded at lower thresholds or exhibited a stronger discharge at suprathreshold stimuli which might correspond to the increased pain sensitivity of bladder contractions experienced during cystitis. SLEEPING NOCICEPTORS

The existence of large numbers of unmyelinated afferent neurones with no sensitivity to physiological or supraphysiological pressure changes in the bladder (so-called “silent afferents”, or “sleeping nociceptors”), raises the possibility that some of these neurones are recruited only in pathophysiological states. This notion has been examined by studying these fibres at the onset of an experimental inflammation. After intraluminal injections of chemical irritants some 10 % of more than 100 “silent” fibres studied in the pelvic nerve were excited and the average discharge rate was 2.8 impulses per second. Some of the units also developed a novel mechanosensitivity in the biologically relevant pressure range. Four times as many unmyelinated afferents can be activated during the pathophysiological state compared with non-inflamed condition. The activation of a numerically significant population of afferents at the onset of an acute inflammation indicates that peripheral afferent mechanisms encoding visceral pain are highly malleable and are strongly affected by the state of the tissue. The importance of the recruitment of mechanically insensitive visceral

138 afferents in inflammatory pain states is substantiated by quantitative estimations of primary afferent activity which also provides further evidence for different primary afferent mechanisms signalling pain from normal and inflamed tissue. One can estimate that in the normal state, severe bladder distension (to 50 mm Hg) induces a total of about 4500 action potentials per second, only 5 % of which will be contributed by unmyelinated afferents. However, after experimental inflammation, the total afferent inflow to the same stimulus is likely to be doubled, and now unmyelinated afferents are carrying the majority of these impulses. It is likely that these novel inputs also contribute to the qualitatively different sensations experienced by patients with cystitis. One might speculate that afferents which do not respond to transient noxious events rather sample the state of the tissue and provide long-term signals that are important for the setting of the excitability in the CNS [109]. SPINAL PROCESSING OF INFORMATION FROM THE INFLAMED BLADDER

Dorsal horn neurones in the rat lumbosacral cord that responded to electrical stimulation of the pelvic nerves were studied before and during the onset of bladder inflammation by McMahon [66]. A number of changes in response properties of these cells were observed. First, the cells frequently showed a progressive increase in the level of their ongoing activity, starting some 5–10 min after the inflammatory stimulus and persisting for the duration of recordings. Second, about 50 % of the neurones showed increased responses to bladder distension. In some cases this took the form of a leftward shift in the normal stimulus-response function, while in others it was seen as the development of an entirely novel mechanosensitivity. This increased responsiveness also began with a relatively short latency (15–30 min). These changes are reminiscent of those seen in primary afferent fibres but they are likely to involve at least a component of central change. This is because, in the presence of inflammation, the neurones showed enhanced responses to electrical stimuli applied to the pelvic nerves (stimuli which bypass the peripheral terminals of the afferents and provide a constant input volley). Additional evidence for a central sensitization comes from the observation that the somatic receptive fields of some neurones are enlarged or show reduced thresholds. The somatic afferents forming these receptive field components are, of course, unaffected by the intravesical irritants. Thus, there is evidence for a slowly developing and maintained increase in the excitability of central neurones following bladder inflammation. The most likely trigger for this change is the afferent barrage induced in the peripheral C-fibres innervating the inflamed bladder. In a number of other tissues, inflammatory stimuli activating C-fibre afferents have been found capable of inducing very similar central sensitizations. In all cases examined to date, the pharmacology of the central change appears similar. The phenomenon can be blocked by the application of antagonists of excitatory amino acids

British Journal of Anaesthesia acting at the N-methyl-D-aspartate (NMDA) receptor [89]. Once the central changes have become established they may become independent of the peripheral input necessary to initiate them. If this is the case it will of course have major implications for therapy aimed at treating inflammatory conditions. However, it is presently unclear whether central sensitization selectively affects sensory discriminative components of pain or other forms of sensory processing, such as afferent control of bladder motility and generalized autonomic responses. In figure 3 we summarize the peripheral and central changes occurring with inflammation of the urinary bladder. It is less clear what aspect of tissue inflammation is capable of promoting these changes in sensory processing. In the final section of this chapter, we provide evidence for an important role of one particular factor, nerve growth factor, which is upregulated in inflamed tissues.

Nerve growth factor as a mediator of persistent visceral pain (table 2) Nerve growth factor (NGF) is a member of a small family of secretory proteins, known as, the neurotrophins. In mammals, the other currently identified members are brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neuretrophin 4/5 (NT-4/5) [56, 105]. The neutotrophins appear to exert their biological action through interactions with specific receptors. One low affinity receptor exists which binds all neurotrophins with equal affinity. This is generally referred to as the p75 receptor. Additionally, there is a small family, of structurally related high-affinity receptors which exhibit considerable specificity for the different neurotrophins. These are part of a larger group of tyrosine kinases and are known as “trks”. Currently three trks have been well characterized, trkA, trkB and trkC [8, 46, 105]. NGF binds selectively to trkA, BDNF and NT4/5 bind to trkB and NT3 binds with highest affinity to trkC. ROLE OF NGF AND OTHER NEUROTROPHINS IN DEVELOPMENT

One important role of neurotrophins, during early stages of ontogeny, is as target-derived trophic factors. That is, these factors are produced by the targets of neurones, in limited amounts, and their uptake and retrograde transport to the neuronal cell

Table 2 Evidence that nerve growth factor (NGF) is a mediator of persistent visceral pain 1. 2. 3. 4. 5. 6. 7.

NGF upregulated in bladder inflammation NGF receptors found on nearly all pelvic afferents Exogenous NGF mimics motility changes seen in inflammation Exogenous NGF mimics extravasation seen in inflammation Exogenous NGF activates and sensitizes pelvic afferents—including “silent” afferents Exogenous NGF upregulates sensory neuropeptides Sequestration of NGF prevents motilitiy changes in inflammation

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body is seen as an important route for influencing neuronal phenotype and, in the extreme, cell survival. This has been demonstrated by a variety of approaches. Recently, a series of transgenic animals with disrupted genes encoding the different neurotrophins and trks has been described. In the case of NGF, these studies show that the molecule is necessary for the survival of sympathetic and small diameter sensory neurones of neural crest origin [18, 99]. The limited availability of neurotrophins in vivo is believed to regulate the density of peripheral innervation of some targets. It is also becoming clear that the neurotrophins may have other roles in development. For instance, the availability of neurotrophins during critical periods of development can influence the phenotypic character of some sensory neurones [16, 49, 50]. ROLE OF NGF IN THE ADULT

In the adult, neurotrophins continue to be expressed by peripheral tissues, at low levels, and neurotrophin receptors are still expressed on peripheral neurones. There are now several careful studies on the expression of high affinity NGF receptors (trkA) on adult primary sensory neurones. Binding studies, in situ hybridization and immunohistochemistry all suggest that about 40 % of L4/L5 sensory neurones express trkA [7, 69, 108]. Moreover, these neurones are predominantly small in diameter. For these somatic afferents, trkA is expressed highly selectively in, neurones containing calcitonin gene-related peptide (CGRP) [69]. We know that these small neurones mostly have unmyelinated axons, are connected to nocieeptive terminals in peripheral targets and that CGRP is released from central terminals with noxious stimuli. Thus, trkA appears to be selectively expressed on nociceptive afferents. We have also studied trk expression in visceral afferents innervating the urinary bladder and projecting through the pelvic nerve. We identified these afferents by retrograde labelling with fluorogold, and then subjected tissue sections of L6/S1 dorsal root ganglia to in situ hybridization with riboprobes specific for different trks [69]. We found, rather surprisingly and in contrast with somatic afferents, that over 90 % of these visceral afferents expressed trkA. Interestingly, these same afferents also expressed trkB. Of course, this very large population must include both A␦ and C afferents to the bladder. Thus, nociceptive afferents in general, and visceral afferents in particular, have the capacity to respond to NGF. NGF SENSITIZES PRIMARY SENSORY AFFERENTS

Since nociceptors express trkA, a logical question is whether NGF activates or sensitizes the peripheral terminals of these fibres. Systemic applications of NGF can produce a cutaneous hyperalgesia in animals and humans [50, 81a]. We have recently injected relatively small doses of human recombinant NGF (0.5 ␮g) into the footpad of rats and similarly seen thermal hyperalgesia which is apparent after 10 min [5]. We have also recently studied the

Figure 4 Average stimulus-response curves of A␦ fibres (A) and unmyelinated C-fibres (B) to isotonic distensions before and at various times after exposure to vehicle of intravesical nerve growth factor (NGF) (0.5 ml of 10 ␮g ml91). Error bars show SEM. Note the marked sensitization (leftward shift) of the response curves after NGF but not after vehicle. The error bars become larger at long times after NGF treatment primarily because progressively smaller numbers of units were followed for these long times (from Dmitrieva and McMahon [24]). (⫻) Control, (") DMSO, (!) 30 min after NGF, (#) 2 h after NGF, ($) 3 h after NGF.

responses of single visceral afferents, projecting to the urinary bladder via the pelvic nerve, to peripheral administration of NGF [24]. A group of 15 A␦ and 12 C-fibres were studied in urethane-anaesthetized adult rats. Their responses to a series of graded distensions of the urinary bladder (through the physiological and into the supraphysiological range) were used to construct stimulus-response functions. The same units were then studied 0.5 to 1 h after exposing the luminal surface of the bladder to a 10 % DMSO solution, the vehicle used to subsequently deliver NGF (10 ␮g/ml) intraluminally. The responses of these rat bladder afferents in the control states were qualitatively similar to those previously seen by us in cat; that is, nearly all A␦-fibres were mechanosensitive, and they encoded both innocuous and noxious levels of distension (fig. 4). Of the Cfibres, nearly half had no significant mechanosensitivity in the control state and the remainder, while encoding innocuous and noxious levels of distension, exhibited significantly lower rates of discharge than the A␦ afferents. DMSO treatment

140 had no significant effects on afferent sensitivity. After intraluminal NGF (in DMSO to aid penetration into bladder tissue), 91 % of units were sensitized to increases of intravesical pressure, both initially mechanosensitive and insensitive afferents (fig. 4). The increased sensitivity was shown by a leftward shift in the stimulus-response function and, in some cases, the development of low levels of ongoing activity when the bladder was empty. The increased sensitivity was apparent by 1 h after NGF treatment, and persisted for the period studied (up to 3 h). The same intravesical NGF treatment (in other experiments) produced significant neurogenic extravasation, measured 1 h after treatment. Since series of bladder contractions does not produce neurogenic extravasation, it is likely that chemosensitive primary afferents mediate that NGFevoked extravasation. Thus it appears that NGF, given exogenously, can activate vesical afferent systems. The rapid onset of effects, and the fact that they are seen with peripheral administration, suggests that they arise because of peripheral sensitization of primary afferents. MECHANISM OF NGF-INDUCED SENSITIZATION

One way in which this might occur, of course, is via an interaction with trkA receptors on the terminals of nociceptive afferents. However, it is also possible that other tissue elements may participate in a cascade that ultimately sensitizes afferents by some other mediator(s). In this context, it is interesting that trkA receptors are found on two other peripheral structures: some forms of mast cell [39] and on the terminals of sympathetic postganglionic axons [105]. It is well recognized that mast cell products such as histamine or serotonin have the capacity to excite and sensitize some nociceptive afferents, and there is some evidence that postganglionic sympathetic fibres contribute to the sensitization of primary afferent neurones [36, 45]. Recently we have demonstrated a contribution of sympathetic and mast cells in the mechanism of NGF sensitization of primary somatosensory and visceral afferents [Dmitrieva and McMahon, unpublished observations]. We used guanethidine and compound 48/80 to impair sympathetic fibres and mast cells function, respectively. We were able to show that plasma extravasation following intravesical injection of NGF was almost abolished in guanethidine pretreated rats and attenuated up to 86 % in 48/80-treated rats. On the other hand, bladder extravasation to NGF was also largely abolished following neonatal capsaicin treatment (which destroys most unmyelinated afferent neurones), suggesting a direct action of NGF through its receptor on primary afferents. CENTRAL SENSITIZATION BY NGF

NGF may also be capable of affecting pain signalling systems by producing central sensitization. Of course, the rapid activation and sensitization of nociceptive afferents is itself likely to trigger increases in central excitability. In fact, we have evidence that this is the case in the somatic nervous

British Journal of Anaesthesia system, where NGF injected into the cutaneous receptive field of convergent dorsal horn neurones produces a rapid enhancement of the neurone's responsiveness to heat stimuli [6]. However, in addition to excitatory effects on the peripheral terminals of sensory neurones, NGF may also indirectly affect central processing of information from afferent neurones. Following binding to receptors, NGF is internalized by neurones and retrogradely transported to neurpnal perikarya. For adult sensory neurones in vivo, it appears that only a restricted population of mainly small sensory neurones (probably just those expressing trkA) can transport NGF retrbgradely [23]. At the level of the cell body, NGF upregulates a number of sensory neuropeptides such as CGRP and substance P. This has been well studied in vitro where the levels of sensory neurone peptides and their controlling mRNAs are closely linked to NGF concentrations in the medium [55]. There are some data available regarding the actions of neurotrophins in vivo [4]. The addition of NGF to peripheral targets for several days can induce an increased expression of peptides such as substance P in sensory neurones [78a]. A role for NGF in regulating neuropeptide expression is further supported by the observation that administration of anti-NGF antibodies in vivo also down-regulates the production of some neuropeptides in dorsal root ganglion cells [78a, 89a]. There is additional support for the hypothesis that these same sensory neuropeptides, when released from the central terminals of sensory neurones, can contribute to the process of central sensitization, whereby the processing of nociceptive inputs is potentiated in dorsal horn neurones [69]. There is, therefore, good circumstantial evidence that NGF availability may contribute indirectly to central sensitization. This is also apparent in recordings from dorsal horn neurones which exhibit an increased responsiveness after chronic application of exogenous NGF [51, 106]. UPREGULATION OF NGF IN INFLAMMATION

Although much of the evidence presented above is consistent with a specific action of NGF on peripheral nociceptive systems, the possibility that endogenously produced neurotrophin may actually fulfil such a role depends in part on the demonstration that it can be upregulated in appropriate pathophysiological states. In fact, there are now data from several different research laboratories showing that in inflammatory states, NGF expression is increased. This has been seen as an increase in NGF protein in the affected tissue [3, 111] and also as an increase in the amount of NGF transported retrogradely in nerves supplying the inflamed tissue [25]. The concentration of NGF in inflamed tissue has been measured by different methods, among which the two-site enzyme-linked immunosorbent assay (ELISA) is the most reliable [111]. Results obtained with this method show an increase of NGF concentration in various inflamed tissues of 10, 100 and even 1000 times normal levels [3, 25, 113]. The NGF produced in inflammation may derive from a

Visceral pain

141

Table 3 Sources of nerve growth factor in inflamed and damaged tissue Cell types

Tissue

References

Lymphocyte-like cells

Synovial tissue of patients with rheumatoid arthritis Various sources Peripheral nerve after axotomy Conditioned cell culture Hypertrophied rat and human bladder Sciatic nerve after transection Nerve sheath after transection of the sciatic nerve and conditioned cell culture

Aloe et al. [3]

T and B lymphocytes Macrophages Peritoneal mast cells Parenchymal cells Fibroblast-like cells Schwann cells

variety of sources (table 3). Macrophages and mast cells are apparently capable of producing NGF. It may also derive from Schwann cells in the nerve trunks in the tissue. There is clear evidence that these cells can produce NGF in some cases (e.g. nerve injury) under the influence of IL-1 [54] which is itself increased in inflamed tissue. In rats with localized Freund's adjuvant arthritis, the increased retrograde delivery of NGF is associated with increased sensory neuropeptide concentrations in these neurones, and importantly, these neurochemical changes are prevented in animals with adjuvant arthritis treated with neutralizing antibodies to NGF [25, 113].

Santambrogio et al. [95] Brown et al. [14] Leon et al. [48] Steers et al. [100] Heumann et al. [38] Heumann et al. [38) Matsuoka et al. [61]

cellular domain of trkA receptor coupled to the Fc moiety of IgG (“trkA receptor body” or trkA-IgG), to block endogenous NGF actions [96]. We have shown that this molecule, given systemically before an experimental inflammation of the bladder is induced, prevents the development of exaggerated bladder reflexes [unpublished observations]. The actions of NGF in inflammatory states may provide new opportunities to understand particularly long-term changes that occur in sensory processing in such states, and also offer possibilities to generate new therapeutic strategies to treat inflammatory pain.

Acknowledgements POTENTIAL ROLE OF NGF IN CHRONIC CYSTITIS

We have used turpentine inflammation of rat urinary bladder as a model of chronic cystitis. This chronic disease, of unknown aetiology, is characterized by well described symptoms: reduced micturition threshold, increased voiding frequency, decrease of bladder compliance and visceral pain [47, 87]. Injection of turpentine into the bladder lumen of animals reproduces these symptoms within a few hours [67, 68]. Interestingly, exogenous NGF given into the bladder significantly increases bladder reflexes with a similar time-course (unpublished observations). We have used in situ hybridization to localize mRNA for NGF in the bladder wall, and found that within a few hours of the turpentine stimulus, the concentrations of NGF mRNA are markedly increased [6]. This increased expression is relatively short lasting in this model and levels return to control within about 24 h, in parallel with the behavioural and reflex changes [67, 68]. SEQUESTRATION OF NGF BLOCKS THE DEVELOPMENT OF INFLAMMATORY CHANGES

A final and perhaps critical piece of evidence to establish NGF as a mediator in inflammatory conditions, is whether pharmacological block can prevent or reverse the consequences of inflammation. It has already been pointed out that anti-NGF treatment blocks some of the histochemical changes occurring in small afferent neurones in adjuvant arthritis [25]. There are also recent reports that the behavioural hyperalgesia that develops in adjuvant arthritis can be reduced by treatment with blocking NGF antibodies [49, 113]. We have recently used a synthetic fusion molecule, composed of the extra-

Some of the work described in this review was supported by grants from the Wellcome Trust, the MRC of Great Britain and the Deutsche Forschungsgemeinschaft (SFB 353). We would also like to thank Genentech Inc. for the kind gift of riboprobes against neurotrophin mRNA and rhNGF.

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