Future trends in pain research

In the future we shall need to modify the classical view that nerve impulses which signal the presence of injury are reliably transmitted by specified and automatic relay cells. We must investigate at least four different modifying mechanisms that are likely to generate chronic intractable pains. With a latency of milliseconds, combinations of afferent signals and of descending controls operate a rapid and powerful gate control. With a latency of minutes, impulses in C fibres change the excitability of peripheral endings and of spinal cord circuits. With a latency of days, chemical transport in C fibres from areas of damage further modifies cord connectivity with a disappearance of inhibitions and an expansion of receptive fields. With a latency of weeks and months, anatomical degeneration produces secondary changes in deafferented cells with atrophy, sprouting and abnormal firing patterns.     

Capsaicin application to central or peripheral vagal fibers attenuates CCK satiety

Capsaicin treatment destroys small primary sensory neurons including a subpopulation of vagal afferents. Intraperitoneal, fourth ventricular or perivagal application of capsaicin attenuated or abolished cholecystokinin (CCK)-induced suppression of food intake. Capsaicin applied to the thoracolumbar spinal cord or to the pyloric region of the stomach did not alter CCK-induced reductions of food intake. Intraperitoneal capsaicin treatment reduced substance P-like immunoreactivity (SPLI) in the spinal dorsal horn and parts of the dorsal hindbrain. SPLI depletion, therefore, served as a histochemical indicator of the spread of capsaicin from its site of application. Capsaicin applied directly to the vagal trunks did not reduce SPLI in the spinal cord or hindbrain. Intraventricular capsaicin reduced SPLI in the hindbrain but not in the spinal cord. These data indicate that localized capsaicin application attenuates CCK-induced suppression of food intake by impairing the function of either central or peripheral portions of vagal afferent neurons. The data also support the conclusion that intraperitoneal capsaicin attenuates CCK-induced suppression of feeding by impairing vagal sensory function.     

Mechanisms of depletion of substance P by capsaicin

Capsaicin is a neurotoxin that can deplete sensory nerves of their content of substance P and interfere with certain sensory functions, such as responses of animals to noxious heat stimuli. In adult guinea pigs, a species that is susceptible to the effects of capsaicin on both substance P content and sensory function, capsaicin induces selective depletion of substance P from dorsal root ganglia and the dorsal spinal cord, sites of the cell bodies and central terminals of primary afferent neurons, respectively. As the onset of thermal analgesia in guinea pigs precedes depletion of substance P, direct neural actions of capsaicin probably account for its effects on sensory function. Capsaicin interferes with the retrograde transport of nerve growth factor (NGF) to the cell bodies of sensory nerves. Decreased availability of NGF at the site of neural protein synthesis leads to decreased synthesis of substance P. After failure of synthesis of substance P, the content of the peptide in sensory nerves gradually decreases until depletion occurs.     

Neurotoxic effect of capsaicin in mammals

Capsaicin is now widely used to explore and/or prove the role of peptide-containing primary afferent neurones in different somato- and viscerosensory functions. The present paper deals with the morphological effects of capsaicin administered according to currently used experimental paradigms. As it has been repeatedly confirmed in the recent literature, administration of capsaicin to newborn mammals results in a highly selective degeneration of a particular population of small sized, B-type primary afferent neurones located in spinal and cranial sensory ganglia. Chemosensitive i.e. capsaicin sensitive primary sensory neurones (CPSNs) correspond to primary sensory ganglion cells which contain neuropeptides. The permanent functional impairments and the decrease in the peptide contents of the sensory neurones observed after neonatal capsaicin treatment may be accounted for an irreversible loss of CPSNs. Direct application of capsaicin to peripheral nerves results in an apparently irreversible functional impairment of unmyelinated afferent fibres implicated in nociceptive, viscerosensory and neurogenic inflammatory mechanisms. Morphological observations indicate that perineural treatment with capsaicin initiates a selective but delayed degeneration process of unmyelinated afferent nerve fibres presumably due to an inhibition of intraneuronal transport mechanisms. In contrast with perineural capsaicin treatment affecting the chemistry and function of the whole sensory neurone, injection of capsaicin into the subarachnoid space results in an irreversible abolition of the "afferent" but not the "efferent" function of CPSNs. Accordingly, noxious thermal or chemical stimuli applied to the peripheral innervation areas of the trigeminal nucleus caudalis or the affected segments of the spinal cord fail to induce nociceptive reflexes because of the degeneration of the central terminals of CPSNs. However, in these same skin areas, application of chemical irritants invariably evoked the neurogenic inflammatory response, indicating that CPSNs deprived of their central terminals maintain their capacity to synthesize and release the peptide(s) responsible for the initiation of that response. In contrast with previous findings, our recent studies furnished evidence for a selective neurodegenerative action of systemically injected capsaicin in adult mammals, as well. Therefore, some of the irreversible functional impairments produced by capsaicin in adult animals may result from the degeneration of a particular subpopulation of CPSNs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Capsaicin-sensitive adrenal sensory fibers participate in compensatory adrenal growth in rats

Compensatory adrenal growth, in which one gland undergoes hyperplasia after removal of the other, is mediated by a neural reflex. In the present studies, a method employing capsaicin to selectively remove adrenal sensory fibers was developed and applied to determine whether adrenal capsaicin-sensitive fibers participate in compensatory adrenal growth. The splanchnic nerves of anesthetized male rats were treated with capsaicin or vehicle. Capsaicin treatment selectively removed adrenal calcitonin gene-related peptide-positive fibers. One week after drug treatment, rats underwent left adrenalectomy or sham surgery and recovered for 5 days. Capsaicin treatment bilaterally or to the left splanchnic nerve alone (i.e., the afferent nerve in the reflex) impaired compensatory adrenal growth at 5 days compared with vehicle controls, whereas capsaicin treatment to the right splanchnic nerve alone did not affect growth. Moreover, left adrenalectomy induced c-Fos immunolabeling in ipsilateral dorsal spinal cord that was prevented by capsaicin treatment. These data suggest that adrenal capsaicin-sensitive afferent nerves participate in compensatory adrenal growth and that this effect is primarily on the afferent limb of the reflex.     

C-fibres provide a source of masking inhibition to primary somatosensory cortex.

Capsaicin was applied to the exposed radial nerve of adult flying foxes (n = 5) and cats (n = 2) while recording in primary somatosensory cortex from a single neuron with a receptive field on digits 1 or 2. Within four minutes of application of capsaicin the borders of these receptive fields dramatically expanded. In a further four flying foxes it was shown, with subcutaneous delivery just proximal to the receptive fields, that capsaicin need affect only afferents from the region of a neuron's receptive field to induce expansion. Capsaicin applied directly to a nerve, or subcutaneously in high concentrations, is a selective neurotoxin that rapidly prevents the propagation of action potentials in most C-fibres. The result provides a partial explanation for experiments involving the specific and complete denervation of receptive fields of neurons in primary somatosensory cortex. Such denervation does not lead to unresponsiveness but to immediate sensitivity to stimulation of areas surrounding the original fields. Thus it appears that some subclass of capsaicin-sensitive C-fibres provides a primary source for the masking inhibition that normally limits the extent of the receptive fields of cortical neurons.     

Effect of capsaicin on neuropeptides in areas of termination of primary sensory neurones

Capsaicin stimulates chemosensitive peripheral pain receptors, and neonatal administration produces degeneration of a population of primary afferent fibres. It has been shown previously that the effects of capsaicin are accompanied by the loss of substance P from areas of primary afferent termination and that enkephalin is not depleted from such areas. However, a number of other peptides are thought to be contained in sensory fibre systems and so we have used immunohistochemistry to examine the effect of capsaicin on the distribution of five different peptides in the substantia gelatinosa of the spinal trigeminal nucleus and spinal cord. Neonatal capsaicin treatment produces a depletion of somatostatin and cholecystokinin immunofluorescence in addition to substance P, but enkephalin and neurotensin immunofluorescence are not depleted. The implications of this result for theories of peptide involvement in nociceptive mechanisms are discussed.     

Effects of capsaicin on inflammation and on the substance P content of nervous tissues in rats with adjuvant arthritis

Capsaicin (20-80 mg/kg, s.c.) reduced the inflammatory response to inoculation with Mycobacterium butyricum in the rat. The effect was apparent within 24 h, was partial, persisted for well over 20 days, and occurred irrespective of whether capsaicin was administered before or after the onset of inflammation, or at the time when the pathology reached peak. Capsaicin also attenuated the increase in substance P content in sciatic nerve, saphenous nerve, dorsal root ganglia, dorsal roots, and dorsal spinal cord (L4, L5) which occurs in rats with adjuvant arthritis. The data are consistent with a possible role of substance P in the peripheral manifestations of adjuvant arthritis.     

The distribution and origin of VIP in the spinal cord of six mammalian species

The distribution of VIP-immunoreactivity was studied in the spinal cord and dorsal root ganglia of 6 mammalian species. Immunoreactive fibres and cell bodies were most apparent in the dorsal horn, dorsolateral funiculus, intermediolateral cell columns and the area around the central canal. The distribution of VIP immunoreactivity was similar in all species studied, mouse, rat, guinea pig, cat, horse and the marmoset monkey. There were fewer VIP fibres in the dorsal horn of cervical and thoracic segments than in lumbosacral segments. Using radioimmunoassay this gradient increase was quantitatively most marked in the sacral spinal cord of the cat. In dorsal root ganglia few nerve cell bodies but numerous fibres were present. A dual origin for VIP in the spinal cord is suggested: (A) Extrinsic, from dorsal root afferent fibres since immunoreactivity was decreased in dorsally rhizotomized animals (cats and rats) and in capsaicin pretreated rats (microinjection of dorsal root ganglia). (B) From local cell bodies intrinsic to the spinal cord which became visible after colchicine pretreatment of rats.     

Selective degeneration of chemosensitive primary sensory neurones induced by capsaicin: Glial changes

Summary Capsaicin treatment of newborn rats results in the degeneration of primary sensory neurones involved in the mediation of chemogenic pain. In the present study glial changes following the pharmacologically-induced degeneration of unmyelinated primary afferent fibres terminating in Rexed's laminae I and II of the spinal cord were investigated. Light microscopy revealed an increase in the number of phagocytic glial cells in this area, reaching a maximum at 24 h after the administration of capsaicin; they had almost completely disappeared by 72 h. At the ultrastructural level these cells were characterized by their elongate or irregular nuclei with a pronounced heterochromatin pattern, a moderately dense cytoplasmic matrix, hour-glass shaped mitochondria and very large numbers of heterogeneous dense bodies and lipid droplets. On the basis of these observations, these cells were considered to represent reactive microglial cells engaged in the phagocytosis of degenerated neuronal debris. The possible origin and mode of elimination of these elements from the central nervous tissue is briefly discussed.Fellow of the Deutscher Akademischer Austauschdienst on leave from the Department of Anatomy, University Medical School, Szeged, Hungary     

The depolarising action of capsaicin on rat isolated sciatic nerve

The effect of capsaicin on the isolated sciatic nerve of rat was studied by extracellular recording of membrane polarisation. Capsaicin depolarised the sciatic nerve, but desensitization occurred rapidly upon repeated administration. Several other neuroactive substances, including substance P, were inactive. The depolarisation was reduced in nerves depleted of unmyelinated fibres by neonatal capsaicin treatment, suggesting that it occurs mainly in C-fibres. This depolarising action of capsaicin could explain the irritant and acute antinociceptive properties of capsaicin.     

Mechanism of action of capsaicin-like molecules on sensory neurons.

Capsaicin produces pain by selectively activating polymodal nociceptive neurons. This involves a membrane depolarization and the opening of a unique, cation-selective, ion channel which can be blocked by ruthenium red. The capsaicin-induced activation is mediated by a specific membrane receptor which can be selectively and competitively antagonised by capsazepine. Repetitive administrations of capsaicin produces a desensitization and an inactivation of sensory neurons. Several mechanisms are involved. These include receptor inactivation, block of voltage activated calcium channels, intracellular accumulation of ions leading to osmotic changes and activation of proteolytic enzyme processes. Systemic and topical capsaicin produces a reversible antinociceptive and antiinflammatory action after an initial undesirable algesic effect. Capsaicin analogues, such as olvanil, have similar properties with minimal initial pungency. Systemic capsaicin produces antinociception by activating capsaicin receptors on afferent nerve terminals in the spinal cord. Spinal neurotransmission is subsequently blocked by a prolonged inactivation of sensory neurotransmitter release. Local or topical application of capsaicin blocks C-fibre conduction and inactivates neuropeptide release from peripheral nerve endings. These mechanisms account for localized antinociception and the reduction of neurogenic inflammation respectively.     

Effect of diaphragm small-fiber afferent stimulation on ventilation in dogs

Little is known regarding the role of diaphragm small-fiber afferents (groups III and IV) in the control of breathing. This study was designed to determine whether activation of these afferents with use of capsaicin affects phrenic efferent activity. Capsaicin injections into the phrenic artery were made in 10 alpha-chloralose-anesthetized dogs after each of the following procedures performed in succession: bilateral cervical vagotomy, C7 spinal cord transection, bilateral cervical dorsal rhizotomy. In six of these animals injections were also made after C2 spinal cord transection and removal of the cervical spinal cord. Injections made in the vagotomized animals were associated with apneusis followed by hyperpnea. C7 spinal transection eliminated the hyperpneic response, but the apneusis remained. Cervical dorsal rhizotomy or C2 spinal cord transection failed to abolish the apneusis in response to injection. No diaphragm response was obtained after removal of the cervical spinal cord. Experiments in three additional animals showed that capsaicin does not have a direct excitatory effect on the muscle cells of the crural diaphragm, nor does it potentiate the release of neurotransmitter in the diaphragm. The results of this study indicate that small-fiber afferents in the diaphragm have an excitatory effect on phrenic motoneurons. There is a segmental component to this reflex, since the response is observed after C2 spinal cord transection. The data also suggest that at least some of these afferents enter the spinal cord through the ventral roots.     

Capsaicin Does Not Change Tissue Levels of Glutamic Acid, Its Uptake, or Release in the Rat Spinal Cord

Capsaicin treatment (50 mg/kg, subcutaneous) of newborn rats resulted in 1 75% decrease of substance P immunoreactivity in the dorsal spinal cord of the adult animal, but failed to affect levels of the proposed sensory neurotransmitter glutamic acid or to alter high-affinity uptake of [3H]glutamic acid into synaptosomes of the same tissue. Furthermore, capsaicin (30 microM) in vitro had no influence on the release of [3H]glutamic acid from spinal cord P2 fractions of untreated adult rats, but induced a marked release of substance P. The results suggest that, in contrast to substance P fibers, neurons containing glutamic acid are not sensitive to capsaicin. Eleven other neurochemical parameters measured in the spinal cord did not appear to be changed by the treatment with capsaicin, suggesting a considerable neurochemical selectivity of the lesion.     

Changes in fibre populations of the rat hairy skin following selective chemodenervation by capsaicin

Perineural application of capsaicin results in a selective and permanent reduction in the sensitivity to noxious chemical and heat stimuli and elimination of the neurogenic inflammatory response. The present quantitative immunohistochemical study has been undertaken to reveal the populations of cutaneous afferent nerves that are affected by perineural capsaicin treatment. Areas of intact and chemodenervated skin were determined with the aid of the vascular labelling technique. In sections taken from intact skin areas, staining with antibodies against protein gene product 9.5 revealed a rich epidermal innervation. Fibres immunoreactive for growth-associated protein 43 were also abundant; nerve fibres immunoreactive for substance P and calcitonin gene-related peptide were less numerous. Somatostatin- and RT97-immunoreactive fibres were seen only in the subepidermal layer. In sections taken from skin areas supplied by the sciatic nerve treated with capsaicin 3 days previously, the number of epidermal nerve fibres immunoreactive to protein gene product 9.5, growth-associated protein 43, substance P and calcitonin gene-related peptide was reduced by 90%, 95%, 97% and 66%, respectively. These changes persisted for at least 42 days. The findings reveal that the majority of epidermal axons are capsaicin-sensitive and comprise a chemically heterogeneous population. Reductions in cutaneous fibre populations following perineural capsaicin treatment may result from both the degeneration of sensory axons and the depletion of neuron-specific macromolecules. In addition, most cutaneous nociceptive axons may not use the major sensory neuropeptides substance P and calcitonin gene-related peptide as afferent neurotransmitters.     

Effects of capsaicin in rat and pigeon on peripheral nerves containing substance P and calcitonin gene-related peptide

Summary Substance P and calcitonin gene-related peptide were immunohistochemically identified in axons innervating the cornea and the ureter of adult rats and pigeons. The two neuropeptides were similarly distributed in both species. Capsaicin pretreatment induced depletion of the immunoreactivity; this was quantitatively and qualitatively different in rats and pigeons. Topical application of capsaicin (1%) reduced the immunoreactivity in the cornea in both species by 50%. Systemic capsaicin treatment completely depleted both peptides from the corneal innervation of rats but reduced the peptide content only by 50% in the cornea of pigeons. In the ureter of rats, capsaicin pretreatment completely depleted the peptide immunoreactivity. In pigeons the peptide depletion was only complete in the outer longitudinal muscle layer. Whereas only a few immunoreactive fibres were observed in the circular muscle layer, about 50% of the peptide remained in the inner longitudinal muscle layer. The results demonstrate that peptidergic afferents in the cornea and ureter of pigeons are sensitive to capsaicin, although birds do not show nociceptive responses to local administration of the drug. The long-term depletion of substance P and calcitonin gene-related peptide by capsaicin is discussed with regard to the possibility that functionally capsaicin receptors may exist in the axon but not at nerve endings.Part of the thesis of Gerhard Harti, to be presented to the Fachbereich Biologie, Justus-Liebig-Universität, Giessen     

Primary afferent depolarization of C fibres in the spinal cord of the cat

The excitability of primary afferent terminals of cutaneous C fibres was tested in the spinal cord of decerebrated cats. C fibre terminal excitability was decreased in the spinal state, and increased by conditioning volleys that activated only A fibres of another cutaneous nerve and by stimulating hair mechanically. It is suggested that C fibre input and therefore nociceptive information to the central nervous system is susceptible to presynaptic control by segmental and suprasegmental mechanisms.     

Galanin-immunoreactive nerves in the female rat paracervical ganglion and uterine cervix: distribution and reaction to capsaicin

Summary The presence and distribution of galanin-immunoreactivity was examined in the uterine cervix and paracervical autonomic ganglia of the female rat. Some animals were treated with capsaicin to determine if galanin-immunoreactivity was present in small-diameter primary afferent nerves. Other animals were treated with the noradrenergic neurotoxin 6-hydroxydopamine to ascertain if galanin-immunoreactivity was present in sympathetic noradrenergic nerves. Galanin-immunoreactive nerve fibers were sparse in the cervical myometrium and vasculature, but numerous in the paracervical ganglion where they appeared to innervate principal neurons. Immunoreactivity was also present in dorsal root ganglia, dorsal horn of spinal cord, and inferior mesenteric ganglia. Capsaicin treatment resulted in a marked reduction of galanin-immunoreactivity in the spinal cord dorsal horn, but not in the dorsal root ganglia, paracervical ganglia, or cervix (although there was a substantial reduction of substance P-, neurokinin A-, and calcitonin gene-related peptide-immunoreactivity in the dorsal horn, dorsal root ganglia, and uterine cervix). 6-Hydroxydopamine treatment did not cause any appreciable change in the galanin-immunoreactivity in any tissues. We conclude that galanin-like immunoreactivity is expressed in nerve fibers innervating the paracervical ganglia and uterine cervix of the female rat. This immunoreactivity is probably present in afferent nerves and could play a role in neuroendocrine reflexes and in reproductive function.     

Calcitonin gene-related peptide (CGRP) in capsaicin-sensitive substance P-immunoreactive sensory neurons in animals and man: distribution and release by capsaicin

The presence of calcitonin-gene related peptide (CGRP)-like immunoreactivity (-LI) in sensory neurons was established by immunohistochemistry and radioimmunoassay (RIA) in combination with high performance liquid chromatography (HPLC). CGRP-immunoreactive (-IR) nerve fibres were present in many peripheral organs including heart, ureter, uterus and gall bladder of guinea-pig and man. The distribution of CGRP-IR nerves in the dorsal horn of the spinal cord, of positive cell bodies in thoracic spinal and nodose ganglia and nerves in peripheral organs was closely related to that of substance P-LI. Double staining experiments revealed that in most cases peripheral CGRP-IR nerve terminals also contained SP-LI. However, different localization of SP- and CGRP-IR neurons was observed in the nucleus of the solitary tract as well as in the ventral horn of the spinal cord. In the heart, CGRP-IR nerves were associated with myocardial cells (mainly atria), coronary vessels, local parasympathetic ganglia as well as with the epi- and endocardia. Three to 4-fold higher levels of native CGRP-LI were observed in the atria than in the ventricles of the heart. HPLC analysis revealed that the major peak of CGRP-LI in the heart of rat and man had the same retention times as the synthetic equivalents. Systemic capsaicin pretreatment and adult guinea-pigs caused a loss of CGRP-IR terminals in the dorsal horn of the spinal cord as well as in peripheral organs including the heart. After capsaicin treatment, the content of CGRP-IR was reduced by 70% in the heart and by 60% in the dorsal part of the spinal cord. In superfusion experiments with slices from the rat spinal cord, a release of CGRP-LI was induced by 60 mM K+ and 3 microM capsaicin in a calcium-dependent manner.     

Corticotropin releasing factor-like immunoreactivity in sensory ganglia and capsaicin sensitive neurons of the rat central nervous system: colocalization with other neuropeptides

Immunohistochemistry and radioimmunoassay (RIA) revealed that corticotropin releasing factor (CRF)-like immunoreactivity was found to be colocalized with substance P (SP)-, somatostatin (SST)- and leu-enkephalin (LENK)-like immunoreactivity in the dorsal root- and trigeminal ganglia, the dorsal horn of the spinal cord (laminae I and II), the substantia gelatinosa, and at the lateral border of the spinal nucleus and in the tractus spinalis of the trigeminal nerve. These peptides were also located in fast blue labeled cells of the trigeminal ganglion following injection of the dye into the spinal trigeminal area. This indicates that there are possible sensory projections of these peptides into the spinal trigeminal area. Capsaicin treatment of neonatal rats resulted in a marked decrease in the density of CRF-, SP-, VIP- and CCK-containing neurons in the above mentioned hindbrain areas, whereas SST- and LENK-immunoreactivity were not changed. RIA revealed that, compared to controls, CRF, SP and VIP concentrations in these areas were decreased in rats pretreated with capsaicin, while SST levels were increased; CCK and LENK levels were unchanged. It is concluded that the primary afferent neurons of the nucleus and tractus spinalis of the trigeminal nerve are richly endowed with a number of peptides some of which are sensitive to capsaicin action. The close anatomical proximity of these peptide containing neurons suggests the possibility of a coexistance of one or more of these substances.