Capsaicin-sensitive afferents are involved in signalling transneuronal effects between cutaneous sensory nerves.

The aim of the present study was to investigate changes in contralateral nerves associated with peripheral nerve injuries. Transection and subsequent regeneration of the saphenous nerve on one side caused a suppression of the ability of the contralateral saphenous nerve to produce a neurogenic plasma extravasation response. This effect was transient, and was first evident two weeks after injury, reaching its maximum at four weeks, but was no longer detectable at eight weeks. This change was paralleled by a decrease in the content of substance P, a neuropeptide involved in neurogenic plasma extravasation, in the contralateral nerve. The neurotoxin capsaicin was used to deplete the nerve of a subclass of C-fibres, namely the polymodal nociceptor afferents. Pretreatment of the nerve to be lesioned with capsaicin was sufficient to significantly attenuate the changes in the plasma extravasation response and substance P content observed on the contralateral side. The effectiveness of the capsaicin treatment was confirmed by histological examination. These results strongly suggest that changes observed at a site distant from the location of the nerve injury are dependent on the integrity of capsaicin-sensitive C-fibre afferents within the injured nerve. Furthermore, given that the contralateral nerve has commonly been used as the control for an injury conducted on the homologous nerve or muscle on the opposite side of the body, the underlying assumption being that the contralateral nerve remained unchanged, the present findings emphasize the need for separate groups of control animals which have undergone no surgical procedures.

Use of protease inhibitors increases the amounts of substance P extracted from small specimens of nerve tissue.

When using radioimmunoassay to measure the levels of peptides from small pieces of nerve tissue it is crucial to maximize the amount of peptides extracted. Here we report on the value of including protease inhibitors in the extraction buffer when extracting substance P (SP) from short lengths of rat saphenous nerve tissue. Nerve segments were removed from terminally anesthetized 13-week-old rats and directly added to acid buffer (including EDTA) either with or without 1 mM 4-(2-amino-ethyl)-benzesulfonyl fluoride-HCl, 2 micrograms/ml aprotinin, 100 microM leupeptin, 1 microgram/ml cystatin, and 1 mM benzamidine. These "direct" samples were then boiled for 10 min. With additional groups of pieces of saphenous nerve tissue the effects of leaving the samples for 10 min in both buffers at room temperature either intact ("delayed") or after mincing the tissue ("minced") were investigated. Addition of protease inhibitors increased the amount of SP extracted in both direct and delayed procedures, although the increase was only significant for the delayed situation (P < or = 0.05). "Delay" in the absence of protease inhibitors resulted in a significantly decreased amount of SP being extracted compared to the direct and minced situations (P < or = 0.05). We recommend use of protease inhibitors be included as part of the standard procedure for extracting neuropeptides from small specimens of nerve tissue for radioimmunoassay.

A study of vasodilator responses evoked by antidromic stimulation of A delta afferent nerve fibers supplying normal and reinnervated rat skin.

Experiments have been carried out on anaesthetized rats to investigate some properties of the vasodilator responses evoked in skin by stimulation of small myelinated, A delta-fibers. Both normal and reinnervated skin in the saphenous nerve field were studied. In unoperated animals it was found that the magnitude and time course of the responses recorded from a particular patch of skin remain stable over a period of hours, but the responses varied in magnitude in different parts of the saphenous nerve territory. A delta-fiber evoked vasodilatation responses recorded from reinnervated skin were generally very similar to those seen in normal skin except that they tend to be smaller. These observations, taken together with previous findings, suggest that this ability of cutaneous A delta-fibers to influence the vasculature in the vicinity of their sensory endings has some physiological significance. Unfortunately, attempts to link these skin blood flow responses with activity of single, identified A delta-fiber units were not successful.

The relationship between unmyelinated afferent type and neurogenic plasma extravasation in normal and reinnervated rat skin.

Electrophysiological experiments have been carried out on rats to examine the relationship between plasma extravasation into skin and antidromic activation of individual unmyelinated afferent fibres supplying cutaneous receptors, particularly polymodal nociceptors. Normal animals and ones in which the skin had been reinnervated after peripheral nerve transection 26-52 weeks before were studied. In the normal animals 12 of 18 (66%) polymodal nociceptor afferents showed evidence of plasma extravasation after antidromic stimulation of the nerve filament from which they were initially recorded. Antidromic stimulation of other types of unmyelinated unit gave no detectable signs of plasma extravasation. The results from the animals with regenerated nerves were similar with 15 of 21 (71%) of the polymodal nociceptor afferents showing clear signs of plasma extravasation after antidromic activation and the other types of unit giving no signs of such a response. These results show that while antidromic stimulation of a cutaneous nerve after intravascular administration of Evans Blue dye can be used to get a general view of the extent of innervation by polymodal nociceptors, it does not necessarily show the entire distribution of these receptors. It is likely that the ability or lack of ability of a polymodal nociceptor afferent to enhance vascular permeability, and so bring about plasma extravasation, is related to its neuropeptide content, and thus it follows that this might be a useful criterion for separating this broad class of cutaneous receptor into sub-groups.

Neonatal Anti-NGF Treatment Reduces the Adelta- and C-Fibre Evoked Vasodilator Responses in Rat Skin: Evidence That Nociceptor Afferents Mediate Antidromic Vasodilatation.

Electrical stimulation of cutaneous nerve Adelta-fibres can lead to increases in skin blood flow (Jänig and Lisney, J. Physiol. (Lond.), 415, 477 - 486, 1989). Here we have examined this phenomenon in adult rats treated neonatally with antisera to nerve growth factor between postnatal days 2 and 14. This treatment forces many Adelta nociceptor afferents to take on the phenotype of low-threshold D-hair afferents (Lewin et al., J. Neurosci., 12, 1896 - 1905, 1992). In animals treated this way we found a parallel decrease in the ability of Adelta-fibres to increase skin blood flow. The increase in blood flow evoked by C-fibre stimulation was also reduced, but no change was seen in the ability of C-fibres to elicit neurogenic extravasation in skin. These data may be taken as evidence that in rats, amongst the cutaneous Adelta-fibres, nociceptor but not D-hair afferents are capable of producing blood flow changes in the skin.

Transneuronal effects triggered by saphenous nerve injury on one side of a rat are restricted to neurones of the contralateral, homologous nerve.

Transection and regeneration of the saphenous nerve on one side of a rat reduces the ability of the contralateral saphenous nerve to evoke plasma extravasation following antidromic nerve stimulation. It is proposed that a transneuronal signal--that is a signal that passes from injured neurones on one side of the body to intact ones on the other via the spinal cord--is involved in triggering this response. The present experiments have shown that the influence of this transneuronal signal is restricted to neurones of homologous nerves on the two sides of an animal, i.e. it passes from saphenous nerve neurones on one side to saphenous nerve neurones on the other; its influence does not seem to extend to neurones in other nerves, for example ones in either the ipsi- or contralateral sural nerves.

A proposed relationship between circumference and conduction velocity of unmyelinated axons from normal and regenerated cat hindlimb cutaneous nerves.

Data on the conduction velocities and circumferences of unmyelinated axons in cat hindlimb nerves obtained in two laboratories, each working independently of the other, have been brought together and compared. Results were obtained from normal, regenerated and deafferented nerves. Matching the conduction velocity and circumference distributions against one another suggested that conduction velocity (theta), in m/s, is linearly related to circumference (s), in microns, according to the equation theta = 0.24s. If this relationship is correct, it applies equally well to both sympathetic postganglionic and unmyelinated afferent axons.

Changes in myelinated and unmyelinated axon numbers in the proximal parts of rat sural nerves after two types of injury.

Counts of myelinated and unmyelinated axon profiles have been made from normal, uninjured rat sural nerves and from nerves injured 6 months earlier in one of two ways. In one group of rats the nerve was simply cut and left to regenerate, leading to the development of a neuroma in continuity, while in the second group the nerve was cut but then ligated as well to prevent regeneration; this led to stump neuroma formation. After nerve transection and regeneration, with subsequent formation of a neuroma in continuity, there was no change in the number of myelinated axon profiles found 25 mm proximal to the old injury site when compared with control, but there was an 18% reduction (P < 0.05) in the number of unmyelinated axon profiles. Immediately proximal to the injury site the picture was similar, with there still being the same number of myelinated axon profiles as in control material but here the reduction in unmyelinated axon numbers was slightly greater at 24% (P < 0.05). In the proximal part of nerves that had been cut and stump neuroma formation induced there was a large increase (33%) in myelinated axon profiles over and above control values (P < 0.001) but the number of unmyelinated profiles was the same as in controls. Closer to the stump neuroma the number of myelinated axon profiles had increased yet further to be 88% (P < 0.001) above control while the number of unmyelinated ones remained no different from control. Our interpretation of these results is that after nerve transection and regeneration there is no loss of peripheral neurons supporting myelinated axons but some loss of those supporting unmyelinated ones. If a cut nerve is prevented from regenerating and a stump neuroma forms, however, a vigorous sprouting response is triggered in neurons with myelinated axons while those supporting unmyelinated axons are possibly prevented from dying. The reaction of peripheral neurons to injury is such that the number of axons they support varies along the nerve as one goes disto-proximally away from the injury site. Thus discrepancies in results from different laboratories have come about because material for axon counting has been taken from different points along the nerve relative to the injury site and also because the material has been taken from nerves injured in different ways.

Success of regeneration of peripheral nerve axons in rats after injury at different postnatal ages.

Electrophysiological experiment have been carried out on rats to see if the age at which a peripheral nerve injury occurs influences the success of regeneration. The assessment was made on the basis of two measures of peripheral nerve regeneration; the extent to which axons manage to grow across the injury site and into the distal stump, and their ability to resupply cutaneous structures with functional endings. Regeneration after nerve transection of both myelinated and unmyelinated axons was studied. The results showed that, apart from rats injured when 2 weeks old, the age at which injury occurred, over the range 4-40 weeks, had little bearing on the overall success of skin reinnervation. The 2-week-old rats showed significantly poorer recovery.

Saphenous nerve injury and regeneration on one side of a rat suppresses the ability of the contralateral nerve to evoke plasma extravasation.

Leakage of Evans blue dye from the circulation into the skin has been used to measure plasma extravasation evoked by antidromic nerve stimulation of the saphenous nerves in anaesthetised rats. Normal animals and ones in which one saphenous nerve had been cut and left to regenerate some time before were studied. Saphenous nerve injury on one side of a rat significantly reduced the ability of the contralateral, uninjured nerve to evoke plasma extravasation compared with the response measured in totally uninjured control animals. This suppression of plasma extravasation was not dependent on activation of nerve fibres in the regenerated nerve. The effect was evident by 6 weeks after injury and persisted for at least another 20 weeks. At the moment the mechanism underlying this suppression of neurogenic plasma extravasation is unknown.

Small diameter myelinated afferents produce vasodilatation but not plasma extravasation in rat skin.

1. Antidromic stimulation of the rat saphenous nerve at intensities sufficient to excite small myelinated (A delta) fibres but not unmyelinated (C) fibres produced evidence of a transient increase in skin blood flow in the saphenous nerve territory. The magnitude and time course of the vasodilatation depended on the number and frequency of stimuli delivered to the nerve. 2. There was no evidence of an accompanying plasma extravasation. 3. The results suggest that A delta afferent fibres are involved in axon reflex/axon response reactions as well as unmyelinated (C) afferent fibres.

Influence of autograft size on peripheral nerve regeneration in cats.

A combination of electrophysiological and histological techniques has been used to study the extent to which the size of an autograft affects myelinated axon regeneration in damaged cat peripheral nerves. The length of the graft and the match between the number of axons and Schwann cell basement membrane tubes in the graft and the repaired nerve were investigated. No difference was found in the success of regeneration through 10, 20 and 30 mm autografts. Changing the number of Schwann cell tubes available to regenerating myelinated axons also had no detectable effect on the success of regeneration.

Reinnervation of sweat glands in the rat hind paw following peripheral nerve injury.

Electrophysiological experiments have been carried out to investigate the time course and extent of sweat gland reinnervation in the rat hind paw. The first evidence of functional reinnervation after nerve transection was obtained at 12 weeks, when the extent of innervation was 20% of that measured in control animals. By 20 weeks, reinnervation had reached almost 50% of control values but then there was no further improvement up to 52 weeks. These results are comparable to those for skin reinnervation by polymodal nociceptor afferents.

An electrophysiological and histological study of myelinated axon regeneration after peripheral nerve injury and repair in the cat.

Electrophysiological and histological methods have been combined to obtain quantitative measures of the success of regeneration of myelinated axons in a cutaneous nerve after injury and repair by a variety of procedures. Following a simple transection injury more axons regenerated successfully when the nerve was repaired by epineurial suturing or stump suturing than when it was left unrepaired; both types of repair gave similar results. After loss of a 10-mm piece of the nerve trunk, repair with an autograft produced more regeneration than when the nerve was left untouched, but repair by stump mobilization with epineurial suturing made matters worse. On the whole, the regenerated afferents had receptive field properties similar to those found in control animals but there was a higher incidence of units that could not be typed using conventional criteria. A small proportion of them had split receptive fields. Fibre diameters and conduction velocities were reduced compared with controls; this was particularly so through the neuroma and in the distal stump. There was also evidence of abnormal interactions, possibly ephaptic, between some regenerated axons.

The proportions of sympathetic postganglionic and unmyelinated afferent axons in normal and regenerated cat sural nerves.

Electrophysiological experiments have been carried out to see if the proportions of sympathetic postganglionic and unmyelinated afferent axons in a cutaneous nerve were changed after injury and regeneration. It seemed possible that an alteration in the relative numbers of the two groups of axons could contribute to the aetiology of reflex sympathetic dystrophy, but the experiments provided no evidence for such a change. There were, however, signs of a decrease in axon numbers in the regenerated nerves.

The numbers of unmyelinated and myelinated axons in normal and regenerated rat saphenous nerves.

Counts have been made of the numbers of unmyelinated and myelinated axons in the proximal and distal stumps of regenerated rat saphenous nerves and from equivalent sites in normal nerves. In the proximal part of normal nerves there were averages of 1,045 myelinated axons and 4,160 unmyelinated ones. Regenerated nerves contained the same number of myelinated axons in their proximal stumps but there was a 40% reduction in the unmyelinated axon count. In the distal stumps of these nerves the myelinated axon count had increased by an average of 620; this comes about because some regenerated myelinated axons support more than one process in the distal stump. In contrast, the number of unmyelinated axons was reduced further, from a mean of 2,476 in the proximal stump to one of 2,219. The sizes of Schwann cell units in the normal and regenerated nerves were also noted. Schwann cell units in the proximal and distal stumps of the regenerated nerves were smaller than those in the normal ones. These changes associated with unmyelinated axons in regenerated nerves are likely to contribute to the sensory, vasomotor and sudomotor abnormalities that sometimes occur after peripheral nerve injury and regeneration.

Functional aspects of the regeneration of unmyelinated axons in the rat saphenous nerve.

Electrophysiological experiments have been carried out to investigate aspects of unmyelinated axon regeneration in a transected cutaneous nerve. Some comparisons with regeneration of myelinated axons in the same nerve have also been made. By 3 months after injury approximately 80% of the unmyelinated axons that had survived in the proximal stump had regenerated into the distal stump. About the same proportion of myelinated axons had regrown into the distal stump by this time. With both groups of axons there was no marked increase in the amount of regeneration across the injury site with longer recovery times. Conduction velocities in the regenerated unmyelinated axons tended to be slower across the injury site than proximally; the proximal conduction velocities did not differ from those in control nerves. The unmyelinated axons seemed to take longer to resupply the skin than did the myelinated ones, but in both cases the extent of skin innervation had reached about 60% control values by 6 months after the injury.

Afterdischarge and interactions among fibers in damaged peripheral nerve in the rat.

Sensory fibers trapped in nerve-end neuromas become abnormally excitable, and produce an ectopic discharge which is believed to contribute to paresthesias and pain associated with chronic nerve injury in man. Here we report that stimulation of injured nerves can alter this discharge, directly by antidromic invasion of active neuroma fibers, and indirectly through interactions with neighboring fibers. Antidromic stimulation of spontaneously active fibers in experimental neuromas in the rat sciatic nerve, using single electrical stimulus pulses, produced time-locking of rhythmic spontaneous firing and of spontaneous impulse bursts. Some initially silent fibers generated a burst of rhythmic afterdischarge when stimulated in this way. Stimulation delivered in brief trains (tetani) produced more prolonged alterations in spontaneous neuroma discharge, including excitation, suppression and combinations of the two. In some cases initially silent fibers were activated for extended periods. These responses to tetanic stimulation occurred even when the active fibers were not themselves stimulated, and reflect a novel form of fiber-fiber interaction in neuromas that we term 'crossed afterdischarge'. This interaction probably results from the accumulation of potassium ions within the extracellular compartment adjacent to active neuroma fibers during activation of their neighbors. It differs fundamentally from the high safety factor ephaptic cross-talk seen in acutely cut nerves and in neuromas of 30 or more days standing.