Experimental Neuromas Research Articles

Immunostaining in whole-mount lipid-cleared peripheral nerves and dorsal root ganglia after neuropathy in mice

Immunohistochemical characterization of primary afferent fibers (intact or after nerve damage) is traditionally performed in thin sections from dorsal root ganglia (DRGs) or in teased fibers, as light scattering in whole-mounts compromises visualization. These procedures are time-consuming, require specific equipment and advanced experimental skills. Lipid-clearing techniques are increasing in popularity, but they have never been used for the peripheral nervous system. We established a modified, inexpensive clearing method based on lipid-removal protocols to make transparent peripheral nerve tissue (inCLARITY). We compared retrograde-labeling and free-floating immunostaining with cryo-sections. Confocal microscopy on whole-mount transparent DRGs showed neurons marked with retrograde tracers applied to experimental neuromas (Retrobeads, Fluoro-ruby, Fluoro-emerald, DiI, and Fluoro-gold). After immunostaining with calcitonin gene-related peptide (peptidergic) or isolectin IB4 (non-peptidergic), nociceptors were visualized. Immunostaining in transparent whole-mount nerves allows simultaneous evaluation of the axotomized branches containing the neuroma and neighboring intact branches as they can be mounted preserving their anatomical disposition and fiber integrity. The goal of our study was to optimize CLARITY for its application in peripheral nerve tissues. The protocol is compatible with the use of retrograde tracers and improves immunostaining outcomes when compared to classical cryo-sectioning, as lack of lipids maximizes antibody penetration within the tissue.

Testing the effectiveness and the contribution of experimental supercharge (reversed) end-to-side nerve transfer.

Supercharge end-to-side (SETS) transfer, also referred to as reverse end-to-side transfer, distal to severe nerve compression neuropathy or in-continuity nerve injury is gaining clinical popularity despite questions about its effectiveness. Here, the authors examined SETS distal to experimental neuroma in-continuity (NIC) injuries for efficacy in enhancing neuronal regeneration and functional outcome, and, for the first time, they definitively evaluated the degree of contribution of the native and donor motor neuron pools. This study was conducted in 2 phases. In phase I, rats (n = 35) were assigned to one of 5 groups for unilateral sciatic nerve surgeries: group 1, tibial NIC with distal peroneal-tibial SETS; group 2, tibial NIC without SETS; group 3, intact tibial and severed peroneal nerves; group 4, tibial transection with SETS; and group 5, severed tibial and peroneal nerves. Recovery was evaluated biweekly using electrophysiology and locomotion tasks. At the phase I end point, after retrograde labeling, the spinal cords were analyzed to assess the degree of neuronal regeneration. In phase II, 20 new animals underwent primary retrograde labeling of the tibial nerve, following which they were assigned to one of the following 3 groups: group 1, group 2, and group 4. Then, secondary retrograde labeling from the tibial nerve was performed at the study end point to quantify the native versus donor regenerated neuronal pool. In phase I studies, a significantly increased neuronal regeneration in group 1 (SETS) compared with all other groups was observed, but with modest (nonsignificant) improvement in electrophysiological and behavioral outcomes. In phase II experiments, the authors discovered that secondary labeling in group 1 was predominantly contributed from the donor (peroneal) pool. Double-labeling counts were dramatically higher in group 2 than in group 1, suggestive of hampered regeneration from the native tibial motor neuron pool across the NIC segment in the presence of SETS. SETS is indeed an effective strategy to enhance axonal regeneration, which is mainly contributed by the donor neuronal pool. Moreover, the presence of a distal SETS coaptation appears to negatively influence neuronal regeneration across the NIC segment. The clinical significance is that SETS should only employ synergistic donors, as the use of antagonistic donors can downgrade recovery.

Research progress on animal models of experimental terminal neuroma and their evaluation methods

Background Terminal neuroma model is an important tool to investigate the mechanism and prevention strategies of post-amputation pain(PAP). There isn′t an agreed method to establish this animal model. Objective Providing references for effective modeling through exploring the methods of establishing terminal neuroma models and related modeling issues. Content This review is to give a brief summary about the animal models of terminal neuroma and their evaluation methods according to the difference of nerve transected. Trend There is not an uniform and standard method to build a terminal neuroma model so far. However, researchers can choose suitable terminal neuroma model depending their various experimental purposes. Key words: Terminal neuroma; Post-amputation pain; Autotomy phenomenon; Animal model

Na V1.7 accumulates and co-localizes with phosphorylated ERK1/2 within transected axons in early experimental neuromas

Peripheral nerve injury can result in formation of a neuroma, which is often associated with heightened sensitivity to normally innocuous stimuli as well as spontaneous dysesthesia and pain. The onset and persistence of neuropathic pain have been linked to spontaneous ectopic electrogenesis in axons within neuromas, suggesting an involvement of voltage-gated sodium channels. Sodium channel isoforms Na V1.3, Na V1.7 and Na V1.8 have been shown to accumulate in chronic painful human neuromas, while, to date, only Na V1.3 has been reported to accumulate within experimental neuromas. Although recent evidence strongly support a major contribution for Na V1.7 in nociception, the expression of Na V1.7 in injured axons within acute neuromas has not been studied. The current study examined whether Na V1.7 accumulates in experimental rat neuromas. We further investigated whether activated (phosphorylated) mitogen-activated protein (MAP) kinase ERK1/2, which is known to modulate Na V1.7 properties, is co-localized with Na V1.7 within axons in neuromas. We demonstrate increased levels of Na V1.7 in experimental rat sciatic nerve neuromas, 2 weeks after nerve ligation and transaction. We further show elevated levels of phosphorylated ERK1/2 within individual neuroma axons that exhibit Na V1.7 accumulation. These results extend previous descriptions of sodium channel and MAP kinase accumulation within experimental and human neuromas, and suggest that targeted blockade of Na V1.7 or ERK1/2 may provide a strategy for amelioration of chronic pain that often follows nerve injury and formation of neuromas.

Cold sensitivity in axotomized fibers of experimental neuromas in mice

Cold allodynia is a common complaint in patients with peripheral neuropathies. However, cold sensitivity of the different types of sensory afferents present in injured nerves is poorly known. We recorded activity evoked by cold in intact sensory fibers of the skin-saphenous nerve preparation and in axotomized sensory fibers of ∼21 days-old neuromas of the saphenous nerve of mice, in vitro. Sixteen percent of the axotomized units responded to cooling with an accelerating discharge, which stopped immediately during rewarming. This response was similar to that observed in the intact cold-sensitive fibers. Temperature threshold distribution was broad in intact and axotomized cold fibers (30.7–22 °C and 34.5–14.5 °C, respectively). One-third of the axotomized cold-sensitive fibers were mechanosensitive and none of them displayed spontaneous activity at baseline temperature. In contrast, 33% of intact cold-sensitive fibers exhibited low rates of ongoing discharges. In 60% of the cold-sensitive, axotomized units, cold threshold was shifted towards warmer values by the TRPM8 agonist l-menthol. Seventy percent of axotomized, cold-insensitive units developed sensitivity to cold when exposed to 4-aminopyridine and their mean cold threshold (∼28 °C) was unaffected by menthol. Their response properties differed greatly from those of cold-sensitive units. In conclusion, the transducing capacity to cold stimuli is substantially recovered in neuromas. Furthermore, axotomized fibers maintain the 4-AP-sensitive, voltage-activated, transient potassium conductance that counteracts the depolarizing effects of cold in the majority of intact, cold-insensitive primary afferents. Our results indicate that injured nociceptors do not develop abnormal cold sensitivity, suggesting that other mechanisms underlie the cold-induced allodynia following peripheral nerve injury.

Ischemia and failed regeneration in chronic experimental neuromas

Neuromas are generally considered to be swollen uniform collections of uncontrolled aberrantly sprouting axons. In early experimental neuromas, there are substantial rises in local blood flow associated with their formation, but human studies of chronic lesions have suggested that neuromas develop ischemia and become impediments to regeneration. The issue is important because traumatically severed human nerves are frequently considered for repair some time after injury, when neuroma formation has occurred. In this work, we examined local perfusion, axon penetration and other characteristics of long-term (6 month) experimental neuromas created by sciatic nerve transection and resection of the distal sciatic nerve and its branches. The scenario was designed to model prior transection in a human nerve, where late surgical reconnection might be contemplated. Local blood flow in the extrinsic plexus of neuromas, examined using a laser Doppler flowmetry probe, declined in distal portions of the stump to values considerably lower than observed in intact nerves. Intrinsic blood flow near the stump tip, examined using microelectrode hydrogen clearance polarography was highly nonuniform and included zones with very low perfusion. Correlated with these findings were nonuniform histological features with zones of absent axons and blood vessels, progressive distal disorganization, marked declines in distal axon penetration, nonremodelled microfascicles and persistent expression of ‘regenerative’ axon and Schwann cell markers. Uncontrolled axon sprouting was not a feature. Longstanding neuromas include zones of relative ischemia and limited axon penetration that develop in the absence of nerve trunk reconnection. These features would limit their suitability for later repair.

Effect of local methylprednisolone on pain in a nerve injury model: A pilot study

Local injections of corticosteroids are frequently used in the treatment of regional pain. The rationale for this is not very clear, since an inflammatory cause of pain is rarely evident. There are few data on the effect of corticosteroids on nociception in experimental animals. However, corticosteroids have been found to suppress ectopic discharges from experimental neuromas and to have a short-lasting suppressive effect on transmission in normal C-fibers. In this study the influence of a locally applied depot form of a corticosteroid on neuropathic pain was investigated in a rat model. Peripheral mononeuropathy was induced with a chronic constriction injury to the left sciatic nerve. This procedure has previously been shown to produce various signs of disturbed sensibility, including heat hyperalgesia, mechanical allodynia, and mechanical hyperalgesia, indicating that a neuropathic pain-like condition has developed. The occurrence of neuropathic pain in these animals was confirmed with behavioral testing after 9 days. The site of injury was then reexposed and treated locally with either a depot form of a corticosteroid (methylprednisolone) or saline. The animals were then tested for another 11 days. The heat hyperalgesia and mechano-allodynia but not the mechano-hyperalgesia were depressed in the animals receiving the corticosteroid but not in those treated with saline. The effect remained during the whole 11-day test period. It is hypothesized that the corticosteroid acts by suppression of ectopic neural discharges from the injured nerve fibers.

Exogenous opioids influence the microcirculation of injured peripheral nerves

Local microvessels of peripheral nerve trunks (vasa nervorum) dilate following capsaicin-induced inflammation or local nerve trunk injury. In previous work, we observed that morphine blocked capsaicin-induced dilation of vasa nervorum presumably through the action of local opioid receptors. In the present work, we studied injury-related hyperemia of the rat sciatic vasa nervorum using laser Doppler and hydrogen clearance microelectrode measurements of local perfusion. Systemic morphine reversed hyperemia by vasoconstricting both extrinsic and intrinsic microvessels supplying 48-h-old "neuroma" preparations or crush zones of peripheral nerve trunks. Morphine did not constrict microvessels of contralateral uninjured or sham exposed but uninjured sciatic nerves. In contrast to the injured nerves, contralateral uninjured nerves exposed to morphine frequently had a rise in local perfusion, indicating vasodilation. The vasoconstrictive actions of morphine were blocked by pretreatment with naloxone and were not mimicked by saline injections alone. Systemic doses of selective opioid agonists to mu-, kappa-, and delta-receptors also selectively constricted microvessels of injured nerves. Local blood flow in older experimental neuromas at 7 days had partial sensitivity to morphine, whereas at 14 days perfusion flow was not influenced by morphine. Exogenous opioids dampen early but not later inflammatory microvasodilation and could have important influences on the nerve regenerative milieu.

Response to intravenous lidocaine infusion predicts subsequent response to oral mexiletine: A prospective study

The local anesthetic sodium-channel blockers lidocaine and mexiletine reduce spontaneous and evoked activity in experimental neuroma models and have been reported to relieve a variety of clinical neuropathic pain conditions. The predictive value of relief from an intravenous lidocaine infusion (IVL) for subsequent relief from a 4-week trial of oral mexiletine was assessed in a prospective study of nine subjects with chronic neuropathic pain of peripheral origin. Subjects received IVL, 2 mg/kg and 5 mg/kg, over 45 min during separate sessions in random order under double-blind conditions. One week later, a 4-week titrating trial of oral mexiletine was initiated. Both doses of IVL significantly reduced pain visual analogue scores (VAS) scores. Although IVL 5mg/kg produced significantly higher pain relief scores than IVL 2 mg/kg, subjects responded in a highly consistent manner to the two IVL. Subsequent response to oral mexiletine was significantly correlated with the average response to the two IVL. Mexiletine dose and blood levels were not correlated with pain relief. The results suggest that IVL may be a valuable tool in selecting patients for oral therapy with analogous drugs.

Evidence for CGRP accumulation and activity in experimental neuromas.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and widely distributed neuropeptide that may participate in the injury response of peripheral nerve. We examined evidence for the presence of CGRP immunoreactivity (IR) and its activity in experimental neuromas of Sprague-Dawley rats created by sectioning the midsciatic nerve with resection of 2-3 cm of its distal portion and branches. CGRP activity was evaluated by measuring local blood flow in neuromas using hydrogen polarography and laser-Doppler flowmetry. At all time points studied after nerve section (24 h, 48 h, 7 days, 14 days) there was a rise in local blood flow in the neuroma stumps. At 48 h the hyperemia was maximum but was reversed by topical application of human CGRP(8--37), a specific CGRP-receptor antagonist. CGRP presence was evaluated by immunohistochemistry and radioimmunoassay (RIA). At 24 and 48 h, CGRP IR was intense and distributed in a globular and diffuse pattern apparently not confined to discrete axonlike profiles. At 7 and 14 days, CGRP IR remained prominent and was associated with disorganized axonlike profiles, sometimes directed in a circumferential pattern around the outside of the neuroma. RIA confirmed rises in CGRP content at 24 and 48 h that accompanied the changes in local blood flow and altered distribution of CGRP IR. CGRP accumulates in a time-related fashion within experimental neuromas, where it induces among other possible actions prominent local vasodilatation. CGRP may be important in the regenerative milieu of injured nerves.

Accumulation and degranulation of mast cells in experimental neuromas

Mast cell accumulation and degranulation occur within the endoneurium of injured peripheral nerves. We investigated the time course of mast cell accumulation and degranulation in experimental neuromas of the sciatic nerve in rats. Mast cell accumulation and degranulation were significant only after the first week following neuroma creation and were prominent at 14 days within the neuroma stump. Mast cell degranulation could account for microvascular changes within neuromas after the first week following injury.

Reduction of experimental neuroma formation with ricin

Twenty rat sciatic nerves were bilaterally transected, one as control, and one intraneurally injected with ricin. At 11 weeks, all controls demonstrated large neuromas. Ten injected nerves had no neuroma or a significantly smaller one, while the other 10 had neuroma formation similar to controls. No effect on distant dorsal root ganglia or spinal cord was seen. Thirteen additional rats underwent nerve injection with I 125-labeled ricin. At one week, most radioactivity was localized to sciatic nerve, surrounding muscle, and thyroid, with trace amounts in dorsal root ganglia and spinal cord. Intraneural ricin can inhibit neuroma formation in transected nerve, but results in unpredictable uptake of ricin by nerve and excess spillage into surrounding tissues.

A fluorescence study of changes in noradrenergic sympathetic fibres in experimental peripheral nerve neuromas

Experimental neuromas were produced in rats by sciatic nerve section and avulsion of the distal stumps. At intervals varying from 3 days to 8 weeks after nerve section, the developing neuromas were resected and processed for noradrenaline (NA) fluorescence microscopy by the sucrose-phosphate-glyoxylic acid (SPG) method. From serial longitudinal sections through the neuromas and the nerve proximally, counts of noradrenergic sympathetic axons were made, together with qualitative observations of axon sprouting and NA content. By 3 days after nerve section there was a massive sprouting of sympathetic axons, with increased NA content, particularly towards the distal tip of the neuroma. Axon counts remained high 1 week following section then fell to below normal levels at 2 weeks, returning towards normal 8 weeks after nerve section. These results are discussed in relation to the known pathophysiological interaction between sympathetic efferent and sensory afferent fibres, which develops in neuromas following nerve section.

Substance P-, CGRP- and NPY-immunoreactive nerve fibers in rat sciatic nerve-end neuromas

Substance P (SP)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-immunoreactive nerve fibers were examined in experimental sciatic nerve-end neuromas in the rat with immunohistochemical techniques. At 1–3 days after nerve ligation and section of the sciatic nerve there was an accumulation of SP-like immunoreactivity (SP-LI). Six days after the lesion there was, however, a marked reduction and the neuromas remained virtually depleted from SP-LI at survival times between 8 days and 3 months. CGRP-LI was strong at 1–5 days post-operatively. By 8 days, CGRP-LI was reduced, but a large number of axons were still immunoreactive, and remained immunolabelled up to 3 months. CGRP-LI nerve fibers formed endbulbs, and appeared to grow in both anterograde and retrograde directions. Fine fibers sprouts were first observed at 8 days, but preterminal branching in neuromas aged less than a month was uncommon. At early stages (1–3 days) after ligation, there was a marked accumulation of NPY-LI proximal to the nerve constriction. NPY-LI was reduced from 5 days and on, but many fibers remained NPY-positive and their growth pattern through proximal and distal neuroma segments could be determined. The present results thus may indicate a differential effect of nerve injury on neuropeptide expression: immunohistochemically detectable SP-LI rapidly disappears from sciatic nerve fibers trapped in nerve-end neuromas, but CGRP-LI and NPY-LI remain and are useful as neuroanatomical markers for two subclasses of sprouting axons. Furthermore, the findings suggest that both CGRP and NPY, but not SP, could be involved in ectopic electrical activity in experimental neuromas.

Carbamazepine inhibits spontaneous activity in experimental neuromas

In eight adult Sprague-Dawley rats the effect of parenteral carbamazepine on spontaneous discharges from saphenous neuromas (7–42 days following nerve section) was tested. Intravenous carbamazepine produced immediate inhibition of spontaneous activity originating in both A- α β and A-δ fibers at doses of 2.51–11.2 (7.9 ± 3.3) mg/kg. In four additional animals, serum levels of carbamazepine were determined following iv administration of the drug. These results indicated that ectopic spontaneous impulse generation from experimental neuromas was inhibited by carbamazepine in the range of serum concentration in which the agent is used to treat trigeminal neuralgia and other painful neuropathies in humans. This implies that the effectiveness of this agent in the treatment of these disorders may result from suppression of peripherally originating ectopic spontaneous activity.

Contrasting time course of catecholamine accumulation and of spontaneous discharge in experimental neuromas in the rat

Catecholamine-containing sympathetic axons in rat sciatic nerve-end neuromas were visualized histochemically. Within a few hours of ligating and sectioning the nerve, axons began to accumulate catecholamine histofluorescence. Density of labelled fibers peaked 2-5 days postoperative, then declined rapidly so that little or no label was observed beyond 12 days. Sympathectomy eliminated staining; neonatal treatment with capsaicin had no effect. Accumulation and dissipation of histofluorescence preceded the rise and fall of electrical hyperexcitability in neuromas by several days respectively.

Contrasting thermal sensitivity of spontaneously active A- and C-fibers in experimental nerve-end neuromas

Injured afferent A- and C-fibers ending in experimental neuromas in the rat sciatic nerve generate a substantial spontaneous discharge. We show that for individual axons the rate and percent incidence of spontaneous discharge are sensitive to neuroma temperature. Within the range of 14–43°C, firing rate of all of the myelinated fibers examined increased as temperature rose, and decreased as temperature fell. For fibers with a tonic rhythmic discharge pattern, Q 10 averaged 1.64 at 34–42°C. Some fibers that were initially silent began to fire as the neuroma was warmed, and some fibers active at baseline temperature fell silent when the neuroma was cooled. Unmyelinated fibers behaved quite differently, showing either no response to temperature changes (44% of fibers sampled), or an increase in discharge rate upon cooling (56%). These effects are probably not secondary to vascular changes, but rather reflect thermal sensitivity of the ectopic neuroma impulse generator sites. This thermal sensitivity may account for the aggravation of phantom limb pain and other neuralgias during cold weather (i.e., post-traumatic cold intolerance).

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.

Has the amount of spontaneous electrical activity in experimental neuromas been overestimated?

Previous studies of experimental neuromas have indicated that some axons terminating in the neuroma exhibit both spontaneous and mechanosensitive discharges. Since these spontaneous discharges appear to occur in potentially nociceptive axons (A delta and C fibers), it has been speculated that this activity may relate to pain that occurs after peripheral nerve injury. Recent results from our laboratory have revealed several possible sources of error in prior electrophysiological studies of neuromas. Most notably, gallamine, a muscle-paralyzing agent that has been used in the majority of previous studies of experimental neuromas, has profound potassium-channel-blocking properties that may increase spontaneous activity in damaged axons. The present study was conducted to re-evaluate the incidence of spontaneous activity in experimental neuromas, and the fiber types involved in these discharges. A group of 44 male Sprague-Dawley rats underwent unilateral saphenous axotomy 1-8 weeks prior to acute neurophysiological recording experiments, and 6 additional rats underwent acute control recording procedures only. Recording was performed in all animals using a modification of the microfilament recording technique to determine the conduction velocities (CVs) and origins of spontaneously discharging axons. A thorough search for spontaneous discharges was made in each nerve both before and after the administration of gallamine. Spontaneous activity was rare in acutely severed saphenous nerve and was not significantly affected by gallamine administration. In rats with 1- to 4-week-old experimental saphenous neuromas, spontaneous activity was rare but was increased by a factor of 12.75 after gallamine treatment. Gallamine administration produced significantly more of both A alpha beta and A delta activity, compared to control recordings. No spontaneous C-fiber activity was found originating in neuromas either before or after gallamine. C-fiber spontaneous discharges in the apparently isolated saphenous nerve segment had receptive fields in fascia, superficial vasculature, and hairy skin of the medial hindlimb. Our conclusions are as follows: (1) Neuromas exhibit only rare spontaneous discharges unless exposed to potassium-channel-blocking agents; (2) all C-fiber activity recorded in saphenous nerve with a distal neuroma is derived from vascular, fascial, and other receptive fields rather than from the neuroma; (3) these data are consistent with known clinical phenomena in that neuromas are not usually spontaneously painful.

Corticosteroids suppress ectopic neural discharge originating in experimental neuromas

Some injured sensory fibers ending in an experimental neuroma in the rat sciatic nerve discharge spontaneously. Furthermore, many become sensitive to a range of physical and chemical stimuli. The resulting afferent barrage is thought to contribute to paresthesias and pain associated with peripheral nerve injury. We report that the development of such ectopic neuroma discharge is largely prevented when the freshly cut nerve end is treated with any of 3 commercially available corticosteroid preparations including two in depot form, triamcinolone hexacetonide (Lederspan) and triamcinolone diacetate (Ledercort), and one in soluble form, dexamethasone (Dexacort). These corticosteroids also produce a rapid and prolonged suppression of ongoing discharge in chronic neuromas that have already become active. The kinetics of corticosteroid suppression of neuroma discharge suggest a direct membrane action rather than an anti-inflammatory action.