Neuropeptide immunoreactivity in ligature-induced neuromas of the inferior alveolar nerve in the ferret

Injury to branches of the trigeminal nerve can sometimes result in persistent dysaesthesia. In an attempt to understand the aetiology of this condition we are currently investigating changes which occur at the injury site. In the present study we have examined the expression of seven neuropeptides, all of which have been implicated in nociceptive transmission, or have previously been shown to have altered expression following nerve injury. In 20 adult ferrets the inferior alveolar nerve was sectioned and ligated, and recovery permitted for 3 days, 8 days, 3 weeks, 6 weeks or 12 weeks. Longitudinal sections of the neuromas were processed using immunohistochemical techniques to quantify the expression of substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, galanin, somatostatin, enkephalin and neuropeptide Y. After 3 days, all of the neuropeptides were expressed at the injury site. In the neuromas examined after longer recovery periods these levels of expression had declined and were similar to those found in the contralateral controls. This initial high level, followed by a decline, parallels the incidence of ectopic neural activity recorded electrophysiologically in the same model. It is, therefore, possible that the accumulation of neuropeptides at the injury site may play a role in the initiation or modulation of ectopic neural activity.     

An evaluation of the incisive nerve block and combination inferior alveolar and incisive nerve blocks in mandibular anesthesia

The purpose of this study was to measure the degree of anesthesia obtained with the incisive nerve block, the inferior alveolar nerve block and a combination of both injections in mandibular teeth. Using a repeated measures design, 40 subjects randomly received an incisive nerve block, a conventional inferior alveolar nerve block, or a combination inferior alveolar nerve block plus an incisive nerve block using either lidocaine or saline (control), at four successive appointments. The mandibular teeth and contralateral canine (+/- controls) were blindly tested with an Analytic Technology pulp tester at 4-min cycles for 60 min. An 80 reading indicated complete pulpal anesthesia. The incisive nerve block alone did not result in successful pulpal anesthesia in the central, lateral, first, and second molars. It was successful in the first and second premolars but the duration was approximately 30 min. The combination inferior alveolar nerve block plus incisive nerve block was successful in the first and second premolars, and enhanced anesthesia for the laterals and first molars.     

Anesthetic efficacy of the supplemental intraosseous injection of 2% lidocaine with 1:100,000 epinephrine in irreversible pulpitis

The purpose of this study was to determine the anesthetic efficacy of a supplemental intraosseous injection of 2% lidocaine with 1:100,000 epinephrine in teeth diagnosed with irreversible pulpitis. Fifty-one patients with symptomatic, vital maxillary, and mandibular posterior teeth diagnosed with irreversible pulpitis received conventional infiltrations or inferior alveolar nerve blocks. Pulp testing was used to determine pulpal anesthesia after "clinically successful" injections. Patients who were positive to the pulp tests, or were negative to the pulp tests but felt pain during endodontic access, received an intraosseous injection using 1.8 ml of 2% lidocaine with 1:100,000 epinephrine. The results demonstrated that 42% of the patients who tested negative to the pulp tests reported pain during treatment and required supplemental anesthesia. Eighty-one percent of the mandibular teeth and 12% of maxillary teeth required an intraosseous injection due to failure to gain pulpal anesthesia. Overall, the Stabident intraosseous injection was found to be 88% successful in gaining total pulpal anesthesia for endodontic therapy. We concluded that, for posterior teeth diagnosed with irreversible pulpitis, the supplemental intraosseous injection of 2% lidocaine (1:100,000 epinephrine) was successful when conventional techniques failed.     

Anatomical regeneration and behavioral recovery following crush injury of the trigeminal root in lamprey

In normal larval lamprey, bilateral application of horseradish peroxidase (HRP) to the dorsal part of the anterior oral hood labeled subpopulations of trigeminal components on both sides of the brain; peripherally projecting motoneurons, medullary dorsal cells (sensory), and spinal dorsal cells (sensory), as well as centrally projecting afferents in the trigeminal descending tracts. Following unilateral crush injury of the right trigeminal root, HRP labeling of sensory and motor trigeminal components on the right side gradually increased with increasing recovery time, between 2 weeks and 12 weeks postcrush (PC). Axons of trigeminal motoneurons appeared to exhibit robust regeneration, whereas restoration of projections in the descending trigeminal tract ipsilateral to the injury was incomplete. Control experiments indicated that motor and sensory axons from the intact side of the oral hood did not sprout across the midline to the denervated side. Several results suggested that regenerated trigeminal sensory fibers made synapses with brain neurons that have direct or indirect inputs to reticulospinal (RS) neurons. Following a unilateral crush injury of the right trigeminal root, escape behavior in response to stimulation of the right side of the oral hood gradually returned to normal. Muscle recordings at various recovery times confirmed that anatomical regeneration of trigeminal sensory axons was functional. In addition, at 8 or 12 weeks PC, brief stimulation of the oral hood ipsilateral or contralateral to the crush injury elicited synaptic responses in RS neurons on either side of the brain, similar to that in normal animals. In the lamprey, compensatory mechanisms probably allow recovery of behavioral function despite incomplete regeneration of trigeminal sensory axons within the central nervous system.     

Transient axonal branching in the developing corpus callosum

During development, there is a transient overproduction of axons in the corpus callosum; this overproduction of axons is due, in part, to a transient excess of neurons that send an axon through the corpus callosum. However, transient axonal branching could also contribute to the developmental overproduction of callosal axons. To investigate this possibility, we filled developing callosal axons in the Syrian hamster with the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil). Light microscopic analysis showed that, indeed, developing callosal axons branch transiently in the hamster: branching was robust on postnatal day 0 (P0) and P3 (P0 = the first 24 hr after birth), less prominent on P6 and P8, and absent by P11. Immature callosal axons branched before or after crossing the midline and at all rostral-caudal and medial-lateral levels within the corpus callosum. The majority of callosal axon collaterals that were contained within individual 100-micron-thick sections were relatively short (mean = 15.1 microns) but some collaterals extended up to approximately 135 microns from the main axon trunk before passing out of the section in which they were observed. Nearly all of the collaterals emanated from the main axon trunk; higher-order collaterals were rare. Some callosal axon trunks had multiple collaterals. Branching callosal axons originated from multiple cortical areas, including area 17. Electron microscopic observations indicated that the processes designated as axon collaterals by light microscopic criteria would have been included in electron microscopic counts of developing callosal axons. Some callosal axon trunks and branches had ultrastructural features that suggested they were degenerating. In cats, developing callosal axons branch on embryonic day 57 (E57; the first 24 hr after conception = E0) and P0. Thus, it is likely that transient branching of immature callosal axons is a generalized feature of mammalian cortical development and that it contributes to the overproduction of callosal axons, albeit perhaps to varying degrees, in multiple species.     

The effect of age on peripheral motor nerve function after crush injury in the rat

OBJECTIVE: To determine the ontogeny of functional recovery after peripheral nerve crush injury. DESIGN: Comparative study in rats of varying ages. MATERIAL AND METHODS: Sixty-second crush injury was performed on the left posterior tibial nerve. Control animals underwent either nerve transection or sham procedure. Nerve function was evaluated 2, 4, and 8 weeks following injury by walking track analysis. Print length ratio (PLR), (ratio of normal right-sided print length to experimental left-sided print length), was used to evaluate functional recovery. MEASUREMENTS AND MAIN RESULTS: Two weeks after crush injury, adult rats experienced significantly greater functional impairment than both 4-day-old and 3-week-old animals (p < 0.05). Four weeks after injury, the difference in function between 4-day-old and adult rats and between 3-week-old and adult rats became insignificant. Complete recovery had been achieved by 8 weeks in all groups. CONCLUSIONS: These results demonstrate faster functional recovery after nerve injury in immature rats than in adults.     

Activation of protein kinase A contributes to the expression but not the induction of long-term hyperexcitability caused by axotomy of Aplysia sensory neurons

Nociceptive sensory neurons (SNs) in Aplysia provide useful models to study both memory and adaptive responses to nerve injury. Induction of long-term memory in many species, including Aplysia, is thought to depend on activation of cAMP-dependent protein kinase (PKA). Because Aplysia SNs display similar alterations in models of memory and after nerve injury, a plausible hypothesis is that axotomy triggers memory-like modifications by activating PKA in damaged axons. The present study disproves this hypothesis. SN axotomy was produced by (1) dissociation of somata from the ganglion [which is shown to induce long-term hyperexcitability (LTH)], (2) transection of neurites of dissociated SNs growing in vitro, or (3) peripheral nerve crush. Application of the competitive PKA inhibitor Rp-8-CPT-cAMPS at the time of axotomy failed to alter the induction of LTH by each form of axotomy, although the inhibitor antagonized hyperexcitability produced by 5-HT application. Strong activation of PKA in the nerve by coapplication of a membrane-permeant analog of cAMP and a phosphodiesterase inhibitor was not sufficient to induce LTH of either the SN somata or axons. Furthermore, nerve crush failed to activate axonal PKA or stimulate its retrograde transport. Therefore, PKA activation plays little if any role in the induction of LTH by axotomy. However, the expression of LTH was reduced by intracellular injection of the highly specific PKA inhibitor PKI several days after nerve crush. This suggests that long-lasting activation of PKA in or near the soma contributes to the maintenance of long-term modifications produced by nerve injury.     

Dental innervation and CGRP in adult p75-deficient mice

Adult dental tissues have unusual neurotrophin biology. Pulpal fibroblasts express nerve growth factor (NGF) and the low-affinity p75 neurotrophin receptor, their sensory nerve fibers express p75 and trk A, and pulpal sympathetic fibers lack p75. Following tooth injury, there is increased pulpal NGF, sprouting of sensory nerve endings, and increased immunoreactivity for the sensory neuropeptide calcitonin gene-related peptide (CGRP). In the present study, we have analyzed tooth structure and innervation of pulp and periodontal ligament in young (6-8 weeks, 3 months) and older (5-12 months) adult mice carrying a null mutation in the p75 gene and compared the results with those of age-matched wild-type controls. Our hypotheses were that tooth structure would be abnormal and that pulpal innervation would be greatly reduced because it consists primarily of nociceptive fibers that have been found to be severely depleted in skin of p75(-/-) mice. Tissues were fixed, X-rayed for gross dental morphology, decalcified, and analyzed for immunoreactivity for CGRP and for a general nerve marker, protein gene product 9.5. Radiographs showed worn-down molar crowns in p75-deficient mice. Light microscopy confirmed the accelerated molar wear and showed intense CGRP immunoreactivity in pulp nerve endings of mutant mice, compared with a gradual decrease in CGRP intensity in controls during normal aging. The CGRP intensity in 5-12-month-old pairs of mice was threefold greater in the mutants (P < 0.03), and in younger mice the mutant always had more CGRP than its matched control. The innervation of molar ligament in all p75-deficient mice was similar to that of controls except there was nerve sprouting near bone loss in mutants. The incisors of mutant mice did not have unusual wear and their pulpal CGRP immunoreactivity remained normal, but their periodontal ligament had fewer thin branched nerve endings at all ages. Thus, most innervation of teeth and their supporting tissues developed normally, and the only neural changes in p75(-/-) mutant mice were the reduction of incisor ligament sensory receptors and increased molar CGRP. Sensory nerves in teeth gradually lose neuropeptide intensity during aging, but that did not happen in the mutant mice, suggesting that the accelerated molar wear stimulated persistent high levels of CGRP.     

Increased spike-frequency adaptation and tea sensitivity in dorsal root fibers after sciatic nerve injury

Excitability of rat dorsal root axons were studied 3 weeks after injury to the sciatic nerve. Whole nerve recordings were obtained from injured and control nerves in a sucrose gap chamber. Constant current depolarization pulses (30-200 ms) applied approximately 50% above the stimulus strength required for maximal amplitude compound action potentials (CAPs) evoked a burst of action potentials in the dorsal root which displayed spike adaptation. The depolarization-induced burst response of the dorsal roots was greatly reduced after crush or transection of the sciatic nerve. However, application of the potassium channel blocker, tetraethylammonium (TEA), restored the burst discharge in injured dorsal root axons. Brief tetanic stimulation of the dorsal root also induced an afterhyperpolarization (AHP) that was twice as large in the transection group as compared to the control group, and which was blocked by TEA. There were no changes seen in the amplitude of the compound action potential, frequency-following characteristics, refractory properties, or 4-AP sensitivity in the dorsal roots after peripheral nerve injury. These results suggest that there is enhanced spike adaptation that occurs at the same time as an increase in the sensitivity to the potassium channel blocker, TEA, in axon regions proximal to the site of nerve injury and have implications for the pathophysiology of nerve injury.     

Procurement, preservation, and transport of cadaver kidneys

Numerous findings support the possibility that highly sulfated proteoglycans are inhibitory molecules which, at high concentration relative to growth-promoting signals, may regulate or guide axonal growth. Although most studies implicate sulfated proteoglycans in the poor regenerative capacity of the central nervous system, inhibitory proteoglycans also may play an important role in the successful regeneration of axons within peripheral nerve. Cultured rat schwannoma and Schwann cells produce chondroitin sulfate proteoglycan (CSPG) which binds to and inhibits the neurite-promoting activity of laminin [Muir et al. (1989) J. Cell Biol. 109:2353]. In the present study, we found a similar neurite-inhibiting activity associated with CSPG isolated from normal adult rat sciatic nerve. Following nerve crush injury, this inhibitory activity was increased sevenfold in regenerating nerve distal to the injury. This increase was largely attenuated by in vivo administration of the proteoglycan synthesis inhibitor beta-D-xyloside. In normal adult nerve, immunolabeling for CSPG core protein was concentrated in slender bands surrounding axon-Schwann cell units and within nodes of Ranvier. Following nerve crush injury, immunolabeling of CSPG and laminin became more intense in distal nerve and CSPG increased within endoneurium and surrounding nerve sheaths. Embryonic dorsal root ganglionic neurons cultured on longitudinal nerve sections extended neurites along the exposed surfaces of Schwann cell basal lamina. The length of neurites was increased 58% on normal nerve sections pretreated with chondroitinase. Even though laminin levels were elevated in basal lamina of injured nerve, neuritic growth on sections of injured nerve was not significant increased unless sections were pretreated with chondroitinase. These results indicate that inhibitory CSPG is up-regulated in injured nerve and plays a role in regulating axonal regeneration.     

Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions

The novel sodium channel PN3/alpha-SNS, which was cloned from a rat dorsal root ganglion (DRG) cDNA library, is expressed predominantly in small sensory neurons and may contribute to the tetrodotoxin-resistant (TTXR) sodium current that is believed to be associated with central sensitization in chronic neuropathic pain states. To assess further the role of PN3, we have used electrophysiological, in situ hybridization and immunohistochemical methods to monitor changes in TTXR sodium current and the distribution of PN3 in normal and peripheral nerve-injured rats. (1) Whole-cell patch-clamp recordings showed that there were no significant changes in the TTXR and TTX-sensitive sodium current densities of small DRG neurons after chronic constriction injury (CCI) of the sciatic nerve. (2) Additionally, in situ hybridization showed that there was no change in the expression of PN3 mRNA in the DRG up to 14 d after CCI. PN3 mRNA was not detected in sections of brain and spinal cord taken from either normal or nerve-injured rats. (3) In contrast, immunohistochemical studies showed that major changes in the subcellular distribution of PN3 protein were caused by either CCI or complete transection of the sciatic nerve. The intensity of PN3 immunolabeling decreased in small DRG neurons and increased in sciatic nerve axons at the site of injury. The alteration in immunolabeling was attributed to translocation of presynthesized, intracellularly located PN3 protein from neuronal somata to peripheral axons, with subsequent accumulation at the site of injury. The specific subcellular redistribution of PN3 after peripheral nerve injury may be an important factor in establishing peripheral nerve hyperexcitability and resultant neuropathic pain.     

Tenascin-R inhibits the growth of optic fibers in vitro but is rapidly eliminated during nerve regeneration in the salamander Pleurodeles waltl

Tenascin-R is a multidomain molecule of the extracellular matrix in the CNS with neurite outgrowth inhibitory functions. Despite the fact that in amphibians spontaneous axonal regeneration of the optic nerve occurs, we show here that the molecule appears concomitantly with myelination during metamorphosis and is present in the adult optic nerve of the salamander Pleurodeles waltl by immunoblots and immunohistochemistry. In vitro, adult retinal ganglion cell axons were not able to grow from retinal explants on a tenascin-R substrate or to cross a sharp substrate border of tenascin-R in the presence of laminin, indicating that tenascin-R inhibits regrowth of retinal ganglion cell axons. After an optic nerve crush, immunoreactivity for tenascin-R was reduced to undetectable levels within 8 d. Immunoreactivity for the myelin-associated glycoprotein (MAG) was also diminished by that time. Myelin was removed by phagocytosing cells at 8-14 d after the lesion, as demonstrated by electron microscopy. Tenascin-R immunoreactivity was again detectable at 6 months after the lesion, correlated with remyelination as indicated by MAG immunohistochemistry. Regenerating axons began to repopulate the distal lesioned nerve at 9 d after a crush and grew in close contact with putative astrocytic processes in the periphery of the nerve, close to the pia, as demonstrated by anterograde tracing. Thus, the onset of axonal regrowth over the lesion site was correlated with the removal of inhibitory molecules in the optic nerve, which may be necessary for successful axonal regeneration in the CNS of amphibians.     

Comparative dose-dependence study of FK506 and cyclosporin A on the rate of axonal regeneration in the rat sciatic nerve

The new immunosuppressant drug FK506 (Tacrolimus) increases the rate of nerve regeneration in vivo (Gold et al., 1994; Gold et al., 1995). In the present study, we have examined the dose-dependence of FK506's ability to enhance nerve regeneration. In the first set of experiments, rats received daily s.c. injections of FK506 (2 mg/kg, 5 mg/kg or 10 mg/kg) for 18 days after a sciatic nerve crush injury. Signs of functional recovery in the hind feet appeared earlier than in saline-treated control rats at all three FK506 dosage; recovery was maximally accelerated in the 5-mg/kg group. Light microscopy at 18 days after nerve crush revealed more regenerating myelinated fibers in FK506-treated rats than in controls; this was most apparent in the 5-mg/kg group. Morphometric analysis of axonal areas in the soleus nerve confirmed that axonal calibers were maximally increased in the 5-mg/kg group. In the second set of experiments, the rate of axonal regeneration was determined by radiolabeling the L5 dorsal root ganglion. Regeneration rate for sensory axons was maximally increased (by 34%) in the 5-mg/kg group. In contrast, cyclosporin A (10 or 50 mg/kg; dosages were selected on the basis of the 1/10 lower potency of cyclosporin A) did not significantly alter the rate of axonal regeneration. Cyclosporin A (50 mg/kg) also failed to increase functional recovery or axonal calibers in the soleus nerve. Because the two drugs share a common mechanism for producing immunosuppression (i.e., calcineurin inhibition), these results indicate that FK506's nerve regenerative property involves a distinct, calcineurin-independent mechanism.     

Granulomatous orchitis. A case report and review of the literature

We developed a method for determination of motor conduction along the mandibular and sensory conduction along the lingual and inferior alveolar nerves in 10 controls and 6 patients with lingual neuropathy following lower wisdom tooth extraction. Patients with lingual neuropathy had reduced/absent or delayed compound sensory action potentials and normal conduction along the fibers of the inferior alveolar nerve and mandibular nerve. The method provides a useful electrophysiological means of evaluating lingual nerve lesions.     

Typing of methicillin-resistant Staphylococcus aureus isolates from Düsseldorf by six genotypic methods

This in vivo double-blind study evaluated the effect of recombinant human glial growth factor 2 (rhGGF2), a Schwann cell mitogen, on the recovery of motor function of rat sciatic nerve following crush injury. Seventy three rats were divided into three groups. Group I (n=5), sham operated; Groups II (n=34) and III (n=34) received a 100 g crush load for 2 h over a 5 mm segment of the sciatic nerve. Group III was treated with 1 mg/kg rhGGF2, via subcutaneous injection one day before nerve crush and daily for the following four days. Group II received an equivalent volume of saline as a control. Motor functional recovery was assessed by calculating the sciatic functional index (SFI) and the recovery rate of tetanic contractile force of the extensor digitorum longus (EDL) muscle. Recovery of nerve function was evident at day 11 after crush in the rhGGF2-treated animals, whereas the nerves in controls were still paralyzed. The rhGGF2-treated animals showed a significant improvement of the SFI between days 11-21 postoperatively when compared to controls. The isometric tetanic contractile force was stronger in the rhGGF2-treated group than in controls, with a significant difference at 40 to 70 Hz stimulus frequencies on day 4. Correlation analysis showed that tetanic contractile force had a linear correlation with the SFI. Histologic assessment indicated that the rhGGF2-treated animals showed less severe degeneration and earlier robust remyelination of axons than controls. The results suggest that treatment with rhGGF2 is effective in promoting nerve regeneration as seen in measurements of functional recovery and qualitative assessment of nerve morphology. The mechanism of GGF's protective effect may be related to its direct action on Schwann cells, stimulating their mitosis as well as inducing neurotrophic factors essential to neuronal maintenance and repair.     

The time-course of abeta-evoked c-fos expression in neurons of the dorsal horn and gracile nucleus after peripheral nerve injury

We have examined the mechanisms underlying Abeta-evoked c-fos expression in the dorsal horn and gracile nucleus following either sciatic nerve section or crush injury. The results indicate that in the spinal cord Abeta-evoked c-fos does not depend on primary afferent sprouting but is associated with the disconnection from the peripheral target since its expression in the dorsal horn reverts to normal after crush injury when regeneration occurs but persists after cut and ligation where regeneration is prevented. In contrast, however, Abeta-evoked c-fos expression in the gracile nucleus may be under some other control since its expression appears independent of peripheral nerve regeneration.     

Neuronal injury increases retrograde axonal transport of the neurotrophins to spinal sensory neurons and motor neurons via multiple receptor mechanisms

We investigated the retrograde axonal transport of 125I-labeled neurotrophins (NGF, BDNF, NT-3, and NT-4) from the sciatic nerve to dorsal root ganglion (DRG) sensory neurons and spinal motor neurons in normal rats or after neuronal injury. DRG neurons showed increased transport of all neurotrophins following crush injury to the sciatic nerve. This was maximal 1 day after sciatic nerve crush and returned to control levels after 7 days. 125I-BDNF transport from sciatic nerve was elevated with injection either proximal to the lesion or directly into the crush site and after transection of the dorsal roots. All neurotrophin transport was receptor-mediated and consistent with neurotrophin binding to the low-affinity neurotrophin receptor (LNR) or Trk receptors. However, transport of 125I-labeled wheat germ agglutinin also increased 1 day after sciatic nerve crush, showing that increased uptake and transport is a generalized response to injury in DRG sensory neurons. Spinal cord motor neurons also showed increased neurotrophin transport following sciatic nerve injury, although this was maximal after 3 days. The transport of 125I-NGF depended on the expression of LNR by injured motor neurons, as demonstrated by competition experiments with unlabeled neurotrophins. The absence of TrkA in normal motor neurons or after axotomy was confirmed by immunostaining and in situ hybridization. Thus, increased transport of neurotrophic factors after neuronal injury is due to multiple receptor-mediated mechanisms including general increases in axonal transport capacity.     

Sex differences in sensory and motor branches of the pudendal nerve of the rat

The morphology of the pudendal nerve was quantified in adult male and female rats. The sensory branch of the pudendal nerve was about three times as large in cross section in males as in females, and the motor branch was about five times as large. Electron microscopy was used to determine the ultrastructural bases of these gross size differences. Differences that were found included greater packing density of both myelinated and unmyelinated axons in females, larger myelinated and unmyelinated axons in males, larger myelin sheaths of sensory axons in males, more numerous myelinated axons in both branches of males, and more numerous unmyelinated axons in the sensory branch of males. There was also some indication that myelinated sensory axons were more likely to branch in the dorsal clitoral nerve of females than in the homologous nerve of males. Morphological differences in the structure of pudendal axons, their associated Schwann cells, and the extracellular matrix as well as differences in sensory and motor axonal number all have potential implications for the sexual differentiation of the central nervous system and behavior.     

Dependence of centriole formation on protein synthesis

Electron microscope observations were made of rat peroneal nerve after crushing using intravenously injected horseradish peroxidase (HRP) as a tracer protein to indicate changes in vascular permeability. At 1/2 h and 2 d after the crush there was gross leakage of HRP from damaged capillaries at the site of injury but none from vessels above or below this. Ultrastructurally vessels at the site of crush showed broken and separated endothelial cells. Proximally and distally there was little abnormal in the vessel walls; vesicles containing HRP were absent and tight-junctions between cells remained intact. Twenty-one days after the crush, leakage of HRP was found both at the site of crush and along the distal segment. The only change in vessel walls was an obvious increase in vesicles filled with HRP. Tracer was also found both in perivascular locations and throughout the endoneurial space.