Arm cycling increases the short-latency reflex from ankle dorsiflexor afferents to knee extensor muscles.

Low-intensity electrical stimulation of the common peroneal nerve (CPN) evokes a short latency reflex in the heteronymous knee extensor muscles (referred to as the CPN reflex). The CPN reflex is facilitated at a heel strike during walking, contributing to body weight support. However, the origin of the CPN reflex increase during walking remains unclear. We speculate that this increase originates from multiple sources due to a body of evidence suggesting the presence of neural coupling between the arms and legs. Therefore, we investigated the extent to which the CPN reflex is modulated during rhythmic arm cycling. Twenty-eight subjects sat in an armchair and were asked to perform arm cycling at a moderate cadence using a stationary ergometer while performing isometric contraction of the knee extensors, such that the CPN reflex was evoked. The CPN reflex was evoked by stimulating the CPN [0.9-2.0× the motor threshold (MT) in the tibialis anterior muscle] at the level of the neck of the fibula. The CPN-reflex amplitude was measured from the vastus lateralis (VL). The biphasic reflex response in the VL was evoked within 27-45 ms following CPN stimulation. The amplitude of the CPN reflex increased during arm cycling compared with that before cycling. The modulation of the CPN reflex during arm cycling was detected only for CPN stimulation intensity around 1.2× MT. Furthermore, CPN-reflex modulation was not observed during the isometric contraction of the arm or passive arm cycling. Our results suggest the presence of neural coupling between the CPN-reflex pathways and neural systems generating locomotive arm movement.NEW & NOTEWORTHY Whether locomotive arm movements contribute to the control of the reflex pathway from ankle dorsiflexor afferents to knee extensor muscles [common peroneal nerve (CPN)-reflex] is an unresolved issue. The CPN reflex in the stationary leg was facilitated only by arm cycling, and not by passive or isometric motor tasks. Our results suggest that the arm locomotor system modulates the reflex pathway from ankle dorsiflexor afferents to the knee extensor muscles.

Histological analysis of single peripheral nerve fiber in acute nerve elongation process.

To observe the histological alterations of single nerve fiber structures after nerve elongation by employing a rabbit peroneal nerve stretching model. 14 rabbits weighing mean 3. 0 kg (2.02-3.31 kg.) were used in the experiment. Two rabbits were used as control when only a sham operation was done (group one, 0% stretch). Acute stretching of the peroneal nerves to elongate them by 10% was done in 6 rabbits (group two, 10% elongation) and by 20% (group three, 20% elongation) in another 6 rabbits. All animals were evaluated by tissue staining technology in a teased-fiber study. The internodal lengths were measured, and nodes of Ranvier and Schmidt-Lanterman notch were observed. The nerve fiber length was increased after stretching. The mean internodal length was 1208.31 microm in group one, 1347.26 microm in group two, and 1411.35 microm in group three. Compared with the control group, mean internodal length was elongated by 11.50% in group two and 16.80% in group three. The difference was statistically significant. The node of Ranvier and Schmidt-Lanterman notch was wider in both group two and group three. Rupture of nerve fiber at the node of Ranvier was observed in group three. The peroneal nerve in rabbits can adapt to mild stretching by internodal length elongation. Elongation by 20% will cause structural rupture and therefore is the limit for nerve elongation.

Neuropathy patterns differ in patients with diabetic complications.

PURPOSE: Diabetic polyneuropathy (PNP) is an important risk factor for foot ulcers. Diabetic dermopathy is more frequent in patients with diabetic neuropathy. We compared clinical and electrophysiological characteristics of PNP localizations/recurrences of foot ulcers, and diabetic dermopathy (DD) between sexes. METHODS: Eighty-eight diabetic patients (44 men, 44 women) had an evaluation regarding detailed history of their diseases, lesion-related data, and clinical examination. Nerve conduction velocities (NCV), compound motor action potentials (CMAP), distal latencies (DL), and sensory nerve action potentials were assessed from the right and left peroneal, right median/ulnar nerves. RESULTS: The presence of DD was more common in men (p < 0.001). The mean NCV of ulnar nerves was slower (p < 0.001); mean CMAP values were lower (p = 0.006); and mean DL was longer in men with compared to women (p = 0.003). Although EMG features of peroneal nerves showed no significant difference, diabetic men had more common and severe peroneal nerve involvement (p = 0.004). Carpal tunnel syndrome was more common in women, though not significant. Patients with right-sided ulcers had lower CMAP amplitudes on the right peroneal nerves in regard to left peroneal nerves (p = 0.009). CONCLUSIONS: Our findings suggest that ulnar nerves are more commonly involved in men, with lower CMAP slower NCV values, and longer DL values.

Reflex sympathetic activity after intravenous administration of midazolam in anesthetized cats.

BACKGROUND: Although intrathecal midazolam has been reported to produce antinociceptive effects mediated by gamma-aminobutyric acid type A-benzodiazepine receptor complexes in the spinal cord, the effects of systemic midazolam on nociception remain unclear. We performed this study to examine the effects of IV-administered midazolam on somatosympathetic Adelta and C reflex discharges in brain-intact cats and decerebrate cats (with transection at midbrain level). METHODS: Somatosympathetic Adelta and C reflexes were elicited in the inferior cardiac sympathetic nerve by electrical stimulation of myelinated (Adelta) and unmyelinated (C) afferent fibers of the superficial peroneal nerve in 28 mature cats. After control somatosympathetic reflex responses were obtained, midazolam was administered IV to four groups of randomly allocated cats as follows: brain-intact cats at a dose of 0.03 mg/kg, brain-intact cats at a dose of 0.1 mg/kg, brain-intact cats at a dose of 0.5 mg/kg, and decerebrate cats at a dose of 0.1 mg/kg. RESULTS: C reflex discharges were significantly augmented at the dose of 0.03 mg/kg and significantly depressed at the dose of 0.1 and 0.5 mg/kg in brain-intact cats. C reflex discharges were also significantly depressed at the dose of 0.1 mg/kg in decerebrate cats. CONCLUSIONS: We have demonstrated that IV midazolam produces dose-related effects on somatosympathetic reflex discharges. The clinical implication of these findings is that the effect of midazolam on nociception depends on its dosage. It also appears that the infra-midbrain region plays a major role in mediating the depressive effects of midazolam on somatosympathetic C reflex discharges.

Fix the problem - A practical guide to whole-mount immunohistochemistry of teased nerve fibres.

BACKGROUND: Immunohistochemical staining of entire nerve fibres allows for studying the molecular composition of functional fibre subunits and may add to the diagnostic value of nerve fibre teasing. NEW METHOD: In this study, we established a sealed-slide method for reproducible immunostaining of deep axoplasmic proteins in permanently straightened nerve fibres. RESULTS: Immunostaining of teased nerve fibres very much is facilitated by tip-fixation with biocompatible glass adhesives. Antibody penetration in fresh nerves can be achieved by thermic and chemical permeabilisation while enzymatic digestion allows for sufficient permeability after aldehyde fixation. COMPARISON WITH EXISTING METHODS: The methods recommended herein are easy to perform and represent a reliable and reproducible way to whole mount immunostaining. CONCLUSIONS: Sealed-slide immunostaining of tip-fixed and permeabilised nerve biopsies will help to validate neurophysiological abnormalities and to screen for target molecules and predictive markers of peripheral nerve disorders such as in inherited neuropathies and Guillain-Barré syndrome.

A device for the electrophysiological recording of peripheral nerves in response to stretch.

The functional consequences of nervous tissue subjected to mechanical loads are of vital importance in successful clinical outcomes and in tissue engineered applications. In this paper, we developed a new ex vivo device that permitted the recording of nerve action potentials while the nerve was subjected to elongation. Experimental results showed guinea pig nerves to possess an inherent tolerance to mild stretch. The mean elongation at which the compound action potential (CAP) amplitude began to decrease was found to be 8.3 +/- 0.56%. The CAP response to stretch was immediate beyond this threshold. After 17.5 +/- 0.74% elongation, the CAP levels decreased to approximately 50% of its uninjured values. When allowed to relax, the CAP recovered almost completely within minutes. Based on the temporal scale of the CAP response and the presence of oxygen during testing, we conclude that the initial mechanism to CAP degradation cannot be ischemia. Since it is inherently difficult to study mechanical damage independent of hemodynamic factors in vivo, the developed model could be used to further elucidate the injury mechanisms of stretch-induced trauma. The design of the ex vivo chamber will also permit the administration and assessment of pharmacological agents on electrical conduction in various deformation conditions.

RetroDISCO: Clearing technique to improve quantification of retrograde labeled motor neurons of intact mouse spinal cords.

BACKGROUND: Quantification of the number of axons reinnervating a target organ is often used to assess regeneration after peripheral nerve repair, but because of axonal branching, this method can overestimate the number of motor neurons regenerating across an injury. Current methods to count the number of regenerated motor neurons include retrograde labeling followed by cryosectioning and counting labeled motor neuron cell bodies, however, the process of sectioning introduces error from potential double counting of cells in adjacent sections. NEW METHOD: We describe a method, retroDISCO, that optically clears whole mouse spinal cord without loss of fluorescent signal to allow imaging of retrograde labeled motor neurons using confocal microscopy. RESULTS: Complete optical clearing of spinal cords takes four hours and confocal microscopy can obtain z-stacks of labeled motor neuron pools within 3-5min. The technique is able to detect anticipated differences in motor neuron number after cross-suture and conduit repair compared to intact mice and is highly repeatable. COMPARISON WITH EXISTING METHOD: RetroDISCO is inexpensive, simple, robust and uses commonly available microscopy techniques to determine the number of motor neurons extending axons across an injury site, avoiding the need for labor-intensive cryosectioning and potential double counting of motor neuron cell bodies in adjacent sections. CONCLUSIONS: RetroDISCO allows rapid quantification of the degree of reinnervation without the confounding produced by axonal sprouting.

Neural tissue engineering: a self-organizing collagen guidance conduit.

We report a novel implantable device that will deliver a tethered aligned collagen guidance conduit containing Schwann cells into a peripheral nerve injury site. Cells (Schwann cells and fibroblasts) incorporated into tethered rectangular collagen gels contracted and resulted in uniaxial alignment. This tissue-engineered construct was tested in three-dimensional culture and demonstrated the ability to guide neurite extension from dissociated dorsal root ganglia. A silicone tube was adapted to provide tethering sites for an implantable construct such that uniaxial cell-generated tension resulted in the formation of a bridge of aligned collagen fibrils, with a resident Schwann cell population. The potential of this device for surgical nerve regeneration was assessed in a 5-mm defect in a rat sciatic nerve model. Neural regeneration through this device was significantly greater than in controls, demonstrating that this system has potential both as a simple robust clinical implant and as a three-dimensional engineered tissue model.

Nerve cross-bridging to enhance nerve regeneration in a rat model of delayed nerve repair.

There are currently no available options to promote nerve regeneration through chronically denervated distal nerve stumps. Here we used a rat model of delayed nerve repair asking of prior insertion of side-to-side cross-bridges between a donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) nerve stump ameliorates poor nerve regeneration. First, numbers of retrogradely-labelled TIB neurons that grew axons into the nerve stump within three months, increased with the size of the perineurial windows opened in the TIB and CP nerves. Equal numbers of donor TIB axons regenerated into CP stumps either side of the cross-bridges, not being affected by target neurotrophic effects, or by removing the perineurium to insert 5-9 cross-bridges. Second, CP nerve stumps were coapted three months after inserting 0-9 cross-bridges and the number of 1) CP neurons that regenerated their axons within three months or 2) CP motor nerves that reinnervated the extensor digitorum longus (EDL) muscle within five months was determined by counting and motor unit number estimation (MUNE), respectively. We found that three but not more cross-bridges promoted the regeneration of axons and reinnervation of EDL muscle by all the CP motoneurons as compared to only 33% regenerating their axons when no cross-bridges were inserted. The same 3-fold increase in sensory nerve regeneration was found. In conclusion, side-to-side cross-bridges ameliorate poor regeneration after delayed nerve repair possibly by sustaining the growth-permissive state of denervated nerve stumps. Such autografts may be used in human repair surgery to improve outcomes after unavoidable delays.

The cell bodies of origin of sympathetic and sensory axons in some skin and muscle nerves of the cat hindlimb.

Cell bodies of sensory and sympathetic axons projecting to skin and skeletal muscle of the cat hindlimb have been labeled retrogradely with horseradish peroxidase (HRP) in order to study location, size, and numbers of the somata of these neurons. HRP was applied to the freshly transected axons of nerves supplying hairy skin (superficial peroneal, SP; sural, Su), hairy and hairless skin of the paw (medial plantar, MP), or skeletal muscle (gastrocnemius-soleus, GS). Serial sections of lumbosacral dorsal root and sympathetic ganglia were studied after standard histochemical processing. Additionally, the numbers of myelinated fibers in the same nerves were determined. All sensory somata and 99.4% of sympathetic cell bodies were located ipsilaterally. Sensory somata were commonly restricted to two adjacent dorsal root ganglia (usually L6-7 for SP, MP; L7-S1 for Su, GS). Although sympathetic somata were more widely distributed rostrocaudally, their maximum frequency always occurred in the segmental ganglia immediately rostral to the sensory outflows, i.e., corresponding to rami communicantes grisei. Dimensions of sympathetic somata varied little between populations projecting to different tissues and were unimodally distributed. The size distributions of sensory somata were characterized by a peak between 10 and 20 microns radius, similar to sympathetic somata, and a varying smaller number of cells ranging up to 60 microns radius. Each nerve had a characteristic distribution profile of afferent somata. A population of very small cells was only present in GS, while the largest sensory somata in GS and MP were bigger than those in SP and Su. Numerical analysis of the data disclosed the characteristic composition of both myelinated and unmyelinated fibers in each nerve studied.

The fate of cryopreserved nerve isografts and allografts in normal and immunosuppressed rats.

Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1-1.5 degrees C/minute down to -40 degrees C at which point the vials were plunged into liquid nitrogen at -196 degrees C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival.

Organization of hindlimb nerve projections to the rat spinal cord: a choleragenoid horseradish peroxidase study.

The aim of the present study has been to investigate the projections of hindlimb muscle afferent fibers to the spinal cord with particular emphasis on the ventral horn and the column of Clarke. Following transections of the appropriate ventral roots, injections of the B-subunit of cholera toxin conjugated to horseradish peroxidase were made into the tibial, peroneal, hamstring, superior gluteal, femoral, and obturator nerves in one group of adult rats. In another group of rats, similar experiments were done with intact ventral roots in order to map the location in the ventral horn of the motoneuron cell columns supplying each investigated nerve. An extensive overlap was found for the different nerve projections to Rexed's laminae V-VII. A somatotopic organization of the nerve projections was seen in the lamina IX cell groups of the ventral horn as well as in the column of Clarke, even though an overlap existed. The densest primary afferent projection from each injected nerve was to its homonymous motoneurons. Only a small to moderate overlap between the projections of the tributary branches of the sciatic nerve was found in the ventral horn, whereas the obturator and femoral nerve projections showed more profound overlap. In the column of Clarke, hindlimb nerves innervating distal muscles projected medially, and nerves innervating proximal muscles projected laterally.

The lack of an effect of applied d.c. electric fields on peripheral nerve regeneration in the guinea pig.

This study was undertaken to provide evidence of enhanced regeneration of mammalian peripheral nerves in response to applied d.c. electric fields. Peroneal nerves of adult guinea-pigs were crushed or transected and anastomosed. Constant current d.c. stimulators (20 microA) were implanted in the flank with platinum/iridium electrodes routed to the ankles. Animals with crush lesions were tested for toe spreading ability from the 14th to the 23rd day following the lesion. Animals with transection lesions were allowed to recover for 40 days and isometric force measurements of toe abduction and foot flexion were made. Both myelinated and unmyelinated fiber densities were determined. There proved to be no difference between legs treated with an anode, a cathode, or a sham electrode as evaluated by: the time to return of the toe spreading reflex, the isometric force of either twitches or tonic contractions, the latency between stimulation and contraction, or the number or density of either myelinated or unmyelinated fibers. These negative results are at variance with other studies that have reported beneficial effects of d.c. electric fields on peripheral nerve regeneration. The stimulation and analysis techniques used in this study were well within the variety of protocols that have yielded reports of highly significant positive effects with smaller numbers of animals than used in this study. The conclusion is that either there is a subtle but highly specific and critical difference between the present protocol and others, or the other studies need to be reevaluated. In either case, it seems that the ability of applied d.c. fields to enhance peripheral nerve regeneration in vivo remains open to question.

Improved functional recovery of denervated skeletal muscle after temporary sensory nerve innervation.

Prolonged muscle denervation results in poor functional recovery after nerve repair. The possible protective effect of temporary sensory innervation of denervated muscle, prior to motor nerve repair, has been examined in the rat. Soleus and gastrocnemius muscles were denervated by cutting the tibial nerve, and the peroneal nerve was then sutured to the transected distal tibial nerve stump either immediately or after two, four or six months. In half of the animals with delayed repair, the saphenous (sensory) nerve was temporarily attached to the distal nerve stump. Muscles were evaluated three months after the peroneal-to-tibial union, and were compared with each other, with unoperated control muscles and with untreated denervated muscles. After four to six months of sensory "protection", gastrocnemius muscles weighed significantly more than unprotected muscles, and both gastrocnemius and soleus muscles exhibited better preservation of their structure, with less fiber atrophy and connective tissue hyperplasia. The maximum compound action potentials were significantly larger in gastrocnemius and soleus muscles following sensory protection, irrespective of the delay in motor nerve union. Isometric force, although less than in control animals and in those with immediate nerve repair, remained reasonably constant after sensory protection, while in unprotected muscles there was a progressive and significant decline as the period of denervation lengthened. We interpret these results as showing that, although incapable of forming excitable neuromuscular junctions, sensory nerves can nevertheless exert powerful trophic effects on denervated muscle fibers. We propose that these findings indicate a useful strategy for improving the outcome of peripheral nerve surgery.

Regrowth of acute and chronic injured spinal pathways within supra-lesional post-traumatic nerve grafts.

The present work investigates the extent to which mature central neurons acutely or chronically axotomized by a spinal lesion still maintained the potential to regenerate an axon following post-traumatic nerve grafting within supra-lesional spinal structures. In adult rats, a C3 cervical hemisection (injury) was made and an autologous segment of the peroneal nerve was implanted 2mm rostrally into the ventrolateral part of the ipsilateral C2 spinal cord. Nerve graft implantations were carried out acutely at the time of injury (group I, acute conditions) or chronically, three weeks post-injury (group II, chronic conditions). Central neurons axotomized by the spinal lesion were labeled by True Blue injected at the lesion site at the time of trauma. Central neurons regenerating axons within the nerve grafts were labeled with either horseradish peroxidase (only in group I, n=4) or Nuclear Yellow (group I, n=3 and group II, n=6) applied two to four months post-grafting to the distal cut end of the nerve grafts. Neurons with dual staining (True Blue/Nuclear Yellow) represented central regenerating neurons which were previously axotomized by the spinal lesion and which had retained the capacity for axonal regeneration for a delayed period after injury. In group I (acute injury conditions), all types of labeled cells were found to be scattered with a clear bimodal distribution within the spinal cord and the brainstem. No labeled cells were found within the motor cortex. There was no statistically significant difference between horseradish peroxidase and all cells containing Nuclear Yellow (Nuclear Yellow and True Blue/Nuclear Yellow). In group II (chronic injury conditions), Nuclear Yellow- and True Blue/Nuclear Yellow-labeled cells had a similar dual distribution to that of group I, but were found to be significantly less represented (P=0.019). These differences are discussed in terms of capacity for cell survival and axonal regrowth after acute and chronic injury. The main conclusion is based on the evidence of dual staining of central neurons in both groups, which demonstrates that brainstem and spinal neurons involved in acute and chronic axotomy after spinal C3 lesion can survive the trauma and still maintain the capacity to regenerate lesioned axons within nerve grafts inserted rostrally (C2 spinal cord) to the primary site of injury. Although exhibited to a lesser extent in chronic than in acute conditions, this capacity was found to occur for as long as three weeks post-injury. These results indicate that supra-lesional post-traumatic nerve grafts may constitute an efficient delayed strategy for inducing axonal regrowth of chronically axotomized adult central neurons. We suggest that surgical intervention, which is not always possible immediately after a spinal cord injury, may be satisfactorily carried out after an appropriate delay.

NGF, BDNF, NT-3, and GDNF mRNA expression in rat skeletal muscle following denervation and sensory protection.

Poor muscle and nerve functional recovery after nerve damage is a serious clinical problem, particularly if there is prolonged delay before nerve-muscle contact is reestablished. Our previous studies showed that sensory nerve cross-anastomosis (sensory protection) provides support to the denervated muscle. In the present study, we analyzed neurotrophic factor mRNA expression by RT-PCR in denervated rat gastrocnemius muscle receiving sensory protection with the saphenous nerve, compared to normal innervated muscle, to denervated muscle, and to denervated muscle repaired immediately with the peroneal (motor) nerve, after periods of 3 days to 3 months. No significant differences in mRNA levels of beta-actin, nerve growth factor, brain-derived neurotrophic factor or neurotrophin-3 were found between the sensory protection treatment and the denervated or the motor repair groups. However, sensory protection resulted in levels of muscle glial cell line-derived neurotrophic factor mRNA expression that were lower than in denervated muscle and higher than in muscle given immediate motor repair. These results demonstrate that glial cell line-derived neurotrophic factor mRNA is elevated following denervation but is partially down-regulated by sensory protection. Our study suggests that sensory protection provides a modified trophic environment by modulating neurotrophic factor synthesis in muscle.

Organization of common peroneal nerve motoneurones in the rat and monkey: a comparative study.

The motoneurones with axons in the common peroneal nerve (CPN) of the rat and monkey were examined using retrograde labelling with horseradish peroxidase (HRP). In both species, the CPN motoneurone pool was localized in the dorsolateral part of the ventral horn of the spinal cord. In the rat, the labelled motoneurones were located between the L3 and L6 spinal segments whereas in the monkey, they extended from the caudal end of L4 to the L6 spinal segments. In both species the majority of the labelled neurones were located within the L5 segment. The mean number of the CPN motoneurones in the rat and monkey was 458 and 1148, respectively. A bimodal size distribution of motoneurones was found in both species.

Functional properties of crossed spinocerebellar tract neurones with cell bodies in the S1 segment.

Functional properties of a crossed spinocerebellar tract with cell bodies located in laminae VII and IX of the S1 segment were investigated using intracellular recording. The neurones were found to be excited by group I and II muscle afferents and afferents of skin, joint and interosseus nerves. Volleys from group II muscle afferents and cutaneous afferents evoked inhibition in these cells. It is concluded that S1 spinocerebellar neurones convey a similar type of information to that of dorsal spinocerebellar tract and ventral spinocerebellar tract neurones but integrate it in a different way.

A method for automatic classification of large and small myelinated fibre populations in peripheral nerves.

The statistical analysis of morphometric data collected from biopsies of human superficial peroneal nerve is complicated by the heterogeneity of the population of myelinated fibres. In order to make separate statistical analyses of the subpopulations of large and small fibres we have developed a computer program (written in PASCAL) for their automatic separation. The method is based on a dynamic centres clustering algorithm and was applied to the multifactorial space defined by the principal component analysis of the morphometric variables: axonal diameter, myelin sheath thickness, circularity index and g-ratio. The classification technique was applied to measurements obtained from 5 control nerves, and to simulated data, and in each case it gave consistent Gaussian subpopulations with no need for the introduction of supplementary variables.

Dual staining assessment of Schwann cell viability within whole peripheral nerves using calcein-AM and ethidium homodimer.

A membrane permeant nucleic acid stain, ethidium homodimer was used in combination with calcein-AM to document the viability of Schwann cells (SCs) in whole nerves after cold storage assays. Segments of peripheral nerves were, (i) kept intact in buffer (viability controls), (ii) thawed after a cryopreservation, according to a protocol which has been previously shown to maintain the integrity of most nerve components [Ruwe and Trumble, J. Reconstr. Microsurg., 1990, 6: 239-244; Gauthier et al., In 3rd International Symposium on Axonal Regrowth in the Mammalian Spinal Cord and Peripheral Nerve, Deauville, France, 1995, p. 24, abstract], (iii) killed by chemical injury, or (iv) by successive freezing-thawing. Teased preparations of nerve fibers were prepared from the various types of nerve segments and incubated with calcein-AM and ethidium homodimer, which stain, respectively, living and dead cells. In control or cryopreserved nerves, staining with calcein-AM resulted in bright green fluorescence in the cytoplasm of SCs, with no red fluorescence of ethidium homodimer. In contrast, in killed nerve preparations, intense ethidium red fluorescence was observed in SC nuclei, with negligible green calcein cytoplasmic fluorescence. Thus, the combination of calcein-AM/ethidium homodimer appeared as an effective tool for assessing the viability of SCs and determine the quality of cold stored nerve preparations used in graft repair procedures. In addition, the generated fluorescence enabled clear visualization of myelinated fibers by confocal imaging.