Regrowth of lesioned dorsal root nerve fibers into the spinal cord of neonatal rats

In postnatal rat pups the L4 and L5 dorsal roots were lesioned. After 3-6 months the spinal cord of the rats was subjected to tracing studies of regenerated dorsal root axons with transganglionically transported horseradish peroxidase (HRP) and immunohistochemistry with antibodies to calcitonin gene-related peptide (CGRP). In rats operated at birth (0-2 days old) HRP-filled profiles as well as CGRP staining were found in the outer lamina of the spinal cord dorsal horn. Signs of dorsal root nerve fiber regrowth in the spinal cord could not be found in rats which had been operated at the end of the first postnatal week or later.     

Nerve fibre regeneration across the peripheral–central transitional zone

Neurons cannot negotiate an elongation across the peripheral (PNS)–central nervous system (CNS) transitional zone and grow into or out of the spinal cord in the mature mammal. The astrocytic rich CNS part of the spinal nerve root is most effective in preventing regeneration even of nerve fibres from transplanted embryonic ganglion cells. Regeneration of severed nerve fibres into the spinal cord occurs when the transition zone is absent as in the immature animal. Before the establishment of a transition zone there is also new growth of neuronal processes from dorsal horn neurons distally to the injured dorsal root. Thus the experimental strategy to reestablish spinal cord to peripheral nerve connectivity has been to delete the transitional region and implant severed ventral or dorsal roots into the spinal cord. Dorsal root implantation resulted in reestablished afferent connectivity by new neuronal processes from secondary sensory neurons in the dorsal horn of the spinal cord extending into the PNS. The ability for plasticity in these cells allowed for a concurrent retention of their original rostral projection. Ventral root implantation into the spinal cord corrected deficit motor function. In a long series of experiments performed in different species, the functional restitution was demonstrated to depend on an initial regrowth of motor neuron axons through spinal cord tissue (CNS). These findings have led to the design of a new surgical strategy in cases of traumatic spinal nerve root injuries.     

Reimplantation of avulsed lumbosacral ventral roots in the rat ameliorates injury-induced degeneration of primary afferent axon collaterals in the spinal dorsal columns

Injuries to the cauda equina/conus medullaris portion of the spinal cord can result in motor, sensory, and autonomic dysfunction, and neuropathic pain. In rats, unilateral avulsion of the motor efferents from the lumbosacral spinal cord results in at-level allodynia, along with a corresponding glial and inflammatory response in the dorsal horn of the spinal cord segments immediately rostral to the lesion. Here, we investigated the fate of intramedullary primary sensory projections following a motor efferent lesion. The lumbosacral (L6 and S1) ventral roots were unilaterally avulsed from the rat spinal cord (VRA; n=9). A second experimental group had the avulsed roots acutely reimplanted into the lateral funiculus (Imp; n=5), as this neural repair strategy is neuroprotective, and promotes the functional reinnervation of peripheral targets. A laminectomy-only group served as controls (Lam; n=7). At 8 weeks post-lesion, immunohistochemical examination showed a 42% reduction (P<0.001) in the number of RT97-positive axons in the ascending tracts of the dorsal funiculus of the L4-5 spinal segment in VRA rats. Evidence for degenerating myelin was also present. Reimplantation of the avulsed roots ameliorated axon and myelin degeneration. Axons in the descending dorsal corticospinal tract were unaffected in all groups, suggesting a specificity of this lesion for spinal primary sensory afferents. These results show for the first time that a lesion restricted to motor roots can induce the degeneration of intramedullary sensory afferents. Importantly, reimplantation of the lesioned motor roots ameliorated sensory axon degeneration. These data further support the therapeutic potential for reimplantation of avulsed ventral roots following trauma to the cauda equina/conus medullaris.     

Human dorsal root ganglion neurons from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone

Following dorsal root crush, the lesioned axons regenerate in the peripheral compartment of the dorsal root, but stop at the boundary between the peripheral and the central nervous system, the dorsal root transitional zone. We have previously shown that fibres from human fetal dorsal root ganglia grafted to adult rat hosts are able to grow into the spinal cord, but were not able to specify the route taken by the ingrowing fibres. In this study we have challenged the dorsal root transitional zone astrocyte boundary with human dorsal root ganglion transplants from 5–8-week-old embryos. By tracing immunolabelled human fibres in serial sections, we found that fibres consistently grow around the dorsal root transitional zone astrocytes in laminin-rich peripheral surroundings, and extend into the host rat spinal cord along blood vessels, either into deep or superficial laminae of the dorsal horn, or into the dorsal funiculus. Human fibres that did not have access to blood vessels grew on the spinal cord surface. These findings indicate, that in spite of a substantial growth capacity by axons from human embryonic dorsal root ganglion cells as well as their tolerance to non-permissive factors in the mature mammalian CNS, these axons are still sensitive to the repellent effects of astrocytes of the mature dorsal root transitional zone. Furthermore, this axonal ingrowth is consistently associated with laminin-expressing structures until the axons reach the host spinal cord.     

Localization of vasoactive intestinal peptide immunoreactivity in human foetus and newborn infant spinal cord

Using an indirect immunofluorescence method the distribution of vasoactive intestinal peptide (VIP) immunoreactivity was studied in human foetus and newborn infant spinal cord and dorsal root ganglia. Further, for comparison some newborn infant brains were also investigated. Vasoactive intestinal peptide-like immunoreactive fibres were exclusively found in the caudal spinal cord and corresponding dorsal root ganglia. No immunoreactive cell bodies were detected. The first appearance of VIP-like immunoreactive fibres in both spinal cord and dorsal root ganglia was suggested during the fourth month of foetal life. Most immunolabelled fibres, concentrated in the sacral segment, were distributed in the Lissauer tract, along the dorsolateral gray border, in the intermediolateral areas and near the central canal in the dorsolateral commissure. A few VIP-like immunoreactive fibres were also seen in the dorsal funiculus and occasionally in the ventral gray horn and ventral roots. Further, a large population of VIP-like immunoreactive fibres occurs longitudinally in dorsal root, in ganglia and in the spinal nerve exit zone. These findings indicate the early appearance of VIP-like immunoreactive fibres in the human foetus spinal cord and corresponding ganglia. Moreover, they emphasize that in both foetus and newborn infant spinal cord VIP-like immunoreactive fibre distribution is limited to the lumbosacral segment.     

Regeneration of dorsal root axons into experimentally altered glial environments in the rat spinal cord

Exposure of the lumbar spinal cord of rats to X-rays 3 days after birth results in changes in the composition of central glia. Shortly after irradiation, there is both retardation of central myelin formation and a loss of integrity of the astrocyte-derived glia limitans on the dorsal surface of the cord. Subsequently, Schwann cells invade, undergo division and myelinate axons in the dorsal funiculi in the irradiated region of the cord, creating there an environment similar to that of peripheral nerve. The present study was undertaken to compare the ability of lesioned dorsal root axons to grow back into the altered glial environments that exist within the spinal cord after irradiation. This regrowth was assessed by injecting Fluoro-Gold into the spinal cord and subsequently examining neurons in the dorsal root ganglia (DRG) for the presence of this label. Numbers of retrogradely labeled neurons were counted in the DRG in both injured and contralateral non-injured sides. Non-irradiated control rats had almost no labeled DRG neurons on the injured side, whereas Fluoro-Gold labeled neurons were observed in substantial numbers in the DRG on the injured side of irradiated rats. There was a definite trend in the data, indicating that the longer the interval between irradiation and root injury, the greater the number of labeled neurons. Since the Fluoro-Gold labeling technique does not allow for visualization of the labeled axons within the spinal cord, a few animals were used to assess anterograde labeling with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP/HRP) from the dorsal root into the spinal cord. HRP-filled regenerating axons were visualized in dorsal white and gray matter of the irradiated spinal cord. Such axons were not present in the non-irradiated spinal cords. Radiation-induced changes in glial populations are discussed, particularly with regard to the temporal sequence of these changes and their possible relationship to the conversion of a normally non-permissive environment into one conducive to axonal regrowth.     

Glial–glial and glial–neuronal interfaces in radiation-induced, glia-depleted spinal cord

This review summarises some of the major findings derived from studies using the model of a glia-depleted environment developed and characterised in this laboratory. Glial depletion is achieved by exposure of the immature rodent spinal cord to x-radiation which markedly reduces both astrocyte and oligodendrocyte populations and severely impairs myelination. This glia-depleted, hypomyelinated state presents a unique opportunity to examine aspects of spinal cord maturation in the absence of a normal glial population. An associated sequela within 2–3 wk following irradiation is the appearance of Schwann cells in the dorsal portion of the spinal cord. Characteristics of these intraspinal Schwann cells, their patterns of myelination or ensheathment, and their interrelations with the few remaining central glia have been examined. A later sequela is the development of Schwann cells in the ventral aspect of the spinal cord where they occur predominantly in the grey matter. Characteristics of these ventrally situated intraspinal Schwann cells are compared with those of Schwann cells located dorsally. Recently, injury responses have been defined in the glia-depleted spinal cord subsequent to the lesioning of dorsal spinal nerve roots. In otherwise normal animals, dorsal nerve root injury induces an astrocytic reaction within the spinal segments with which the root(s) is/are associated. Lesioning of the 4th lumbar dorsal root on the right side in irradiated or nonirradiated animals results in markedly different glial responses with little astrocytic scarring in the irradiated animals. Tracing studies reveal that these lesioned dorsal root axons regrow rather robustly into the spinal cord in irradiated but not in nonirradiated animals. To examine role(s) of glial cells in preventing this axonal regrowth, glial cells are now being added back to this glia-depleted environment through transplantation of cultured glia into the irradiated area. Transplanted astrocytes establish barrier-like arrangements within the irradiated cords and prevent axonal regrowth into the cord. Studies using other types of glial cultures (oligodendrocyte or mixed) are ongoing.     

Reactive changes in dorsal roots and dorsal root ganglia after C7 dorsal rhizotomy and ventral root avulsion/replantation in rabbits

Current surgical treatment of spinal root injuries aims at reconnecting ventral roots to the spinal cord while severed dorsal roots are generally left untreated. Reactive changes in dorsal root ganglia (DRGs) and in injured dorsal roots after such complex lesions have not been analysed in detail. We studied dorsal root remnants and lesioned DRGs 6 months after C7 dorsal rhizotomy, ventral root avulsion and immediate ventral root replantation in adult rabbits. Replanted ventral roots were fixed to the spinal cord with fibrin glue only or with glue containing ciliary neurotrophic factor and/or brain-derived neurotrophic factor. Varying degrees of degeneration were observed in the deafferented dorsal spinal cord in all experimental groups. In cases with well-preserved morphology, small myelinated axons extended into central tissue protrusions at the dorsal root entry zone, suggesting sprouting of spinal neuron processes into the central dorsal root remnant. In lesioned DRGs, the density of neurons and myelinated axons was not significantly altered, but a slight decrease in the relative frequency of large neurons and an increase of small myelinated axons was noted (significant for axons). Unexpectedly, differences in the degree of these changes were found between control and neurotrophic factor-treated animals. Central axons of DRG neurons formed dorsal root stumps of considerable length which were attached to fibrous tissue surrounding the replanted ventral root. In cases where gaps were apparent in dorsal root sheaths, a subgroup of dorsal root axons entered this fibrous tissue. Continuity of sensory axons with the spinal cord was never observed. Some axons coursed ventrally in the direction of the spinal nerve. Although the animal model does not fully represent the situation in human plexus injuries, the present findings provide a basis for devising further experimental approaches in the treatment of combined motor/sensory root lesions.     

A single re-implanted ventral root exerts neurotropic effects over multiple spinal cord segments in the adult rat

Spinal cord injuries, particularly traumatic injuries to the conus medullaris and cauda equina, are typically complex and involve multiple segmental levels. Implantation of avulsed ventral roots into the spinal cord as a repair strategy has been shown to be neuroprotective and promote axonal regeneration by spinal cord neurons into an implanted root. However, it is not well known over what distance in the spinal cord an implanted ventral root can exert its neurotropic effect. Here, we investigated whether an avulsed L6 ventral root acutely implanted into the rat spinal cord after a four level (L5–S2) unilateral ventral root avulsion injury may exert neurotropic effects on autonomic and motor neurons over multiple spinal cord segments at 6 weeks postoperatively. Using retrograde labeling techniques and stereological quantification methods, we demonstrate that autonomic and motor neurons from all four lesioned spinal cord segments, spanning more than an 8 mm rostro-caudal distance, reinnervated the one implanted root. The rostro-caudal distribution suggested a gradient of neurotropism, where the axotomized neurons closest to the implanted site had the highest probability of root reinnervation. These results suggest that implantation of a single ventral root may provide neurotropic effects to injured neurons at the site of lesion as well as in the adjacent spinal cord segments. Our findings may be of translational research interest for the development of surgical repair strategies after multi-level conus medullaris and cauda equina injuries, in which fewer ventral roots than spinal cord segments may be available for implantation.     

Effect of embryonal nerve tissue transplantation on regeneration of dorsal roots of the rat spinal nerves

Using cobalt salts axonal ionophoresis posttraumatic regeneration of TXII dorsal roots nerve fibres in the zone of hemisection in conditions of 14 wks embryo spinal cord transplantation into the zone of trauma of spinal cord. Regro Invasion of dorsal roots nerve fibres into recipients posterior cords and Lissawers tract through the transitional zone "spinal cord--dorsal roots" was observed on posttransplantation d 14-120. It was show that afferent axons predominantly spread in substantia alba and substantia grisea caudal to the level of spinal cord transection with only individual fibres invading rostrad through the neuronal plate. In the transplants neurons were encountered up to d 120 of the observation although transplant neuropil was limited from recipient tissue brain by a glial and connective tissue scar. The influence of embryonal nervous tissue transplantation on intraspinal regeneration of dorsal roots afferents was discussed.     

Schwann cell invasion of the central nervous system of the myelin mutants

Schwann cells are excluded from the CNS during development by the glial limiting membrane, an area of astrocytic specialisation present at the nerve root transitional zone, and at blood vessels in the neuropil. This barrier, however, can be disrupted and, with the highly migratory nature of Schwann cells, can result in their invasion and myelination of the CNS in many pathological situations. In this paper we demonstrate that this occurs in a number of myelin mutants, including the myelin deficient ( md ) and taiep rats and the canine shaking ( sh ) pup. While it is still relatively uncommon in the rodent mutants, the sh pup shows extensive Schwann cell invasion along the neuraxis. This invasion involves the spinal cord, brain stem, and cerebellum and increases in amount and distribution with age. In situ hybridisation studies using a P₀ riboprobe suggest that the likely origin of these cells in the sh pup is the nerve roots, primarily the dorsal roots. Paradoxically, Schwann cell myelination of the CNS increases with time in the sh pup despite a marked, progressive gliosis involving the glia limitans and neuropil. Thus the mechanism by which these cells migrate into the CNS through the gliosed nerve root transitional zone or from vasa nervorum remains unknown. Extensive Schwann cell CNS myelination may have therapeutic significance in human myelin disease.     

Microsurgical Anatomy of Lumbosacral Nerve Rootlets for Highly Selective Rhizotomy in Chronic Spinal Cord Injury

It is known that selective sacral roots rhizotomy is effective for relieving the neurogenic bladder associated with spinal cord injury. The goal of this study is to review the surgical anatomy of the lumbosacral nerve rootlets and to provide some morphological bases for highly selective sacral roots rhizotomy. Spinal cord dissections were performed on five cadavers under surgical microscope. At each spinal cord segment, we recorded the number, diameter and length of the rootlets, subbundles and bundles from the L1 to S2 spinal segments, and the length of the dorsal/ventral root entry zone. Peripheral nervous system myelin was examined by immunohistochemistry. We found: (1) the ventral or the dorsal root of the lumbosacral segment of the spinal cord was divided into one to three nerve bundles and each bundle was subdivided into one to three subbundles. Each subbundle further gave out two to three rootlets connected with the spinal cord; (2) there were no significant differences in the number of rootlets within the L1 to S2 segments, but the size of rootlets and the length of nerve roots varied (P < 0.05); and (3) the more myelinated fibers a rootlet contained, the larger transection area it had. The area of peripheral nervous system myelin positive cells and the total area of rootlets were correlated (P < 0.001). Thus, during highly selective sacral roots rhizotomy, the ventral and dorsal roots can be divided into several bundles of rootlets, and we could initially distinct the rootlets by their diameters. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve

Summary We have conducted experiments in the adult rat visual system to assess the relative importance of an absence of trophic factors versus the presence of putative growth inhibitory molecules for the failure of regeneration of CNS axons after injury. The experiments comprised three groups of animals in which all optic nerves were crushed intra-orbitally: an optic nerve crush group had a sham implant-operation on the eye; the other two groups had peripheral nerve tissue introduced into the vitreous body; in an acellular peripheral nerve group, a frozen/thawed teased sciatic nerve segment was grafted, and in a cellular peripheral nerve group, a predegenerate teased segment of sciatic nerve was implanted. The rats were left for 20 days and their optic nerves and retinae prepared for immunohistochemical examination of both the reaction to injury of axons and glia in the nerve and also the viability of Schwann cells in the grafts. Anterograde axon tracing with rhodamine-B provided unequivocal qualitative evidence of regeneration in each group, and retrograde HRP tracing gave a measure of the numbers of axons growing across the lesion by counting HRP filled retinal ganglion cells in retinal whole mounts after HRP injection into the optic nerve distal to the lesion. No fibres crossed the lesion in the optic nerve crush group and dense scar tissue was formed in the wound site. GAP-43-positive and rhodamine-B filled axons in the acellular peripheral nerve and cellular peripheral nerve groups traversed the lesion and grew distally. There were greater numbers of regenerating fibres in the cellular peripheral nerve compared to the acellular peripheral nerve group. In the former, 0.6–10% of the retinal ganglion cell population regenerated axons at least 3–4 mm into the distal segment. In both the acellular peripheral nerve and cellular peripheral nerve groups, no basal lamina was deposited in the wound. Thus, although astrocyte processes were stacked around the lesion edge, a glia limitans was not formed. These observations suggest that regenerating fibres may interfere with scarring. Viable Schwann cells were found in the vitreal grafts in the cellular peripheral nerve group only, supporting the proposition that Schwann cell derived trophic molecules secreted into the vitreous stimulated retinal ganglion cell axon growth in the severed optic nerve. The regenerative response of acellular peripheral nerve-transplanted animals was probably promoted by residual amounts of these molecules present in the transplants after freezing and thawing. In the optic nerves of all groups the astrocyte, microglia and macrophage reactions were similar. Moreover, oligodendrocytes and myelin debris were also uniformly distributed throughout all nerves. Our results suggest either that none of the above elements inhibit CNS regeneration after perineuronal neurotrophin delivery, or that the latter, in addition to mobilising and maintaining regeneration, also down regulates the expression of axonal growth cone-located receptors, which normally mediate growth arrest by engaging putative growth inhibitory molecules of the CNS neuropil.     

An immunohistochemical study of the topography and cellular localization of three neural proteins in the sheep nervous system.

The peroxidase-anti-peroxidase (PAP) method was used to determine the topography and cellular localization of glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) and carbonic anhydrase II (CAII) in the central nervous system (CNS), dorsal root ganglia and dorsal and ventral spinal nerve roots of the sheep. Parallel studies of mouse brain provided comparative data. Several fixatives were compared for their relative merits in preserving marker protein expression: GFAP was well preserved irrespective of the fixative employed; MBP was best preserved in formal sublimate and CAII was best preserved in Carnoy's fluid. In sheep, GFAP expression was seen in protoplasmic and fibrous astrocytes, Bergmann glial cells, a proportion of ependymal cells, amphicytes of spinal ganglia and in a proportion of presumed Schwann cells of dorsal and ventral spinal nerve roots. MBP expression was seen in mature and developing myelin sheaths of the central nervous system and in the cytoplasm of sparse myelinating oligodendroglia of the sub-cortical white matter of the cerebrum. CAII expression was seen in choroid plexus epithelium in all ages of sheep studied and, in a young lamb and an adult sheep, in glia and neuropil of ventral horn grey matter of the spinal cord and in the cytoplasm of white matter glia, presumed fibrous astrocytes, throughout the CNS. Compared with sheep brain, mouse brain showed the following differences in marker protein localization. GFAP was weakly expressed by protoplasmic astrocytes and not expressed in ependyma, oligodendroglia expressing intracytoplasmic MBP were frequent and widespread in neonatal mouse brain, CAII was expressed in myelin and oligodendroglia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-range projections of Adelta primary afferents in the Lissauer tract of the rat

Electrical microstimulation has been used to activate fine myelinated primary afferents running within the Lissauer tract. Stimulation of the tract at the L2/L3 border produced antidromic volleys which were recorded on the dorsal roots of more caudal spinal segments. Antidromic volleys were present in all cases for roots as far caudal as the S2 segment (L3, n=12; L4, n=6; L5, n=6; L6, n=9; S1, n=3; S2, n=6; observations in a total of 15 rats). These fibres were collaterals of primary afferents with conduction velocities in the dorsal root of up to 17.3+/-2.3 ms(-1) (mean+/-S.D., n=6; range 14-20 ms(-1)). Conduction velocities within the Lissauer tract were slower; the fastest contributing fibres had conduction velocities of 9.2+/-2.2 ms(-1) (range 6-12 ms(-1)). Lesions of the Lissauer tract caudal to the stimulation site abolished the volleys on roots lying caudal to the lesion. Most previous works have suggested that primary afferents project in the Lissauer tract for only one or two spinal segments. The present study shows that some fibres project rostrally for up to seven spinal segments (L2-S2).     

The physiology and anatomy of long ranging afferent fibres within the spinal cord.

The caudal extent of the terminal arborizations of dorsal root afferents was determined in adult cats. The method used micro-electrode stimulation within the dorsal horn and the recording on a distant dorsal root filament of the antidromic action potentials evoked by the stimulation of axons within the spinal cord. 2. It was found that all filaments examined in the L2, 3 and 4 dorsal roots contained axons which projected at least as far as the S1 segment. The axons descended in or near the dorsal columns and from there penetrated into the grey matter. 3. The course of single fibres was followed to their apparent terminals. Thresholds, latencies and relative and absolute refractory periods were measured for single axons. These measurements confirmed that continuous axons ran from dorsal roots to distant segments and that the action potentials recorded were not dorsal root reflexes. 4. The majority of fibres with long range central arborizations were shown to have normal receptive fields in the dermatome of their parent dorsal root. They were not aberrant fibres leaving the spinal cord. 5. The long range afferents exist in substantial numbers since fifteen of eighty axons isolated by micro-electrode recording in the L2 dorsal root sent their axons as far as the S1 segment. The presence of these afferents from five segments away does not fit the data published on the inhibitory and excitatory receptive fields or dorsal horn cells which appear adequately explained by afferents arriving over nearby dorsal roots up to two segments away.     

Distribution of histamine in the developing peripheral nervous system

The presence and ontogenetic distribution of histamine was studied in the developing peripheral nervous system of the rat by using an indirect immunofluorescence technique and a specific rabbit anti-histamine antiserum. Histamine immunoreactivity (IR) first appeared in peripheral nerves on embryonic day 14. The number and intensity of histamine-immunoreactive nerves was highest on embryonic days 16–18. During development starting from embryonic day 14, motoneurones in ventral horns of the spinal cord at cervical, thoracic and lumbar levels contained histamine IR. A subpopulation of sensory neurones in dorsal root ganglia exhibited histamine IR. Histamine IR was also present in nerve fibres of ventral and dorsal roots of spinal cord, as well as in spinal nerves. Population of neurones and nerve fibres in sympathetic and pelvic ganglia as well as in myenteric ganglia of the intestine were also labelled with the histamine antiserum. In peripheral target organs, histamine IR was observed in nerve fibres around bronchi of the lungs, in the atria of the heart, in the adrenal gland, in the intestinal wall, in muscular tissues and in subepithelial tissue of the skin.The results of this study indicate that histamine is widely distributed in different types of neurones and nerve fibres of the developing peripheral nervous system.     

Acellular nerve graft re-seeded by Schwann cells migrating from the nerve stump can stimulate spinal motoneurons for functional reinnervation of the rat muscle

The acellular nerve graft was utilised to restore a functional reinnervation of the biceps brachii muscle from the motoneuron pool of the cervical spinal cord. The musculocutaneous nerve stump was sutured to an acellular nerve graft, the opposite end of which was inserted into the cervical spinal cord cranial to the avulsed C5 ventral root. The acellular nerve graft was repopulated by Schwann cells heavily immunostained for NGFr within 90 days. The Schwann cells migrating from the nerve stump reached the spinal cord grey matter, where they stimulated the motoneurons to send axonal sprouts. The functional reinnervation of the biceps brachii muscle was assessed by means of the behavioural (grooming) test and EMG, the presence of myelinated and unmyelinated axons was demonstrated by light and electron microscopy. The axonal reconnection of the musculocutaneous nerve stump was verified by horseradish peroxidase retrograde labelling of the spinal motoneurons. Moreover, the motoneurons on the operated side of the C5 spinal segment displayed increased immunostaining for GAP-43 in comparison to the contralateral side, whereas the pattern of AChE histochemical reaction was similar on both the operated and contralateral side, of the C5 segment 150 days after acellular graft implantation. The regenerated axons bridged a 4-cm long originally acellular nerve graft to reach and reinnervate the biceps brachii muscle. The reinnervation of the neuromuscular junctions was morphologically determined by immunofluorescence for neurofilaments. The number of myelinated axons in the acellular nerve graft was significantly higher than those growing over the cellular graft, but their diameter was smaller. The results of experiments presented here demonstrate functional recovery of the biceps muscle reinnervation through the acellular nerve graft repopulated by migrating Schwann cells. The process of reinnervation by acellular nerve graft is however delayed and worse in comparison with the cellular graft.     

脊神经前根吻合重建脊髓损伤大鼠的股四头肌功能

背景：如何重建脊髓损伤肢体运动功能对截瘫患者具有重要的意义。 目的：探索利用脊髓损伤平面以上健存的脊神经前根与支配股四头肌的腰神经建立神经通路,恢复脊髓损伤后股四头肌的神经支配和肌收缩功能。 方法：对清洁级SD大鼠L₁神经根的前根与L3神经根的前根进行显微吻合。经一段时间(6个月)的轴突再生后,期望建立新的肌肉收缩功能。神经缝合后6个月,在破坏L₂ 脊髓节段前后,分别进行神经电生理检测,观察股四头肌神经支配情况。 结果与结论：在同侧L₂半切脊髓前后,电刺激移植神经干时可记录到股四头肌的收缩肌电图。在同侧L₂半切脊髓前后,电刺激L₁感觉根时可同样在股四头肌记录到肌电图。说明利用脊髓损伤平面以上健存的L₁神经根前根与L₃神经前根移植吻合能重建新的股四头肌神经支配反射通路,并使股四头肌低级反射中枢上移。     

Enkephalin and capsaicin-resistant substance P-like immunoreactivities in intra-ocular grafts of different fetal spinal cord areas

Fetal spinal cord tissue was grafted to the anterior chamber of the eye of adult rats in order to evaluate survival and distribution of substance P- and enkephalin-immunoreactive neurons. Capsaicin treatment was used to evaluate any possible contribution of host iris-derived substance P fibres to the innervation of the grafts and to check for capsaicin sensitivity of graft substance P-positive systems. Substance P- and enkephalin-immunoreactive nerve fibres were present in grafts of half-transverse segments of the spinal cord and were clearly co-distributed throughout the grafts. Areas with a high density of substance P- and enkephalin-positive fibres resembling substantia gelatinosa were seen. Grafts of the dorsal horn alone had a moderate to high density of substance P- and enkephalin-positive fibres, while ventral horn grafts contained a low amount of such fibres. Capsaicin eliminated the substance P innervation of the host iris and the dorsal root ganglion-derived substance P innervation of the host spinal cord, while sparing the intrinsic substance P innervation of both host spinal cord and spinal cord grafts. These experiments show that intra-ocular grafts of defined spinal cord areas express relatively organotypic amounts of substance P- and enkephalin-positive nerve fibres, and thus emphasize the importance of intrinsic genetic determinants for spinal cord development.