Induced expression of polysialic acid in the spinal cord promotes regeneration of sensory axons

After spinal cord injury axonal regeneration is prevented by glial scar formation. In this study we examined whether induced expression of polysialic acid (PSA) in the lesion site would render the glial scar permissive to axonal regeneration after dorsal column transection. PSA was induced by lentiviral vector-mediated expression of polysialyltransferase (LV/PST). PSA expression increased astrocyte infiltration and permitted the penetration of regenerating axons across the caudal border of the lesion and into the lesion cavity. In LV/PST-injected animals with a peripheral nerve-conditioning lesion, 20 times more axons grew into the lesion cavity than those LV/GFP-injected plus conditioning lesion, and some axons grew across the cavity and extended to the rostral cord, while in LV/GFP group most ascending axons terminated at the caudal border of the lesion. Our result suggests that induced expression of PSA can provide a favorable environment for axonal regeneration.     

Olivocerebellar Axon Regeneration and Target Reinnervation Following Dissociated Schwann Cell Grafts in Surgically Injured Cerebella of Adult Rats

The ability of Schwann cells to induce the regeneration of severed olivocerebellar and Purkinje cell axons across an injury up to their deafferented targets was tested by transplanting freshly dissociated cells from newborn rat sciatic nerves into surgically lesioned adult cerebella. The grafted glial cells consistently filled the lesion gap and migrated into the host parenchyma. Transected olivocerebellar axons vigorously regenerated into the graft, where their growth pattern and direction followed the arrangement of Schwann cell bundles. Although some of these axons terminated within the transplant, many of them rejoined the cerebellar parenchyma beyond the lesion. Here, their fate depended on the territory encountered. No growth occurred in the white matter. Numerous fibres penetrated into the granular layer and formed terminal branches that remained confined within this layer. A few of them, however, regenerated up to the molecular layer and formed climbing fibres on Purkinje cell dendrites. By contrast, the growth of transected Purkinje cell axons into the grafts was very poor. These results underscore the different intrinsic responsiveness of Purkinje cell and olivocerebellar axons to the growth-promoting action of Schwann cells, and show that the development and outcome of the regenerative phenomena is strongly conditioned by the spatial organization and specific features of the environmental cues encountered by the outgrowing axons along the course they follow. However, Schwann cells effectively bridge the lesion gap, induce the regeneration of olivocerebellar axons, and direct their growth up to the deafferented host cortex, where some of them succeed in reinnervating their natural targets.     

Schwann cells transduced with a lentiviral vector encoding Fgf‐2 promote motor neuron regeneration following sciatic nerve injury

Fibroblast growth factor 2 (FGF‐2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF‐2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF‐2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF‐2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF‐2 (LV‐FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF‐2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV‐FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV‐FGF2 into the rat sciatic nerve resulted in increased expression of FGF‐2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF‐2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV‐FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV‐FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow‐up. GLIA 2014;62:1736–1746     

GDNF modifies reactive astrogliosis allowing robust axonal regeneration through Schwann cell-seeded guidance channels after spinal cord injury

Reactive astrogliosis impedes axonal regeneration after injuries to the mammalian central nervous system (CNS). Here we report that glial cell line-derived neurotrophic factor (GDNF), combined with transplanted Schwann cells (SCs), effectively reversed the inhibitory properties of astrocytes at graft–host interfaces allowing robust axonal regeneration, concomitant with vigorous migration of host astrocytes into SC-seeded semi-permeable guidance channels implanted into a right-sided spinal cord hemisection at the 10th thoracic (T10) level. Within the graft, migrated host astrocytes were in close association with regenerated axons. Astrocyte processes extended parallel to the axons, implying that the migrated astrocytes were not inhibitory and might have promoted directional growth of regenerated axons. In vitro, GDNF induced migration of SCs and astrocytes toward each other in an astrocyte–SC confrontation assay. GDNF also enhanced migration of astrocytes on a SC monolayer in an inverted coverslip migration assay, suggesting that this effect is mediated by direct cell–cell contact between the two cell types. Morphologically, GDNF administration reduced astrocyte hypertrophy and induced elongated process extension of these cells, similar to what was observed in vivo. Notably, GDNF treatment significantly reduced production of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs), two hallmarks of astrogliosis, in both the in vivo and in vitro models. Thus, our study demonstrates a novel role of GDNF in modifying spinal cord injury (SCI)-induced astrogliosis resulting in robust axonal regeneration in adult rats.     

Promoting survival, migration, and integration of transplanted Schwann cells by over-expressing polysialic acid

The poor survival and migration of transplanted Schwann cells (SCs) are major drawbacks for their clinical application in cell therapy for neurotrauma. To overcome such drawbacks we genetically modified SCs to over-express polysialic acid (PSA) by lentiviral delivery of polysialyltransferase (PST) to study whether over-expression of PSA could enhance their survival, migration, and integration when transplanted into the spinal cord. It was found that more PSA-expressing SCs (PST/SCs) survived than GFP-expressing SCs (GFP/SCs) after transplantation, although cell loss was still quite significant. PSA expression did not enhance the motility of transplanted SCs in uninjured spinal cord. However, in a spinal cord crush injury model PST/SCs transplanted caudal to the lesion showed that increased number of PST/SCs migrated to the injury site compared with that of GFP/SCs. Induced expression of PSA in spinal cord can further facilitate the infiltration of PST/SCs into the lesion site. PST/SCs were also shown to intermingle well with host spinal cells while GFP/SCs formed boundaries with host tissue. This was confirmed by an in vitro confrontation assay showing that more PST/SCs crossed over to astrocyte territory than GFP/SCs. Furthermore, PST/SCs induced much less expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycan in the surrounding tissues than GFP/SCs, indicating that expression of PSA on SCs do not cause significant stress response of astrocytes. These results demonstrate that expression of PSA on SCs significantly changes their biological properties and makes them more feasible for neural repair after neurotrauma.     

Regeneration of nigrostriatal dopaminergic axons after transplantation of olfactory ensheathing cells and fibroblasts prevents fibrotic scar formation at the lesion site

The fibrotic scar formed after central nervous system injury has been considered an obstacle to axonal regeneration. The present study was designed to examine whether cell transplantation into a damaged central nervous system can reduce fibrotic scar formation and promote axonal regeneration. Nigrostriatal dopaminergic axons were unilaterally transected in rats and cultures of olfactory-ensheathing cells (OECs), and olfactory nerve fibroblasts were transplanted into the lesion site. In the absence of transplants, few tyrosine hydroxylase-immunoreactive axons extended across the lesion 2 weeks after the transection. Reactive astrocytes increased around the lesion, and a fibrotic scar containing type IV collagen deposits developed in the lesion center. The immunoreactivity of chondroitin sulfate side chains and core protein of NG2 proteoglycan increased in and around the lesion. One and 2 weeks after transection and simultaneous transplantation, dopaminergic axons regenerated across the transplanted tissues, which consisted of p75-immunoreactive OECs and fibronectin-immunoreactive fibroblasts. Reactive astrocytes and chondroitin sulfate immunoreactivity increased around the transplants, whereas the deposition of type IV collagen and fibrotic scar formation were completely prevented at the lesion site. Transplantation of meningeal fibroblasts similarly prevented the formation of the fibrotic scar, although its effect on regeneration was less potent than transplantation of OECs and olfactory nerve fibroblasts. The present results suggest that elimination of the inhibitory fibrotic scar is important for neural regeneration.     

Enhanced axonal regeneration following combined demyelination plus schwann cell transplantation therapy in the injured adult spinal cord.

We have treated spinal cord injured rats with demyelination plus Schwann cell transplantation and assessed neurite outgrowth in a quantifiable model of axonal regeneration. Axonal injuries of differing severity were induced in the dorsal funiculus of adult rats using a micromanipulator-controlled Scouten knife. Demyelinated regions were produced so as to overlap with the injury site by the injection of galactocerebroside antibodies plus complement one segment cranial to the axonal injury site. Schwann cells were isolated from the sciatic nerve, expanded in vitro, and transplanted into the injury site 1 day later. Animals were killed after an additional 7 days. Schwann cells were evenly distributed throughout the region of demyelination, which extended 6-7 mm cranial to the axonal injury site. The severity of axonal injury was quantified by counting degenerate axons in transverse resin sections. The degree of axonal regeneration was assessed by an electron microscopic analysis of growth cone frequency and distribution relative to the site of axonal injury. Quantification of growth cones at a distance from the site of axonal injury indicated a strong linear relationship (P < 0.001) between the number of growth cones and the number of severed axons; the ratio of growth cones to severed axons was increased by 26.5% in demyelinated plus transplanted animals compared to demyelinated animals without a transplant. Furthermore, only the demyelinated plus transplanted animals contained growth cones associated with myelin in white matter immediately outside of the region of complete demyelination. Growth cones were absent in transplanted-only animals at a distance from the site of axonal injury. These findings indicate that combined demyelination plus Schwann cell transplantation therapy enhances axonal regeneration following injury and suggests that growth cones are able to overcome myelin-associated inhibitors of neurite outgrowth in the presence of trophic support.     

Tanycytes transplanted into the adult rat spinal cord support the regeneration of lesioned axons

During past years a number of therapeutic strategies have been developed in order to stimulate axonal regeneration after traumatic injuries of the spinal cord. Recently, encouraging data have been obtained by grafting specific glial cells such as Schwann cells or olfactory ensheathing glial cells, known to support the regeneration of peripheral or central axons, respectively. In a recent series of studies, we have shown that tanycytes, a particular glial cell type present in the mediobasal hypothalamus, were able to support the regeneration of a variety of axons innervating this region. The aim of the present study was to determine whether tanycytes could also support the regeneration of lesioned spinal axons. Cultured hypothalamic tanycytes and cortical astrocytes were prelabeled with Fast blue (FB) and grafted into the thoracic spinal cord of adult rats. Three weeks after the transplantation, the animals were fixed and spinal cord sections treated for multiple fluorescence detection of the FB-labeled transplanted cells on the one hand and of various glial and neuronal markers on the other hand. We show here that in all the spinal cords examined, transplanted tanycytes or astrocytes formed large spherical clusters of about 0.5 mm in diameter, located in the mediolateral spinal cord layer. The immunodetection of glial markers showed that transplanted astrocytes exhibited intense immunostaining for both glial fibrillary acidic protein (GFAP) and vimentin (VIM), whereas transplanted tanycytes were intensely immunostained for VIM, but GFAP negative. The immunodetection of axonal markers showed that contrasting with astrocyte transplants, tanycyte transplants were invaded by numerous axonal fibers. These data indicate that tanycyte transplants may represent a useful therapeutic tool for the reparation of the lesioned spinal axons.     

Enhanced regenerative axon growth of multiple fibre populations in traumatic spinal cord injury following scar-suppressing treatment

We analysed the effect of scar‐suppressing treatment (anti‐scarring treatment; AST) on augmenting axonal regeneration of various neuronal populations following spinal cord injury (SCI) in adult rat. AST included local iron chelator (2,2′‐dipyridine‐5,5′‐dicarboxylic acid) injection and 8‐bromo‐cyclic adenosine monophosphate application to the lesion core. In previous studies, this treatment promoted long‐distance regeneration of cut corticospinal tract axons, neuroprotection of projecting cortical neurons and functional improvement of treated rats [N. Klapka et al. (2005) Eur. J. Neurosci., 22 , 3047–3058]. Treatment yielded significantly enhanced regrowth of descending serotonergic (5‐HT), catecholaminergic (tyrosine hydroxylase; TH), corticospinal and rubrospinal axons into the lesion zone, as assessed by anterograde tracing and immunohistochemistry followed by quantification of axon profiles at 5 and 12 weeks post‐injury. In addition, the determination of axons crossing the proximal borderline from uninjured tissue into fibrous scar area revealed a significant AST‐promoted increase of intersecting fibres for 5‐HT, TH and calcitonin gene‐related peptide containing ascending sensory fibres. For a prolonged time period after lesion, the delayed (secondary) scar developing in treated rats is significantly more permeable for all analysed axon tracts than the initial (primary) scar forming in injured control animals lacking treatment. Furthermore, enhanced outgrowth of descending axons from fibrous scar into distal healthy spinal tissue was achieved in treated animals, and is in line with previous functional studies [S. Hermanns et al. (2001) Restor. Neurol. Neurosci., 19 ,139–148; N. Klapka et al. (2005) Eur. J. Neurosci., 22 , 3047–3058]. Our findings indicate that AST exerts a prolonged beneficial effect on fibrous scarring allowing enhanced axonal regrowth of different fibre tracts in SCI regardless of their distinct regenerative demands.     

The role of transplanted astrocytes for the regeneration of CNS axons]

Long tract axons in the mammalian CNS do not normally regenerate for appreciable distance after they transected. But we reported transplantation of Schwann cells(SCs) or olfactory ensheathing cells induced regeneration of transected rat dorsal column (DC) axons and improved the conduction. Scar formation(gliosis), for which astrocytes(ACs) play an important role, may be one of strong and physical barriers for the regeneration of CNS axon. Oligodendrocyte and myelin associated protein or products also inhibit the regeneration of the axons, as chemical barriers. To investigate how effective the promotion or the reduction of scar or myelin formation may be for axonal regeneration, we transplanted AC into transected DCs, or radiated(X-ray) the DCs, and compared to normal DCs or regenerated DCs following by SC transplantation. DCs of adult rats were transected at Th 11 and transplanted with SCs(6 x 10(4)) of adult rats or ACs(6 x 10(4)) of neonatal rats. Five to six weeks later, the spinal cords were removed and pinned in a recording chamber, and compound action potentials (CAPs) along the DC through the transected lesion were recorded, to investigate conduction properties(conduction velocity and response after high frequency stimulations). Following transplantation of SCs or ACs, histological examination revealed regenerated axons with SC-like patterns of remyelination in transected DCs. X-ray irradiation did not enhance the regeneration of DC axons. SC transplantation improved the conduction properties of transected DCs and increased the number of regenerated axons, compared to transected DCs without cell transplantation. AC transplantation resulted in improvement of the conduction properties, but the number of regenerated axons was similar to that of transected DCs without the transplantation. X-ray irradiation (40 Gy) three days before DC transection and AC transplantation prevented the electrophysiological continuity of axons through the transected lesion. This evidence revealed that AC transplantation secondarily enhanced the regeneration of axons, probably endogeneous SCs of dorsal roots migrated into the transected lesion and enhanced the axonal regeneration.     

Neutralization of ciliary neurotrophic factor reduces astrocyte production from transplanted neural stem cells and promotes regeneration of corticospinal tract fibers in spinal cord injury

Transplantation of neural stem cells (NSC) into lesioned spinal cord offers the potential to increase regeneration by replacing lost neurons or oligodendrocytes. The majority of transplanted NSC, however, typically differentiate into astrocytes that may exacerbate glial scar formation. Here we show that blocking of ciliary neurotrophic factor (CNTF) with anti-CNTF antibodies after NSC transplant into spinal cord injury (SCI) resulted in a reduction of glial scar formation by 8 weeks. Treated animals had a wider distribution of transplanted NSC compared with the control animals. The NSC around the lesion coexpressed either nestin or markers for neurons, oligodendrocytes, or astrocytes. Approximately 20% fewer glial fibrillary acidic protein-positive/bromodeoxyuridine (BrdU)-positive cells were seen at 2, 4, and 8 weeks postgrafting, compared with the control animals. Furthermore, more CNPase(+)/BrdU(+) cells were detected in the treated group at 4 and 8 weeks. These CNPase(+) or Rip(+) mature oligodendrocytes were seen in close proximity to host corticospinal tract (CST) and 5HT(+) serotonergic axon. We also demonstrate that the number of regenerated CST fibers both at the lesion and at caudal sites in treated animals was significantly greater than that in the control animals at 8 weeks. We suggest that the blocking of CNTF at the beginning of SCI provides a more favorable environment for the differentiation of transplanted NSC and the regeneration of host axons.     

Schwann cell‐derived exosomes enhance axonal regeneration in the peripheral nervous system

Axonal regeneration in the peripheral nervous system is greatly supported by Schwann cells (SCs). After nerve injury, SCs dedifferentiate to a progenitor‐like state and efficiently guide axons to their original target tissues. Contact and soluble factors participate in the crosstalk between SCs and axons during axonal regeneration. Here we show that dedifferentiated SCs secrete nano‐vesicles known as exosomes which are specifically internalized by axons. Surprisingly, SC‐derived exosomes markedly increase axonal regeneration in vitro and enhance regeneration after sciatic nerve injury in vivo. Exosomes shift the growth cone morphology to a pro‐regenerating phenotype and decrease the activity of the GTPase RhoA, involved in growth cone collapse and axon retraction. Altogether, our work identifies a novel mechanism by which SCs communicate with neighboring axons during regenerative processes. We propose that SC exosomes represent an important mechanism by which these cells locally support axonal maintenance and regeneration after nerve damage. GLIA 2013;61:1795–1806     

Axon regeneration through scars and into sites of chronic spinal cord injury

Cellular and extracellular inhibitors are thought to restrict axon growth after chronic spinal cord injury (SCI), confronting the axon with a combination of chronic astrocytosis and extracellular matrix-associated inhibitors that collectively constitute the chronic "scar." To examine whether the chronically injured environment is strongly inhibitory to axonal regeneration, we grafted permissive autologous bone marrow stromal cells (MSCs) into mid-cervical SCI sites of adult rats, 6 weeks post-injury without resection of the "chronic scar." Additional subjects received MSCs genetically modified to express neurotrophin-3 (NT-3), providing a further local stimulus to axon growth. Anatomical analysis 3 months post-injury revealed extensive astrocytosis surrounding the lesion site, together with dense deposition of the inhibitory extracellular matrix molecule NG2. Despite this inhibitory environment, axons penetrated the lesion site through the chronic scar. Robust axonal regeneration occurred into chronic lesion cavities expressing NT-3. Notably, chronically regenerating axons preferentially associated with Schwann cell surfaces expressing both inhibitory NG2 substrates and the permissive substrates L1 and NCAM in the lesion site. Collectively, these findings indicate that inhibitory factors deposited at sites of chronic SCI do not create impenetrable boundaries and that inhibition can be balanced by local and diffusible signals to generate robust axonal growth even without resecting chronic scar tissue.     

Canadian Association of Neuroscience review: axonal regeneration in the peripheral and central nervous systems--current issues and advances

Injured nerves regenerate their axons in the peripheral (PNS) but not the central nervous system (CNS). The contrasting capacities have been attributed to the growth permissive Schwann cells in the PNS and the growth inhibitory environment of the oligodendrocytes in the CNS. In the current review, we first contrast the robust regenerative response of injured PNS neurons with the weak response of the CNS neurons, and the capacity of Schwann cells and not the oligodendrocytes to support axonal regeneration. We then consider the factors that limit axonal regeneration in both the PNS and CNS. Limiting factors in the PNS include slow regeneration of axons across the injury site, progressive decline in the regenerative capacity of axotomized neurons (chronic axotomy) and progressive failure of denervated Schwann cells to support axonal regeneration (chronic denervation). In the CNS on the other hand, it is the poor regenerative response of neurons, the inhibitory proteins that are expressed by oligodendrocytes and act via a common receptor on CNS neurons, and the formation of the glial scar that prevent axonal regeneration in the CNS. Strategies to overcome these limitations in the PNS are considered in detail and contrasted with strategies in the CNS.     

Transplantation of olfactory ensheathing cells promotes axonal regeneration in a rat model of spinal cord injury

BACKGROUND:Transplantation of olfactory ensheathing cells (OECs) into the injured spinal cord has been shown to promote axonal regeneration and functional recovery.However,the mechanisms underlying the effects of OEC transplantation remain controversial.OBJECTIVE:To observe fibrotic scar formation and axonal regeneration in the damaged spinal cord following OEC transplantation,and to determine whether OEC transplantation promotes neural regeneration by attenuating fibrotic scar formation.DESIGN,TIME AND SETTING:A randomized,controlled animal experiment was performed at the Department of Developmental Morphology,Tokyo Metropolitan Institute for Neuroscience,Fuchu,Japan and at the Department of Human Anatomy,College of Basic Medical Sciences,China Medical University,China between April 2007 and May 2009.MATERIALS:OECs were obtained from olfactory nerves and olfactory bulbs of male,4-week-old,Sprague Dawley rats.Rabbit anti-serotonin polyclonal antibody,rabbit anti-calcitonin gene-related peptide polyclonal antibody,rabbit anti-glial fibrillary acidic protein polyclonal antibody,rabbit anti-type IV collagen polyclonal antibody,and mouse anti-rat endothelial cell antigen-1 monoclonal antibody were used.METHODS:Male,Sprague Dawley rats aged 8 weeks were randomly divided into three groups:sham-surgery (n = 3),surgery (n = 9),and OEC transplantation (n = 11).Spinal cord transection at the T9-10 level was performed and the rats were transplanted with a 2-μL (1 × 105 cells) cell suspension.MAIN OUTCOME MEASURES:Formation of glial and fibrotic scars was examined using immunohistochemistry for glial fibrillary acidic protein and type IV collagen.Serotonin-positive and calcitonin gene-related peptide-positive axons were visualized by immunohistochemistry,respectively.Double immunofluorescence for type IV collagen and rat endothelial cell antigen-1 was also performed to determine co-localization of type IV collagen deposition and blood vessels.RESULTS:At 1 week after spinal cord injury,numerous glial cells were observed around the lesion site.Formation of fibrotic scar was determined by a large amount of type IV collagen deposition in the lesion center,and descending serotonin- or ascending calcitonin gene-related peptideconiaining axons stopped at the fibrotic scar that was formed in the lesion site.At week after transplantation,the formation of fibrotic scar was significantly inhibited.In addition,the fibrotic structure was partly formed and centralized in the blood vessel,and serotonergic and calcitonin gene-related peptide-containing axons were regenerated across the lesion site.CONCLUSION:OEC transplantation into the injured spinal cord attenuated fibrotic scar formation and promoted axon regeneration.     

Laminin-coated multifilament entubulation,combined with Schwann cells and glial cell line-derived neurotrophic factor,promotes unidirectional axonal regeneration in a rat model of thoracic spinal cord hemisection

Biomaterial bridging provides physical substrates to guide axonal growth across the lesion. To achieve efficient directional guidance, combinatory strategies using permissive matrix, cells and trophic factors are necessary. In the present study, we evaluated permissive effect of poly (acrylonitrile-co-vinyl chloride) guidance channels filled by different densities of laminin-precoated unidirectional polypropylene filaments combined with Schwann cells, and glial cell line-derived neurotrophic factor for axonal regeneration through a T10 hemisected spinal cord gap in adult rats. We found that channels with filaments significantly reduced the lesion cavity, astrocytic gliosis, and inflammatory responses at the graft-host boundaries. The laminin coated low density filament provided the most favorable directional guidance for axonal regeneration which was enhanced by co-grafting of Schwann cells and glial cell line-derived neurotrophic factor. These results demonstrate that the combinatorial strategy of filament-filled guiding scaffold, adhesive molecular laminin, Schwann cells, and glial cell line-derived neurotrophic factor, provides optimal topographical cues in stimulating directional axonal regeneration following spinal cord injury. This study was approved by Indiana University Institutional Animal Care and Use Committees (IACUC #:11011) on October 29, 2015.Key Words: axonal regeneration, extracellular molecule, filament density, hemisection, laminin, neurotrophic factor, Schwann cell, spinal cord injury, thoracic, transplantation

Chinese Library Classification No. R453; R741; R364.5     

Effects of Axonal Injury on Astrocyte Proliferation and Morphologyin Vitro:Implications for Astrogliosis

Mechanisms inducing gliosis following injury in the central nervous system are poorly understood. We evaluated the effect of axonal injury on astrocyte and Schwann cell proliferation and morphologyin vitro.Purified rat dorsal root ganglion neurons grown on monolayers of rat neonatal cortical astrocytes (N-AS^neonatalcultures) or sciatic nerve-derived Schwann cells (N-SC cultures) were mechanically injured. Noninjured cultures served as controls. Cell proliferation near lesions was monitored by autoradiography 1, 2, 4, and 8 days postinjury. Axonal injury caused a significant transient increase in astrocyte proliferation immediately proximal and distal to the lesion. The lesion did not induce marked changes in the intensity of glial fibrillary acidic protein (GFAP) immunoreactivity. However, processes from GFAP-positive cells usually arranged in random fashion in noninjured cultures were aligned perpendicularly to the cut distal to lesions. Ultrastructural analysis in lesioned N-AS^neonatalcultures indicated that proximal to the lesion filament-filled astrocytes were intermingled with axons. Distal to the lesion astrocyte processes formed layers, between which an increased amount of collagen-like material appeared with time postlesion. Axons distal to the lesion degenerated by 2 days, coinciding with the early disappearance of neurofilament immunoreactivity. In noninjured and proximally in injured N-SC cultures, Schwann cells extended processes, engulfing some axons. Distal to the lesion, Schwann cells appeared more rounded and neurites remained until 4 days postinjury. Media conditioned by injured or noninjured N-AS^neonatalcultures did not affect neuron-induced Schwann cell proliferation. These findings demonstrate that axonal injury and degeneration cause a transient increase in astrocyte proliferation and induce morphological changes in astrocytes consistent with the onset of gliosis.     

Increased expression of the putative axon growth-repulsive extracellular matrix molecule,keratan sulphate proteoglycan,following traumatic injury of the adult rat spinal cord

Keratan sulphate proteoglycan (KSPG) is a developmentally regulated barrier molecule, directing axonal growth during central nervous system (CNS) formation. The possible re-expression and functional significance of KSPG in preventing axon regeneration following spinal cord injury (SCI) is poorly understood. In the present investigation, the spatio-temporal expression of KSPG was studied following experimental SCI. There was no indication of sparing of axons at the lesion epicentre following severe compression injury. By 7 days post operation (p.o.) a diffuse increase of KSPG immunoreactivity (KSPG-IR) was observed in the parenchyma surrounding the lesion. This was followed by a delayed (21-28 days p.o.) and largely heterogeneous increase of KSPG-IR in the lesion epicentre, which revealed both cellular and extracellular matrix-like distribution patterns. Although no re-growth of anterogradely labelled corticospinal axons was observed, many 200-kDa neurofilament (NF)-positive axons could be detected growing into the connective tissue scar. This phase of spontaneous axonal re-growth was closely associated with a framework of glial cells (including Schwann cells from damaged local spinal nerve roots) that had migrated into the lesion site. The spontaneous nerve fibre re-growth could be detected in both KSPG-rich and KSPG-poor territories. The present data suggest that the lesion-induced up-regulation of KSPG-IR may have contributed to the lack of corticospinal axon re-growth. However, the lack of any direct spatio-temporal correlation between the distribution of raised KSPG-IR and spontaneous NF-positive axonal regeneration suggests that at least some populations of axons can resist the putative inhibitory effects of this extracellular matrix molecule.     

Purkinje Cell Transplants inShakerMutant Rats with Hereditary Purkinje Cell Degeneration and Ataxia

Shakermutant rats are characterized by the adult-onset degeneration of cerebellar anterior lobe Purkinje cells and temporally correlated development of ataxia and tremor. Normal E-13 Purkinje cells were transplanted into the anterior cerebellum in adultshakermutant rats to study donor/host interactions in an animal with adult-onset heredodegeneration. Donor Purkinje cells from extraparenchymal transplant sites migrated radially into the host molecular layer and differentiated. Donor Purkinje cell dendrites expanded to fill the host molecular layer, spinous processes were apparent, and axonal projections into the host gray and white matter were observed. Donor Purkinje cells remaining in the extraparenchymal transplant sites differentiated if they were located relatively close to the host cerebellum. Donor Purkinje cells located intraparenchymally in the host white matter or granule cell layer survived, but were stunted in their development. The orthogonal movement of donor Purkinje cells away from transplant sites in the host cerebellum was spatially restricted. The findings from this study indicate that host cerebellar cortex with adult-onset heredodegeneration of Purkinje cells supports the survival and differentiation of transplanted normal embryonic Purkinje cells.