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Immature spinal cord, unlike adult, has an ability to repair itself following injury. Evidence for regeneration, structural repair and development of substantially normal locomotor behaviour comes from studies of marsupials due to their immaturity at birth. We have compared morphological, cellular and molecular changes in spinal cords transected at postnatal day (P)7 or P14, from 3 h to 2 weeks post-injury, in South American opossums (Monodelphis domestica). A bridge between severed ends of cords was apparent 5 days post-injury in P7 cords, compared to 2 weeks in P14. The volume of neurofilament (axonal) material in the bridge 2 weeks after injury was 30% of control in P7- but < 10% in P14-injured cords. Granulocytes accumulated at the site of injury earlier (3 h) in P7 than in P14 (24 h)-injured animals. Monocytes accumulated 24 h post-injury and accumulation was greater in P14 cords. Accumulation of GFAP-positive astrocytes at the lesion occurred earlier in P14-injured cords. Neurites and growth cones were identified ultrastructurally in contact with astrocytes forming the bridge. Results using mouse inflammatory gene arrays showed differences in levels of expression of many TGF, TNF, cytokine, chemokine and interleukin gene families. Most of the genes identified were up-regulated to a greater extent following injury at P7. Some changes were validated and quantified by RT-PCR. Overall, the results suggest that at least some of the greater ability to recover from spinal cord transection at P7 compared to P14 in opossums is due to differences in inflammatory cellular and molecular responses.

To foster axonal growth from a Schwann cell bridge into the caudal spinal cord, spinal cells caudal to the implant were transduced with adeno-associated viral (AAV) vectors encoding for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (AAV-NT-3). Control rats received AAV vectors encoding for green fluorescent protein or saline. AAV-BDNF- and AAV-NT-3-transduced 293 human kidney cells produced and secreted BDNF or NT-3, respectively, in vitro. The secreted neurotrophins were biologically active; they both promoted outgrowth of sensory neurites in vitro. In vivo, transgene expression was observed predominantly in neurons for at least 16 weeks after injection. Compared with controls, a modest though significant improvement in hind-limb function was found in rats that received AAV-BDNF and AAV-NT-3. Retrograde tracing demonstrated that twice as many neurons with processes extending toward the Schwann cell graft were present in the second lumbar cord segment of AAV-BDNF- and AAV-NT-3-injected animals compared with controls. We found no evidence, however, for growth of regenerated axons from the Schwann cell implant into the caudal cord. Our results suggest that AAV vector-mediated overexpression of BDNF and NT-3 in the cord caudal to a Schwann cell bridge modified the local lumbar axonal circuitry, which was beneficial for locomotor function.

Here we present evidence for spontaneous and long-lasting regeneration of CNS axons after spinal cord lesions in adult rats. The length of 200 kD neurofilament (NF)-immunolabeled axons was estimated after photochemically induced ischemic spinal cord lesions using a stereological tool. The total length of all NF-immunolabeled axons within the lesion cavities was increased 6- to 10-fold at 5, 10, and 15 wk post-lesion compared with 1 wk post-surgery. In ultrastructural studies we found the putatively regenerating axons within the lesion to be associated either with oligodendrocytes or Schwann cells, while other fibers were unmyelinated. Immunohistochemistry demonstrated that some of the regenerated fibers were tyrosine hydroxylase- or serotonin-immunoreactive, indicating a central origin. These findings suggest that there is a considerable amount of spontaneous regeneration after spinal cord lesions in rodents and that the fibers remain several months after injury. The findings of tyrosine hydroxylase- and serotonin-immunoreactivity in the axons suggest that descending central fibers contribute to this endogenous repair of ischemic spinal cord injury.

Clinically, high-dose treatment with the glucocorticosteroid, methylprednisolone (MP), within 8 h after spinal cord injury, has been shown to improve neurological recovery. The current standard of care is to administer MP as a bolus of 30 mg/kg followed by a 23-h infusion of 5.4 mg/kg/h to spinal cord injured patients. To better understand the role of MP in neuroprotection, we have studied how MP administration affects macrophage accumulation, tissue loss, and axonal dieback at 1, 2, 4 and 8 weeks after a complete transection of the eighth thoracic spinal cord in the adult rat. A 30 mg/kg dose of MP was administered intravenously at 5 min, and 2 and 4 h after injury. The number of ED1 (antibody against microglia/macrophages) -positive cells was quantified in a 500-micrometer-wide strip of tissue directly adjacent and parallel to the transection. At all time points, MP treatment led to a significant decrease in the number of ED1-positive cells in both rostral and caudal stumps. Over the 2-month post-transection period, the average MP-induced reduction in the number of ED1-positive cells was 82% in the rostral cord stump and 66% in the caudal stump. Using a computerized image analysis system, it was observed that MP treatment resulted in a significant reduction in tissue loss in both cord stumps at 2, 4 and 8 week post-injury. Over the 2-month post-lesion period, the average MP-induced reduction in tissue loss in the caudal cord stump was higher than that in the rostral stump; 48 versus 37%, respectively. Immunostaining for neurofilaments and growth-associated protein-43 (GAP-43) revealed the presence of numerous axons near and in the lesion site. Anterograde neuronal tracing with biotinylated dextran amine showed that, in MP-treated animals, dieback of vestibulospinal fibres, but not of corticospinal fibres, was significantly diminished at all time points studied. In addition, with MP administration, 1 and 2 weeks after injury, an increase in the number of vestibulospinal fibres was found at 1 and 2 mm from the transection, suggesting transient regenerative sprouting of these fibres. The results demonstrate that treatment with MP shortly after spinal cord transection in the adult rat led to a long-term reduction of ED1-positive cells and spinal tissue loss, reduced dieback of vestibulospinal fibres, and a transient sprouting of vestibulospinal fibres near the lesion at 1 and 2 weeks post-lesion. The possible relationships between the inflammatory changes, spinal tissue sparing, and axonal survival and sprouting are complex and need to be further explored.

Future surgical strategies to restore neurological function in the damaged human spinal cord may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. We have studied the in vitro and in vivo degradability of various aliphatic polyesters as well as their effects on rat Schwann cells in vitro and on spinal cord tissue in vivo. In vitro, cylinders made of poly(D,L-lactic-co-glycolic acid) 50:50 (PLA25GA50) started to degrade at 7 days, compared with 28 days for cylinders made of poly(D,L-lactic acid) (PLA50). This faster degradation of PLA25GA50 was reflected by a much higher absorption of water. In vivo, after implantation of PLA25GA50 or PLA50 cylinders between the stumps of a completely transected adult rat spinal cord, the decrease in molecular weight of both polymers was similar to that found in vitro. In vitro degradation of poly(L-lactic acid) (PLA100) mixed with increasing amounts of PLA100 oligomers also was determined. The degradation rate of PLA100 mixed with 30% oligomers was found to be similar to that of PLA50. In vitro, PLA25GA50 and the breakdown products had no adverse effect on the morphology, survival, and proliferation of cultured rat Schwann cells. In vivo, PLA25GA50 cylinders were integrated into the spinal tissue 2 weeks after implantation, unlike PLA50 cylinders. At all time points after surgery, the glial and inflammatory response near the lesion site was largely similar in both experimental and control animals. At time points later than 1 week, neurofilament-positive fibers were found within PLA25GA50 cylinders or the remains thereof. Growth-associated protein 43, which is indicative of regenerating axons, was observed in fibers in the vicinity of the injury site and in the remains of PLA25GA50 cylinders. The results suggest that poly(alpha-hydroxyacids) are likely candidates for application in spinal cord regeneration paradigms involving Schwann cells.

The lack of axonal regeneration in the injured adult mammalian spinal cord leads to permanent functional impairment. To induce axonal regeneration in the transected adult rat spinal cord, we have used the axonal growth-promoting properties of adult olfactory bulb ensheathing glia (EG). Schwann cell (SC)-filled guidance channels were grafted to bridge both cord stumps, and suspensions of pure (98%) Hoechst-labeled EG were stereotaxically injected into the midline of both stumps, 1 mm from the edges of the channel. In EG-transplanted animals, numerous neurofilament-, GAP-43-, anti-calcitonin gene-related peptide (CGRP)-, and serotonin-immunoreactive fibers traversed the glial scars formed at both cord-graft interfaces. Supraspinal serotonergic axons crossed the transection gap through connective tissue bridges formed on the exterior of the channels, avoiding the channel interior. Strikingly, after crossing the distal glial scar, these fibers elongated in white and periaqueductal gray matter, reaching the farthest distance analyzed (1.5 cm). Tracer-labeled axons present in SC grafts were found to extend across the distal interface and up to 800 microm beyond in the distal cord. Long-distance regeneration (at least 2.5 cm) of injured ascending propriospinal axons was observed in the rostral spinal cord. Transplanted EG migrated longitudinally and laterally from the injection sites, reaching the farthest distance analyzed (1.5 cm). They moved through white matter tracts, gray matter, and glial scars, overcoming the inhibitory nature of the CNS environment, and invaded SC and connective tissue bridges and the dorsal and ventral roots adjacent to the transection site. Transplanted EG and regenerating axons were found in the same locations. Because EG seem to provide injured spinal axons with appropriate factors for long-distance elongation, these cells offer new possibilities for treatment of CNS conditions that require axonal regeneration.

The infusion of BDNF and NT-3 into Schwann cell (SC) grafts promotes regeneration of brainstem neurones into the grafts placed in adult rat spinal cord transected at T8 (Xu et al., 1995b). Here, we compared normal SCs with SCs genetically modified to secrete human BDNF, grafted as trails 5 mm long in the cord distal to a transection site and also deposited in the transection site, for their ability to stimulate supraspinal axonal regeneration beyond the injury. SCs were infected with the replication-deficient retroviral vector pL(hBDNF)RNL encoding the human preproBDNF cDNA. The amounts of BDNF secreted (as detected by ELISA) were 23 and 5 ng/24 h per 106 cells for infected and normal SCs, respectively. Biological activity of the secreted BDNF was confirmed by retinal ganglion cell bioassay. The adult rat spinal cord was transected at T8. The use of Hoechst prelabelled SCs demonstrated that trails were maintained for a month. In controls, no SCs were grafted. One month after grafting, axons were present in SC trails. More 5-HT-positive and some DbetaH-positive fibres were observed in the infected vs. normal SC trails. When Fast Blue was injected 5 mm below the transection site (at the end of the trail), as many as 135 retrogradely labelled neurones could be found in the brainstem, mostly in the reticular and raphe nuclei (normal SCs, up to 22, mostly in vestibular nuclei). Numerous neurones were labelled in the ventral hypothalamus (normal SCs, 0). Also, following Fast Blue injection, a mean of 138 labelled cells was present in dorsal root ganglia (normal SCs, 46) and spinal cord (39 vs. 32) rostral to the transection. No labelled spinal neurones rostral to the transection were seen when SCs were not transplanted. Thus, the transplantation of SCs secreting increased amounts of BDNF improved the regenerative response across a transection site in the thoracic cord. Moreover, the enhanced regeneration observed with infected SCs may be specific as the largest response was from neurones known to express trkB.

Two strategies have been shown by others to improve CST regeneration following thoracic spinal cord injury: 1) the administration of a monoclonal antibody, IN-1, raised against a myelin-associated, neurite growth inhibitory protein, and 2) the delivery of acidic fibroblast growth factor (aFGF) in fibrin glue in association with peripheral nerve grafts. Because autologous transplantation of human Schwann cells (SCs) is a potential strategy for CNS repair, we evaluated the ability of these two molecular agents to induce CST regeneration into human SC grafts placed to span a midthoracic spinal cord transection in the adult nude rat, a xenograft tolerant strain. IN-1 or control (HRP) antibodies were delivered to the injury/graft region by encapsulated hybridoma cells ("IN-1 ravioli") or daily infusion of hybridoma culture supernatant; aFGF-fibrin glue was placed in the same region in other animals. Anterograde tracing from the motor cortex using the dextran amine tracers, Fluororuby (FR) and biotinylated dextran amine (BDA), was performed. Thirty-five days after grafting, the CST response was evaluated qualitatively by looking for regenerated CST fibers in or beyond grafts and quantitatively by constructing camera lucida composites to determine the sprouting index (SI), the position of the maximum termination density (MTD) rostral to the GFAP-defined host/graft interface, and the longitudinal spread (LS) of bulbous end terminals. The latter two measures provided information about axonal die-back. In control animals (graft only), the CST did not enter the SC graft and underwent axonal die-back [SI = 1.4 +/- 0.1, MTD = 2.0 +/- 0.2, LS = 1.3 +/- 0.3, (n = 3)]. Results of IN-1 delivery from ravioli did not differ from controls, but injections of IN-1-containing supernatant resulted in a significant degree of sprouting but did not prevent axonal die-back [SI = 1.9 +/- 0.1, MTD = 1.5 +/- 0.2, LS = 1.1 +/- 0.1, (n = 7)] and traced fibers did not enter grafts. Acidic FGF dramatically reduced axonal die-back and caused sprouting [SI = 2.0 +/- 0.1 (n = 5), MTD = 0.5 +/- 0.04 (n = 6), LS = 0.4 +/- 0.1 (n = 6)]. Some traced fibers entered SC grafts and in 2/6 cases entered the distal interface. We conclude that 1) human SC grafts alone do not support the regeneration of injured CST fibers and do not prevent die-back, 2) grafts plus IN-1 antibody-containing supernatant support some sprouting but die-back continues, and 3) grafts plus aFGF-fibrin glue support regeneration of some fibers into the grafts and reduce die-back.

Advances in the purification and expansion of Schwann cells (SCs) from adult human peripheral nerve, together with biomaterials development, have made the construction of unique grafts with defined properties possible. We have utilized PAN/PVC guidance channels to form solid human SC grafts which can be transplanted either with or without the channel. We studied the ability of grafts placed with and without channels to support regeneration and to influence functional recovery; characteristics of the graft and host/graft interface were also compared. The T9-T10 spinal cord of nude rats was resected and a graft was placed across the gap; methylprednisolone was delivered acutely to decrease secondary injury. Channels minimized the immigration of connective tissue into grafts but contributed to some necrotic tissue loss, especially in the distal spinal cord. Grafts without channels contained more myelinated axons (x = 2129 +/- 785) vs (x = 1442 +/- 514) and were larger in cross-sectional area ( x = 1.53 +/- 0.24 mm2) vs (x = 0.95 +/- 0.86 mm2). The interfaces formed between the host spinal cord and the grafts placed without channels were highly interdigitated and resembled CNS-PNS transition zones; chondroitin sulfate proteoglycans was deposited there. Whereas several neuronal populations including propriospinal, sensory, motoneuronal, and brainstem neurons regenerated into human SC grafts, only propriospinal and sensory neurons were observed to reenter the host spinal cord. Using combinations of anterograde and retrograde tracers, we observed regeneration of propriospinal neurons up to 2.6 mm beyond grafts. We estimate that 1% of the fibers that enter grafts reenter the host spinal cord by 45 days after grafting. Following retrograde tracing from the distal spinal cord, more labeled neurons were unexpectedly found in the region of the dextran amine anterograde tracer injection site where a marked inflammatory reaction had occurred. Animals with bridging grafts obtained modestly higher scores during open field [(x = 8.2 +/- 0.35) vs (x = 6.8 +/- 0.42), P = 0.02] and inclined plane testing (x = 38.6 +/- 0. 542) vs (x = 36.3 +/- 0.53), P = 0.006] than animals with similar grafts in distally capped channels. In summary, this study showed that in the nude rat given methylprednisolone in combination with human SC grafts, some regenerative growth occurred beyond the graft and a modest improvement in function was observed., (Copyright 1997 Academic Press.)

Insulin-like growth factor-I (IGF-I) promotes axonal regeneration in the peripheral nervous system and this effect is enhanced by platelet-derived growth factor (PDGF). We decided, therefore, to study the effects of these factors on axonal regeneration in the adult rat spinal cord. Semipermeable polymer tubes, closed at the distal end, containing Matrigel mixed with cultured rat Schwann cells and IGF-I/PDGF, were placed at the proximal stump of the spinal cord after removal of the thoracic T9-11 segments. Control animals received implants of only Matrigel and Schwann cells or only Matrigel and IGF-I/PDGF. Four weeks after implantation, electron microscopic analysis showed that the addition of IGF-I/PDGF resulted in an increase in the myelinated:unmyelinated fiber ratio from 1:7 to 1:3 at 3 mm in the Schwann cell graft, and that myelin sheath thickness was increased 2-fold. The reduced number of unmyelinated axons was striking in electron micrographs. These results suggested that IGF-I/PDGF enhanced myelin formation of regenerated axons in Schwann cell implants, but there was a 36% decrease in the total number of myelinated axons at the 3 mm level of the graft. This finding and the altered myelinated:unmyelinated fiber ratio revealed that the overall fiber regeneration into Schwann cell implants was diminished up to 63% by IGF-I/PDGF. Histological evaluation revealed that there were more larger cavities in tissue at the proximal spinal cord-graft interface in animals receiving a Schwann cell implant with IGF-I/PDGF. Such cavitation might have contributed to the reduction in axonal ingrowth. In sum, the results indicate that whereas the combination of IGF-I and PDGF enhances myelination of regenerating spinal cord axons entering implants of Matrigel and Schwann cells after midthoracic transection, the overall regeneration of axons into such Schwann cell grafts is diminished.

Transplantation of cellular components of the permissive peripheral nerve environment in some types of spinal cord injury holds great promise to support regrowth of axons through the site of injury. In the present study, Schwann cell grafts were positioned between transected stumps of adult rat thoracic spinal cord to test their efficacy to serve as bridges for axonal regeneration. Schwann cells were purified in culture from adult rat sciatic nerve, suspended in Matrigel: DMEM (30:70), and drawn into polymeric guidance channels 8 mm long at a density of 120 x 10(6) cells ml-1. Adult Fischer rat spinal cords were transected at the T8 cord level and the next caudal segment was removed. Each cut stump was inserted 1 mm into the channel. One month later, a bridge between the severed stumps had been formed, as determined by the gross and histological appearance and the ingrowth of propriospinal axons from both stumps. Propriospinal neurons (mean, 1064 +/- 145 SEM) situated as far away as levels C3 and S4 were labelled by retrograde tracing with Fast Blue injected into the bridge. Near the bridge midpoint there was a mean of 1990 +/- 594 myelinated axons and eight times as many nonmyelinated, ensheathed axons. Essentially no myelinated or unmyelinated axons were observed in control Matrigel-only grafts. Brainstem neurons were not retrogradely labelled from the graft, consistent with growth of immunoreactive serotonergic and noradrenergic axons only a short distance into the rostral end of the graft, not far enough to reach the tracer placed at the graft midpoint. Anterograde tracing with PHA-L introduced rostral to the graft demonstrated that axons extended the length of the graft but essentially did not leave the graft. This study demonstrates that Schwann cell grafts serve as bridges that support (1) regrowth of both ascending and descending axons across a gap in the adult rat spinal cord and (2) limited regrowth of serotonergic and noradrenergic fibers from the rostral stump. Regrowth of monoaminergic fibres into grafts was not seen in an earlier study of similar grafts placed inside distally capped rather than open-ended channels. Additional intervention will be required to foster growth of the regenerated axons from the graft into the distal cord tissue.

Schwann cell (SC) grafts support the regeneration of axons of numerous spinal cord neurons when placed into transected adult rat midthoracic spinal cord. Clinically, methylprednisolone (MP) has been shown to be neuroprotective if administered within 8 h after spinal cord injury. We investigated whether axonal regrowth into SC grafts is enhanced when MP is administered at the time of spinal cord transection and SC implantation. SCs from adult rat sciatic nerves were purified in culture, suspended in Matrigel, and drawn into semipermeable polymeric channels. MP (30 mg/kg) or vehicle (control) was administered intravenously at 5 min, 2 h, and 4 h to adult Fischer rats after transection at T8 and removal of the next three caudal segments. The rostral cord stump was inserted 1 mm into the channel; the distal end of the channel was capped. Thirty to forty-five days later, the SC/MP group showed large tissue cables in the channels and host cord tissue retained in the rostral end of the channels. Significantly more myelinated axons (1159 +/- 308) were present at the 5-mm level in SC/MP grafts (n = 6) than in SC/vehicle cables (355 +/- 108, n = 5). More unmyelinated than myelinated axons (approximately 4:1, n = 3) were resolved in the cables by electron microscopy. In the SC/MP group, unlike the SC/vehicle group, serotonergic and noradrenergic fibers were detected immunocytochemically 2.5 and 2.0 mm respectively, into the graft; astrocytes were also identified at similar distances from the interface. Fast Blue retrograde tracing (SC/MP, n = 4; SC/vehicle, n = 3) showed that more spinal cord neurons (1116 +/- 113 vs 284 +/- 88, respectively) and spinal cord neurons more distant from the graft (C8 vs C5) responded by extending axons into the graft in the presence of MP. Also, very significantly, supraspinal brain stem neurons extended axons into the graft only when MP was administered (mean 46 vs 0, n = 3). These results indicate that MP improves axonal regenerationn from both spinal cord and brain stem neurons into thoracic SC grafts, possibly by reducing secondary host tissue loss adjacent to the graft.

We previously demonstrated that Schwann cells (SCs) in semipermeable guidance channels promote axonal regeneration in adult rat spinal cord transected at the mid-thoracic level. Propriospinal but not supraspinal axons grew into these channels. Here, we tested the ability of exogenous brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) to promote axonal regeneration in this novel model. The two neurotrophins were delivered simultaneously into the channel by an Alzet minipump at a rate of 12 micrograms/day for each neurotrophin for 14 of 30 days tested; phosphate-buffered saline, the vehicle solution, was used as a control. Significantly more myelinated nerve fibers were present in SC/neurotrophin grafts than in SC/vehicle grafts (1523 +/- 292 vs 882 +/- 287). In the graft, at least 5 mm from the rostral cord-graft interface, some nerve fibers were immunoreactive for serotonin, a neurotransmitter specific to raphe-derived axons in rat spinal cord. Fast blue retrograde tracing from SC/neurotrophin grafts revealed labeled neurons in 10 nuclei of the brain stem, 67% of these being in the lateral and spinal vestibular nuclei. The mean number of labeled brain stem neurons in the SC/neurotrophin group (92; n = 3) contrasted with the mean in the SC/vehicle group (6; n = 4). Our results clearly demonstrate that BDNF and NT-3 infusion enhanced propriospinal axonal regeneration and, more significantly, promoted axonal regeneration of specific distant populations of brain stem neurons into grafts at the mid-thoracic level in adult rat spinal cord.

Schwann cells (SC) have been shown to promote regeneration in both the peripheral and central nervous systems. In this study we tested the ability of SC to enhance axonal regeneration in adult rat spinal cord by grafting SC-seeded guidance channels into transected cords. SC were purified in culture from adult inbred rat sciatic nerves, suspended in Matrigel, and seeded into semipermeable PAN/PVC channels (2.6 mm I.D. x 10 mm long) at a final density of 120 x 10(6) cells/ml. Channels filled with Matrigel alone served as controls. Adult isologous rat spinal cords were transected at the T8 level, and segments T9-T11 were removed. The rostral stump was inserted 1 mm into channels with capped distal ends. One month after grafting, a vascularized tissue cable was present within the channel in all animals. In SC-seeded channels (n = 14), a mean of 501 myelinated axons was found in the cable, and many axons extended 9-10 mm. Electron microscopy revealed typical SC ensheathment and myelination of axons with four times more unmyelinated than myelinated axons. Control channels (n = 8) contained fewer myelinated axons (mean = 71). When SC were prelabeled in culture with a nuclear dye, labeled nuclei were observed at 30 days, confirming SC survival. Astrocytes identified by glial fibrillary acidic protein staining did not migrate far into the cable, and prelabeled SC did not enter the cord. Lack of immunostaining for serotonin and dopamine beta-hydroxylase indicated that supraspinal axons did not regenerate into the cable. Fast Blue injections into the middle of the cable (n = 3) marked spinal cord interneurons (mean = 306) as far as nine segments rostral (25 mm, C7) extending axons into the graft; fewer dorsal root ganglion neurons were retrogradely labeled. In conclusion, purified populations of SC transplanted within channels promote both propriospinal and sensory axonal regeneration in the adult rat thoracic spinal cord.

This study characterized by light and electron microscopy 49 photochemically induced lesions in adult rat spinal cord at 16 time intervals from 2 days to 17 months after lesioning. Vascular thrombosis, resulting from an intravascular photochemical reaction induced by a rose bengal/laser beam interaction, led within a few days to an extensive area of tissue deterioration. This area, termed the "lesion cavity" in contrast to the "secondary cavity" observed later, was at least 6 mm long and, at the epicenter, extended across most of the spinal cord width and from the dorsal surface to a level near the central canal. The area of spared tissue, 43% of the spinal cord cross-section at 2 days, did not change significantly between 2 and 56 days. Large numbers of macrophages populated the degenerating area by 5 days. This necrotic area was surrounded by a thin peripheral rim of largely intact white matter dorsally and laterally except at the epicenter where the white matter degenerated dorsomedially. In these peripheral regions, demyelination and, by 14 days, remyelination by both oligodendrocytes and Schwann cells (SCs) were evident. By 28 days, far more SCs (and meningeal cells) had entered the dorsal spinal cord, typically at the epicenter where meningeal thickening was most striking, and had migrated farther into the lesion cavity. These SCs and the axons they myelinated remained prominent in dorsal regions for many months, particularly at the epicenter; the proportion of SC to oligodendrocyte myelin diminished away from the epicenter. By 8 weeks, the lesion cavity was considerably diminished in size and thereafter it contained scattered macrophages, SC-myelinated axons, and blood vessels, primarily medially owing to flattening into clefts bilaterally. The cavity was partly bordered by astrocytes whose surfaces toward the lesion cavity were highly irregular and coated with basal lamina. Bare axons, consistently seen by electron microscopy at 5 days to 6 months, were typically ensconced among astrocytes starting at 28 days. Also by this time large, smoothly contoured, empty secondary cavities appeared, usually rostral and caudal to the epicenter; they did not increase in size or number with time. From 28 days to 17 months postlesion they occurred in 68% of the lesioned spinal cords. The secondary cavity border was composed of cells thought to be astrocytes but, surprisingly, the luminal surface was smooth and lacked basal lamina, in contrast to the primary lesion cavity border. Thus, two types of cavities formed after photochemical lesioning. This lesioning technique may provide an appropriate milieu to better understand aspects of the vexing problem of post-traumatic syringomyelia in the human.

Polymerized collagen rolls enclosing Schwann cells (SCs) raised in culture were grafted into cystic cavities formed after lesioning the thoracic spinal cord of adult rats. Axons were already present within the graft by 14 days after implantation and both ensheathed and myelinated axons were numerous by 28 days. This axonal ingrowth was maintained over longer survival periods. The axons within the graft always appeared related to Schwann cells. Acellular collagen rolls did not show axonal ingrowth. These Schwann cell-collagen implants resemble peripheral nerve grafts in their ability to induce axonal regeneration into the graft.

Neurofilament proteins from brain and spinal cord of immature rat (20-35 days of age) and rabbit (15-17) days of age) were prepared by an axonal flotation technique. Examination of rat filament preparations by electron microscopy revealed a preponderance of 10 nm diameter filaments that were usually loosely aggregated although some bundles of more tightly packed filaments were present as well. The neurofilament triplet proteins of the rat and rabbit central nervous system were found to be phosphorylated 24 hr after the intracerebral injection of [32P]orthophosphate when examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography. Examination of each eluted neurofilament protein from both species showed that [32P]phosphate was retained after reelectrophoresis and fluorography. Evidence is presented that the [32P]phosphate is covalently linked to the purified neurofilament proteins by phosphoester bonds.

This report presents ultrastructural observations on the cytological events that attend myelin formation occurring in the wake of demyelination in adult cat spinal cord. Lesions were induced in subpial cord by cerebrospinal fluid (c.s.f.) exchange (1, 2). Tissue from eleven cats at nine intervals from 19 to 460 days was fixed in situ by replacing c.s.f. with buffered OsO(4) and embedded in Araldite. After demyelination, axons are embraced by sheet-like glial processes. An occasional myelin sheath is first seen at 19 days; by 64 days, all axons are at least thinly myelinated. The cytoplasm of the myelin-forming cells, unlike that of either oligodendrocyte or fibrous astrocyte in normal cord, is dense with closely packed organelles and fine fibrils. Many of the myelinogenic cells become scarring astrocytes and at 460 days the lesion teems with their fibril-filled processes. Oligodendrocytes appear in the lesion after remyelination is under way. Phagocytes disappear gradually. A myelin sheath is formed by spiral wrapping of a sheet-like glial process around an axon. Where the first turn of the spiral is completed, a mesaxon is formed. As cytoplasm is lost from the process, the plasma membrane comes together along its outer and cytoplasmic surfaces to form compact myelin. Only a small amount of cytoplasm is retained; it is confined to the paramesaxonal region and, on the sheath exterior, to a longitudinal ridge which appears in profile as a small loop. This outer loop has the same rotational orientation as the inner mesaxon. These vestiges of spiral membrane wrapping are also found in normal adult and new-born cat cord. Nodes are present in all stages of remyelination and in normal adult cat and kitten cord. These observations suggest that myelin is reformed in the lesion in the same way it is first formed during normal development. The mechanism of myelin formation is basically similar to that proposed for peripheral nerve and amphibian and mammalian optic nerve; it does not agree with present views on the mechanism of myelinogenesis in mammalian brain and cord. This is the first demonstration of remyelination in adult mammalian central nervous tissue.

Plaques of subpial demyelination were induced in adult cat spinal cords by repeated withdrawal and reinjection of cerebrospinal fluid. Peripheral cord was fixed by replacing cerebrospinal fluid available at cisternal puncture with 3 per cent buffered OsO(4). Following extirpation, surface tissue was further fixed in 2 per cent buffered OsO(4), dehydrated in ethanol, and embedded in araldite. Normal subpial cord consists mainly of myelinated axons and two types of macroglia, fibrous astrocytes and oligodendrocytes. Twenty-nine hours after lesion induction most myelin sheaths are deteriorating and typical macroglia are no longer visible. Phagocytosis of myelin debris has begun. In 3-day lesions, axons are intact and their mitochondria and neurofibrils appear normal despite continued myelin breakdown. All axons are completely demyelinated by 6 days. They lack investments only briefly, however, for at 10 and 14 days, macroglial processes appear and embrace them. These macroglia do not resemble either one of the normally occurring glia; their dense cytoplasm contains fibrils in addition to the usual organelles. It is proposed that these macroglia, which later accomplish remyelination, are the hypertrophic or swollen astrocytes of classical neuropathology. The suggestion that these astrocytes possess the potential to remyelinate axons in addition to their known ability to form cicatrix raises the possibility of pharmacological control of their expression.

Explants prepared from 17- to 18-day fetal rat spinal cord were allowed to mature in culture; such preparations have been shown to differentiate and myelinate in vitro (61) and to be capable of complex bioelectric activity (14-16). At 23, 35, or 76 days, the cultures were fixed (without removal from the coverslip) in buffered OsO(4), embedded in Epon, sectioned, and stained for light and electron microscopy. These mature explants generally are composed of several strata of neurons with an overlying zone of neuropil. The remarkable cytological similarity between in vivo and in vitro nervous tissues is established by the following observations. Cells and processes in the central culture mass are generally closely packed together with little intervening space. Neurons exhibit well developed Nissl bodies, elaborate Golgi regions, and subsurface cisternae. Axosomatic and axodendritic synapses, including synaptic junctions between axons and dendritic spines, are present. Typical synaptic vesicles and increased membrane densities are seen at the terminals. Variations in synaptic fine structure (Type 1 and Type 2 synapses of Gray) are visible. Some characteristics of the cultured spinal cord resemble infrequently observed specializations of in vivo central nervous tissue. Neuronal somas may display minute synapse-bearing projections. Occasionally, synaptic vesicles are grouped in a crystal-like array. A variety of glial cells, many apparently at intermediate stages of differentiation, are found throughout the otherwise mature explant. There is ultrastructural evidence of extensive glycogen deposits in some glial processes and scattered glycogen particles in neuronal terminals. This is the first description of the ultrastructure of cultured spinal cord. Where possible, correlation is made between the ultrastructural data and the known physiological properties of these cultures.

Purpose: This study investigated whether enrichment improves hindlimb movement following complete spinal cord transection and transplantation of olfactory ensheathing glia (OEG), with or without a Schwann cell (SC) bridge. Methods: Motor activity was encouraged through provision of motor enrichment housing (MEH); a multi-level cage containing ramps, textured surfaces and rewards. Hindlimb joint movement was assessed weekly for 22 weeks starting one week post-surgery, comparing rats housed in MEH to those in basic housing (BH). Transganglionic tracer was injected into the crushed right sciatic nerve three days prior to sacrifice, allowing sensory axons in the dorsal columns to be visualized by immunolabeling. Serotonergic axons and glial cells expressing low affinity nerve growth factor receptor were identified by immunolabeling. Results: All rats, having received transplants, recovered some hindlimb movement. Rats housed in BH progressively lost recovered hindlimb function whereas recovered hindlimb movements were sustained in most rats in MEH. In rats transplanted with SCs and OEG, effects of MEH were first significant 14 weeks after injury. In rats transplanted with OEG, a trend was seen from 14 weeks after injury, but this did not reach significance. In all rats, traced sensory axons died back from sites of transplantation and did not regenerate rostrally. Further, in no rat were serotonergic axons observed regenerating into, around or beyond transplants. Conclusions: Transection and transplantation of SC/OEG or OEG induced recovery of hindlimb function. This recovered hindlimb movement was sustained in rats housed in MEH but was progressively lost in rats housed in BH. Because benefits of MEH were not observed until 14 weeks after injury, long-term assessment of behavior is recommended. BH conditions are not conducive to maintenance of recovered hindlimb function, and MEH should be used in studies of recovery of function following spinal cord injury. [ABSTRACT FROM AUTHOR]