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Mechanical spinal cord injury (SCI) initiates a cascade of pathochemical and pathophysiological events, collectively known as the secondary injury. There has been a long-standing interest in understanding the activation and involvement of proteases in this secondary injury process. Several proteases including the calpains, caspases and matrix metalloproteinases are activated by perturbations to the spinal cord and have been linked to cell death following SCI and in other models of CNS disease and insult. Cathepsin B (Cath B), a potent lysosomal protease, has also been implicated in the pathology of CNS diseases including brain tumors, Alzheimer's disease, amyotrophic lateral sclerosis and stroke. Previously, we reported significant increases in Cath B mRNA and protein expression following contusion-SCI. This characterization of Cath B continues with the experiments reported herein, which were designed to examine Cath B enzymatic activity and cellular localization following contusion-SCI in the rat. Cath B enzymatic activity was significantly increased in the injury epicenter at 5 and 7 days post-injury and was highly correlated with increases in the active forms of the Cath B protein reported earlier. Furthermore, the immunohistochemical analyses revealed that the post-injury increases in expression and enzymatic activity at the injury epicenter were due to the presence of a large and diverse population of inflammatory cells. However, in areas adjacent to the injury epicenter, it appears that parenchymal neurons may also contribute to these increases. Our findings coupled with the documented role of Cath B in other CNS pathologies make this potent protease an attractive candidate for involvement in the tissue destruction associated with the secondary injury cascade following SCI.

Aggrecan is a chondroitin sulfate (CS)/keratan sulfate (KS)-substituted proteoglycan (PG) abundant in cartilage which is also present within the mammalian embryonic, adult, and injured adult central nervous system (CNS). Although its role within the CNS is not clear, cell culture studies show that when substituted with CS, aggrecan inhibits neurite extension. To better understand the inhibitory effect of aggrecan on injured adult axons in vivo, we developed a model to independently test intact aggrecan and CS-depleted aggrecan core glycoprotein. Acute rat spinal cord hemisection cavities were filled with a growth-promoting matrix, Matrigel, and severed dorsal rootlets were placed into this matrix. This created an assay in which axons readily grew. The extent of ingrowth in this baseline assay was compared to the ingrowth in Matrigel loaded with intact aggrecan or the purified core glycoprotein of aggrecan. Our results show that both intact aggrecan and equivalent concentrations of the core glycoprotein component significantly inhibit axonal growth in this model system. These results confirm that aggrecan can inhibit the growth of adult axons in vivo and suggest that the inhibitory effects of aggrecan may be mediated, at least in part, by structures located on the core glycoprotein in the absence of the bulk of the CS chains.

Extrinsic factors appear to contribute to the lack of regeneration in the injured adult spinal cord. It is likely that these extrinsic factors include a group of putative growth inhibitory molecules known as chondroitin sulfate proteoglycans (CSPGs). The aims of this study were to determine: (1) the consequences of spinal cord contusion injury on CSPG expression, (2) if CSPGs can be degraded in vivo by exogenous enzyme application, and (3) the effects of intraspinal transplantation on the expression of CSPGs. Chondroitin 6-sulfate proteoglycan immunoreactivity (CSPG-IR) dramatically increased following spinal cord contusion injury both at and adjacent to the injury site compared to normal controls (no surgical procedure) and laminectomy-only controls by 4 days postinjury. The dramatic increase in CSPG-IR persisted around the lesion and in the dorsal one-half to two-thirds of the spinal cord for at least 40 days postinjury. Glial fibrillary acidic protein (GFAP)-IR patterns were similarly intensified and spatially restricted as CSPG-IR patterns. These results suggest that: (1) CSPGs may contribute to the lack of regeneration following spinal cord injury and (2) astrocytes may contribute to the production of CSPGs. In addition, our results show that CSPGs could be cleaved in vivo with exogenous chondroitinase ABC application. This demonstration of cleavage may the basis for a model to directly assess CSPGs' role in growth inhibition in vivo (studies in progress) and hold potential as a therapeutic approach to enhance growth. Interestingly, the robust, injury-induced CSPG-IR patterns were not altered by intraspinal grafts of fetal spinal cord. The CSPG expression profile in the host spinal cord was similar to time-matched contusion-only animals. This was also true of GFAP-IR patterns. Furthermore, the fetal spinal cord tissue, which was generally CSPG negative at the time of transplantation, developed robust CSPG expression by 30 days posttransplantation. This increase in CSPG expression in the graft was paired with a moderate increase in GFAP-IR. CSPG-IR patterns suggest that these molecules may contribute to the limited regeneration seen following intraspinal transplantation. In addition, it suggests that the growth permissiveness of the graft may change overtime as CSPG expression develops within the graft. These correlations in the injured and transplanted spinal cord support CSPGs' putative growth inhibitory effect in the adult spinal cord.

The cell death suppressors bcl-2 and bcl-x are developmentally regulated and may modulate physiologic cell death in the central nervous system (CNS). However, little data are currently available on the expression patterns of these polypeptides in the human CNS. We examined the ontogeny of bcl-2 and bcl-x in 12 human spinal cords of gestational ages (GA) between 5 and 39 weeks and in 3 adult cords. Paraffin sections were probed by immunohistochemistry using well-characterized, commercially available antibodies that had been raised against poorly conserved epitopes of these homologous proteins. Between 5 and 10 weeks GA, bcl-2 immunoreactivity was identified in primitive neuroepithelial cells of the ventricular zone. Individual cells of the mantle zone were stained including clusters of early anterior horn cells. Bcl-x immunoreactivity was most prominent in differentiating neurons of the mantle zone and less pronounced in the ventricular zone. Between 10 and 14 weeks GA, bcl-2 staining was observed in cells lining the central canal, neurons of the dorsal horn (especially laminae I and II), and in anterior horn cells. The latter exhibited a range of staining intensities from moderate to nondetectable. Bcl-2 immunoreactivity became markedly reduced between 15 and 25 weeks GA, persisting only in ependymal cells. In contrast, strong bcl-x staining was observed in most neurons throughout development and into adulthood. The period of apparent bcl-2 down-regulation overlaps with a peak in physiologic motoneuron death and the establishment of functional neuromuscular synapses in the human spinal cord. These findings suggest that bcl-2 and bcl-x may both be required for survival of early postmitotic neurons before appropriate synaptic connections have been established. Continued neuronal survival (after bcl-2 is down-regulated) may require persistent bcl-x expression in addition to target-derived neurotrophic factors made available through the formation of appropriate synapses.

The present study evaluated the growth potential and differentiation of human fetal spinal cord (FSC) tissue in the injured adult rat spinal cord under different lesion and grafting conditions. Donor tissue at 6-9 weeks of gestational age was obtained through elective abortions and transplanted either immediately into acute resection (solid grafts) or into chronic contusion (suspension and solid grafts) lesions (i.e., 14-40 days after injury) in the thoracic spinal cord. The xenografts were then examined either histologically in plastic sections or immunocytochemically 1-3 months postgrafting. Intraspinal grafts in acute lesions demonstrated an 83% survival rate and developed as well-circumscribed nodules that were predominantly composed of immature astrocytes. Solid-piece grafts in chronic contusion lesions exhibited a 92% survival rate and also developed as nodular masses. These grafts, however, contained many immature neurons 2 months postgrafting. Suspension grafts in chronic contusion lesions had an 85% survival rate and expanded in a nonrestrictive, diffuse pattern. These transplants demonstrated large neuronally rich areas of neural parenchyma. Extensive neuritic outgrowth could also be seen extending from these grafts into the surrounding host spinal cord. These findings show that human FSC tissue reliably survives and differentiates in both acute and chronic lesions. However, both the lesion environment and the grafting techniques can greatly influence the pattern of differentiation and degree of host-graft integration achieved., (Copyright 1997 Academic Press.)

Contusion injuries of the rat thoracic spinal cord were made using a standardized device developed for the Multicenter Animal Spinal Cord Injury Study (MASCIS). Lesions of different severity were studied for signs of endogenous repair at times up to 6 weeks following injury. Contusion injuries produced a typical picture of secondary damage resulting in the destruction of the cord center and the chronic sparing of a peripheral rim of fibers which varied in amount depending upon the injury magnitude. It was noted that the cavities often developed a dense cellular matrix that became partially filled with nerve fibers and associated Schwann cells. The amount of fiber and Schwann cell ingrowth was inversely related to the severity of injury and amount of peripheral fiber sparing. The source of the ingrowing fibers was not determined, but many of them clearly originated in the dorsal roots. In addition to signs of regeneration, we noted evidence for the proliferation of cells located in the ependymal zone surrounding the central canal at early times following contusion injuries. These cells may contribute to the development of cellular trabeculae that provide a scaffolding within the lesion cavity that provides the substrates for cellular infiltration and regeneration of axons. Together, these observations suggest that the endogenous reparative response to spinal contusion injury is substantial. Understanding the regulation and restrictions on the repair processes might lead to better ways in which to encourage spontaneous recovery after CNS injury., (Copyright 1997 Academic Press.)

Although previous work has demonstrated the usefulness of magnetic resonance imaging (MRI) for visualizing intraspinal transplants in vivo, the degree to which MRI can differentiate viable fetal neural tissue from evolving spinal cord pathology has not been investigated. Thus, the present study assessed whether MRI performed at earlier postgrafting intervals (0-20 weeks) could document the survival of fetal neural transplants in the injured cat spinal cord. Twelve adult female cats received a hemisection injury at the L1 level, followed immediately by implantation of either embryonic cat spinal cord or neocortex into the cavity. The spinal cords of three control animals were hemisected but received no transplant. Each animal was subsequently imaged at 4 and 8 weeks postoperative. Selected animals from each group were also studied at additional time points ranging from immediately postoperative to 20 weeks. Multislice T2-weighted and intermediate T1-weighted spin-echo images of the lesion or graft site were obtained. Correlative postmortem histological analyses revealed viable donor tissue in 6 of 12 transplant recipients. Spinal cords from the remaining hosts and the control animals all contained cysts at the surgical site that were devoid of donor neural tissue. The graft sites with viable tissue tended to exhibit a slightly hyperintense signal on both intermediate T1-weighted (T1WI) and T2-weighted images (T2WI) throughout the entire experiment. Control cats and cats with failed transplants also were slightly bright on T1WI, but were very hyperintense on T2WI.(ABSTRACT TRUNCATED AT 250 WORDS)

This article reviews recent findings concerning the feasibility, basic neurobiology, and potential functional benefits of fetal CNS tissue grafts into acute and chronic lesions of the adult spinal cord. In the rat, neuro-anatomical observations suggest that transplants into resection cavities establish neuritic projections that could functionally reunite separated rostral and caudal segments of the host spinal cord. Furthermore, some complementary electrophysiological evidence has been obtained for synaptic connectivity between host and graft neurons. In these studies, extracellular single-unit activity was evoked in fetal spinal cord (FSC) transplants by stimulating host dorsal roots that had been juxtaposed to donor tissue at the time of transplantation. In other investigations, we examined whether grafts could also establish axonal projections to appropriate areas of gray matter in the chronically injured spinal cord. For this purpose, fetal serotoninergic (5-HT) neurons were injected caudal to complete spinal cord transections that had been made 1-3 months earlier. Immunocytochemistry revealed that these cells projected their axons into gray matter regions normally innervated by bulbospinal 5-HT neurons. To investigate transplantation in a more clinically relevant lesion model, a third group of experiments involved injection of dissociated cell suspensions into acute [less than 24 h postinjury (p.i.)]), subchronic (7-10 days p.i), and chronic (greater than or equal to one month, p.i.) contusion lesions. Such grafts routinely filled areas that otherwise would have been regions of cavitation extending rostral-caudal distances of approximately 7 mm. FSC transplants in such injuries also appeared to influence some aspects of motoneuron excitability and hindlimb locomotion. More recent studies of the cat spinal cord have extended these findings in the rat by showing long-term survival (greater than 2 years) of fetal CNS allografts in recipients with either subtotal transection or compression lesions. Preliminary studies of connectivity have also shown host-graft projection patterns similar to those seen in the rat. Behavioral analyses are currently underway to examine the effects of fetal grafts in cats with chronic postcompression lesions. These observations in the rat and cat are discussed in the general context of basic biological and clinical issues relevant to the long-term objective of promoting functional improvement in the damaged spinal cord.