We have investigated the temporal and spatial profiles of apoptotic cells in an experimental transection spinal cord injury by the terminal deoxynucleotidyl transferase-mediated biotin-16-2'-deoxyuridine-5'-triphosphate nick-end labeling (TUNEL) method. Twenty-four hours postinjury, a numerous TUNEL-positive cells appeared both rostrally and caudally to the transection site. Those positive cells, however, gradually diminished in number by several days postinjury. In contrast, other TUNEL-positive cells were found scattered within the white matter remote from the lesion by the third day postinjury. These cells were typically embedded in or among vacuolated fibers, where they were identified in close proximity to the vacuolated space enclosed by myelin basic protein (MBP)-positive structures confirmed by TUNEL-MBP double staining. Because of their linear arrangement, these TUNEL-positive cells were considered interfascicular oligodendrocytes, a fact that was confirmed by the finding that some TUNEL-positive cells were also stained with CCI, a cell marker for oligodendrocyte. Electron microscopic studies revealed that the cells expressing apoptotic morphology were invariably encased in a space formed by myelin splitting. Although the biological significance of apoptotic interfascicular oligodendrocytes in the process of wallerian degeneration is yet to be determined, the finding of such profiles localized within degenerating myelin structures suggests that; oligodendrocytes may be "trapped" within rapidly swollen and disintegrating myelin lamellae, which isolates and perhaps predisposes them to death.

This study sought to experimentally clarify time-dependent, differential microglial activation at various spinal cord locations in response to injury. The spinal cords of Wistar rats were either sharply transected at the Th 11 or subjected to compression at the same site. Immediately to 4 weeks after injury, each spinal cord was fixed and cut into longitudinal frozen sections, and was immunostained with OX42 for resident and activated microglia, OX-6 for activated microglia, GFAP for activated astrocytes, and biotinylated BS-I, a lectin for both resident and activated microglia. From three to 24 hours after injury, we observed a narrow belt around the transection site in which OX42 positive microglia were dramatically reduced in number, or often absent. BS-I labeling of the zone disclosed the rapid transformation of those microglia possessing typical antler-like processes to macrophage-like cells. At day 1 and thereafter, the zone of reduced OX42 immunoreactivity was gradually replaced by macrophage-like OX42-positive round cells, and the lesion itself was ultimately capped by fibrogliotic scar tissue. By 2-4 weeks postinjury, another phase of microglial activation was observed in those white matter tracts undergoing Wallerian degeneration. These microglia characterized by the presence of newly-expressed MHC class II antigens. We posit that the decreased OX42 immunoreactivity suggests that CR3 is quickly saturated by activated iC3b and internalized, but not down-regulated. The trigger for this transformation most likely occurs through signaling by iC3b-saturated CR3. In contrast, microglia activation along those degenerating tracts undergoing Wallerian degeneration does not appear to be CR3-related, as the CR3 is upregulated. These observations indicate microglia have at least two different spatial and temporal patterns of activation. One is rapid and most likely involves the blood-borne complement activating system. The other accompanies Wallerian degeneration and is independent of the blood-borne complement system.

Glutamine synthetase (GS), localized to astrocyte is a key enzyme in the glutamate-glutamine pathway in the brain. 3-Nitropropionic acid (3-NPA) is an irreversible inhibitor of succinate dehydrogenase in the tricarboxylic-acid cycle, and provides ischemic tolerance to the brain. So far, there have been no reports on the relationship of astrocytic GS and ischemic tolerance by chemical preconditioning. In order to test the hypothesis that astrocytes serve a pivotal role in 3-NPA-induced chemical preconditioning, we have investigated the temporal profile of GS expression in astrocyte parallel with those of glial fibrillary acidic protein and heat-shock protein 70 (HSP70). In our rat model of permanent focal ischemia, preconditioning with 3-NPA singnificantly reduced the subsequent neurological deficits and infarct volume within 24-72 hours after treatment. Immunohistochemically, protoplasmic astrocytes in the cortex and striatum were activated in terms of upregulation of GS and more abundant protoplasmic processes with 3-NPA preconditioning, however, HSP70 expression could not be induced. Thus, the activation of astrocytes and upregulation of GS play an important role in 3-NPA-induced preconditioning but HSP70 does not. In view of glutamate being imposed on the cerebral ischemic damage, the astrocytic GS may contribute to 3-NPA-induced ischemic tolerance.

No abstract

Alterations of nodal and paranodal axolemma of the rat sciatic nerve were investigated in antigalactocerebroside serum-induced demyelination. A ferric ion-ferrocyanide (FeFCN) stain that appears to stain the regions with a high sodium channel density in nerve fibers was applied. When acute conduction block was initiated 20 to 180 minutes after the antiserum injection, myelin terminal loops began to be detached from the paranodal axolemma and reaction product of FeFCN stain originally localized at the nodes decreased in density and extended to the paranodal axolemma. By the time that complete conduction block was established, 5 hours after the injection, FeFCN stain was barely detectable around the nodal area. The loss of staining was associated with detachment and vesiculovacuolar degeneration of the paranodal myelin. This rapid deterioration and disappearance of normal cytochemical characteristics of the axolemma in the presence of only modest paranodal demyelination could be a morphological correlate of the loss of excitability of the axon membrane.