Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat.

A number of studies have provided evidence that neuronal cell loss after stroke involves programmed cell death or apoptosis. In particular, recent biochemical and immunohistochemical studies have demonstrated the expression and activation of intracellular proteases, notably caspase-3, which act as both initiators and executors of the apoptotic process. To further elucidate the involvement of caspases in neuronal cell death induced by focal stroke we developed a panel of antibodies and investigated the spatial and temporal pattern of both caspase-8 and caspase-3 expression. Our efforts focused on caspase-8 because its "apical" position within the enzymatic cascade of caspases makes it a potentially important therapeutic target. Constitutive expression of procaspase-8 was detectable in most cortical neurons, and proteolytic processing yielding the active form of caspase-8 was found as early as 6 hr after focal stroke induced in rats by permanent middle cerebral artery occlusion. This active form of caspase-8 was predominantly seen in the large pyramidal neurons of lamina V. Active caspase-3 was evident only in neurons located within lamina II/III starting at 24 hr after injury and in microglia throughout the core infarct at all times examined. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, gel electrophoresis of DNA, and neuronal cell quantitation indicated that there was an early nonapoptotic loss of cortical neurons followed by a progressive elimination of neurons with features of apoptosis. These data indicate that the pattern of caspase expression occurring during delayed neuronal cell death after focal stroke will vary depending on the neuronal phenotype.

Osteopontin and its integrin receptor alpha(v)beta3 are upregulated during formation of the glial scar after focal stroke.

BACKGROUND AND PURPOSE: Microglia and astrocytes in the peri-infarct region are activated in response to focal stroke. A critical function of activated glia is formation of a protective barrier that ultimately forms a new glial-limiting membrane. Osteopontin, a provisional matrix protein expressed during wound healing, is induced after focal stroke. The present study was performed to determine the spatial and temporal expression of osteopontin and its integrin receptor alpha(v)beta3 during formation of the peri-infarct gliotic barrier and subsequent formation of a new glial-limiting membrane. METHODS: Spontaneously hypertensive rats (n = 19) were subjected to permanent occlusion of the middle cerebral artery and killed 3, 6, and 24 hours and 2, 5, and 15 days after occlusion. The spatial and temporal expression of osteopontin mRNA was determined by in situ hybridization, and that of osteopontin ligand and its integrin receptor alpha(v)beta3 was determined by immunohistochemistry. RESULTS: Osteopontin mRNA was expressed de novo in the peri-infarct region from 3 to 48 hours; by 5 days osteopontin mRNA expression was restricted to the infarct. Osteopontin protein was expressed by peri-infarct microglia beginning at 24 hours and by microglia/macrophages at 48 hours in the infarct. Integrin receptor alpha(v)beta3 was expressed in peri-infarct astrocytes at 5 and 15 days. CONCLUSIONS: Early microglial/macrophage expression of osteopontin mRNA defines the borders and final infarct area at 24 hours. At 5 days osteopontin ligand is at a distance from the peri-infarct astrocytes expressing integrin receptor alpha(v)beta3. By 15 days astrocytes expressing integrin receptor alpha(v)beta3 are localized in an osteopontin-rich region concomitant with formation of the new glial-limiting membrane. The de novo expression and interaction of osteopontin ligand with its receptor integrin alpha(v)beta3 suggest a role in wound healing after focal stroke.

The trkC receptor is transiently localized to Purkinje cell dendrites during outgrowth and maturation in the rat.

In vivo studies of granule cell gene expression during corticocerebellar development and in vitro studies of Purkinje cell neurite outgrowth suggest that neurotrophin-3 may influence growth of Purkinje cell dendrites. To determine whether neurotrophic substances affect the growth of specific neuronal processes (i.e. axons and dendrites) or nonspecifically cause process development by exerting a trophic influence upon neuronal physiology we performed an immunohistochemical examination of trkC protein expression during early postnatal development of the rat cerebellum. Our findings indicate that Purkinje cells begin to synthesize trkC protein coincident with the onset of dendritic outgrowth. Robust immunostaining was evident throughout the entire somatodendritic domain of Purkinje cells during dendritic development but became faint and restricted to the cell body subsequent to the completion of dendritogenesis. These results suggest that growth and maturation of the Purkinje cell dendritic arbor may be influenced by neurotrophin-3 activation of trkC receptors distributed within developing dendrites.