An accurate, reproducible method for determining the infarct volumes of gray matter structures is presented for use with presently available image analysis systems. Areas of stained sections with optical densities above that of a threshold value are automatically recognized and measured. This eliminates the potential error and bias inherent in manually delineating infarcted regions. Moreover, the volume of surviving normal gray matter is determined rather than that of the infarct. This approach minimizes the error that is introduced by edema, which distorts and enlarges the infarcted tissue and surrounding white matter.

Poly(ADP-ribose)-1 (PARP-1) is a key mediator of cell death in excitotoxicity, ischemia, and oxidative stress. PARP-1 activation leads to cytosolic NAD⁺depletion and mitochondrial release of apoptosis-inducing factor (AIF), but the causal relationships between these two events have been difficult to resolve. Here, we examined this issue by using extracellular NAD⁺to restore neuronal NAD⁺levels after PARP-1 activation. Exogenous NAD⁺was found to enter neurons through P2X₇-gated channels. Restoration of cytosolic NAD⁺by this means prevented the glycolytic inhibition, mitochondrial failure, AIF translocation, and neuron death that otherwise results from extensive PARP-1 activation. Bypassing the glycolytic inhibition with the metabolic substrates pyruvate, acetoacetate, or hydroxybutyrate also prevented mitochondrial failure and neuron death. Conversely, depletion of cytosolic NAD⁺with NAD⁺glycohydrolase produced a block in glycolysis inhibition, mitochondrial depolarization, AIF translocation, and neuron death, independent of PARP-1 activation. These results establish NAD⁺depletion as a causal event in PARP-1-mediated cell death and place NAD⁺depletion and glycolytic failure upstream of mitochondrial AIF release.

Glutamate excitotoxicity,oxidative stress,and acidosis are primary mediators of neuronal death during ischemia and reperfusion.Astrocytes influence these processes in several ways. Glutamate uptake by astrocytes normally prevents excitotoxic glutamate elevations in brain extracellular space,and this process appears to be a critical determinant of neuronal survival in the ischemic penumbra.Conversely,glutamate efflux from astrocytes by reversal of glutamate uptake, volume sensitive organic ion channels,and other routes may contribute to extracellular glutamate elevations.Glutamate activation of neuronal N-methyl-D-aspartate (NMDA)receptors is modulated by glycine and D-serine:both of these neuromodulators are transported by astrocytes,and D-serine production is localized exclusively to astrocytes.Astrocytes influence neuronal antioxidant status through release of ascorbate and uptake of its oxidized form,dehydroascorbate,and by indirectly supporting neuronal glutathione metabolism.In addition,glutathione in astrocytes can serve as a sink for nitric oxide and thereby reduce neuronal oxidant stress during ischemia.Astrocytes probably also influence neuronal survival in the post-ischemic period.Reactive astrocytes secrete nitric oxide, TNF α ,matrix metalloproteinases,and other factors that can contribute to delayed neuronal death,and facilitate brain edema via aquaporin-4 channels localized to the astrocyte endfoot-endothelial interface.On the other hand erythropoietin,a paracrine messenger in brain,is produced by astrocytes and upregulated after ischemia.Erythropoietin stimulates the Janus kinase-2 (JAK-2)and nuclear factor-kappaB (NF-kB)signaling pathways in neurons to prevent programmed cell death after ischemic or excitotoxic stress.Astrocytes also secrete several angiogenic and neurotrophic factors that are important for vascular and neuronal regeneration after stroke.