Astrocytic demise precedes delayed neuronal death in focal ischemic rat brain

Active neuronal-glial interaction is important in the maintenance of brain homeostasis and is vital for neuronal survival following brain injury. The time course of post-ischemic astroglial dysfunction and neuronal death was studied in the spontaneously hypertensive rat (SHR) brain following permanent middle cerebral artery occlusion (MCAO). In situ hybridization with ³⁵S-labeled riboprobes for GFAP and GLUT3 was used to monitor mRNA expression in glia and neurons. Astrocytic proteins GFAP, vimentin, S100, Glutathione-S-Transferase Y(b) (GST Y(b)) and neuronal protein TG2 were detected by immunofluorescence. Cells were co-stained with in situ end labeling (ISEL) to detect DNA fragmentation, a hallmark of cell death. GFAP mRNA expression declined rapidly in the ischemic region of the cortex and was almost absent by 12 h. Immunohistochemical studies revealed a parallel decline in the corresponding protein: a reduction in GFAP staining was apparent in the infarct after 3 h and by 24 h, there was essentially no remaining GFAP. Three other glial proteins (vimentin, S100 and GST Y(b)) disappeared from infarct over a similar time course. A few ISEL positive cells were observed in the infarct at 6 h, but maximal detection was not seen until 24-48 h. Most of the ISEL-positive cells were neurons, identified by co-staining with the neuronal marker TG2. Few cells expressing GFAP or other glial markers were positive at any time point. Neuronal GLUT3 mRNA declined more slowly than GFAP mRNA in the ischemic core and disappeared during the period of neuronal death. Concurrent with the loss of GFAP mRNA and protein expression in the infarct, there was a rapid rise in GFAP mRNA in the peri- infarct region of ipsilateral hemisphere and proximal region of the contralateral hemisphere. This was followed by the enhanced GFAP protein expression characteristic of reactive astrocytes, but over a significantly slower time course. These studies show that MCAO leads to a rapid decline of GFAP mRNA and glial proteins, which appears to precede the decline in neuronal mRNA and neuronal death within the infarct. Early astroglial dysfunction may play a critical role in determining the outcome of acute hypoxic-ischemic injury by compromising neuronal-glial interactions.