Glial fibrillary acidic protein immunohistochemistry of spinal cord astrocytes after induction of ischemia or anoxia in culture

The effects of ischemia (removal of oxygen and glucose for 4 h) and anoxia (removal of oxygen alone) on astrocytes were studied in dissociated cultures of E14 spinal cord containing both neurons and astrocytes. In addition, a group of cultures was treated with a low Na⁺, low Ca²⁺, and high K⁺ medium during the 4‐h ischemic period (ischemia‐protected group), a process that protects neurons from ischemic damage under identical conditions. Astrocytes were examined immunohistochemically using glial fibrillary acidic protein (GFAP) antiserum 24 h after insult. Densitometry and statistical analysis (1‐way analysis of variance [ANOVA], a priori; 2‐tailed Tukey‐t, a posteriori) of the digitized images of the somata and processes of astrocytes in the anti‐GFAP reacted cultures showed significant differences between the groups; a significant increase (P<0.01) in the GFAP‐positive reaction in the somata of ischemic astrocytes and a significant decrease (P<0.01) in the GFAP‐positive reaction in the processes of ischemic, ischemia‐protected, and anoxic astrocytes. There were no significant differences in the GFAP immunoreactivity of somata between control, ischemia‐protected, and anoxic astrocytes or of processes from ischemic, ischemia‐protected, and anoxic astrocytes. These data show that following ischemia cultured astrocytes increase somatic GFAP immunoreactivity compared to all other groups tested whereas the staining intensity for GFAP was decreased in the processes of all three experimental groups compared to controls. Ischemia protection resulted in the absence of the enhancement of somatic GFAP immunoreactivity. The relationship of the astrocytic response and the type of cellular stress is discussed.