Fig. 2.

OGD causes complete neurodegeneration of all neuronal layers in organotypic hippocampal slices, whereas glucose deprivation alone does not. Cultures were maintained in BSS containing 2 μg/ml PI and exposed to anoxia/aglycemia + 2 mm 2DG (B, D, F) or to aglycemia + 2 mm 2DG without anoxia (A, C, E). After 24 hr, PI fluorescence was digitally imaged in the slices (A, B), after which they were fixed in 4% paraformaldehyde and stained with toluidine blue/acid fuchsin (C–F). No significant PI staining was observed in slices challenged with glucose deprivation alone (A), whereas OGD produced a significant increase in PI fluorescence in all neuronal layers (B). Similarly, glucose-deprived cultures exhibited normal neuronal morphology, whereas OGD-challenged slices sustained widespread neuronal loss at both low (4× objective; C, D) and high (40× objective; E, F) magnification. Thus, anoxic damage is reflected by intense PI staining (B), loss of neuronal cell layers at low magnification (D), and the replacement of neuronal outlines with pyknotic nuclei at higher magnification (F). A andB are digital, 8-bit/pixel pseudocolor images of PI fluorescence intensity, with purple andred representing low and high intensities, respectively (color bar). C–F are true-color images of the slices in A and B after staining with toluidine blue/acid fuchsin. The pale bluebackground in C and D is produced by toluidine blue staining of the membrane, on which the slices are cultured. Scale bars: 500 μm in A–D (shown inA); 75 μm in E and F(shown in E).