Fig. 1.

Characterization of organotypic hippocampal slice culture model. A, Anoxic vulnerability is maximal in 12 DIV or older cultures exposed to OGD, achieved by combining anoxia with aglycemia and 2DG. Cultures were exposed to 60 min of anoxia in the presence or absence of either d-glucose (11 mm) or 2DG (2 mm; n = 3 slices/group for 7–9 DIV, 5 slices/group for 12 DIV, 9 slices/group for 12 DIV + 2DG). Note that 60 min OGD in 12 DIV cultures caused PI fluorescence to rise to 35–40% of the maximum achieved by complete cell killing (neuronal and non-neuronal, see Materials and Methods). Sixty minutes OGD produced consistent near-total neuronal loss in the hippocampal cell layers by histological criteria (see Fig. 2). Inset, Time course of the decline in oxygen concentration after the onset of anoxia as measured with a Clarke oxygen electrode immersed in a culture well containing BSS. The electrode was calibrated at room air (20% oxygen) and in an anoxic atmosphere obtained by a 25 min flush with 95% nitrogen/5% CO₂ with analyzed oxygen content of 7 parts/million in the gas phase (nominally zero oxygen). O₂concentration at 3 min dropped to below 0.3%. B, PI fluorescence intensity is linearly related to the magnitude of neuronal loss (r = 0.95, p < 0.0001). Cultured 12 DIV hippocampal slices (n = 3) were exposed to OGD and fixed with 4% paraformaldehyde at 0, 5, and 24 hr after the insult. The number of pyknotic nuclei was counted in 16 high-power fields in the neuronal layers, and the counts were plotted against the PI fluorescence intensity obtained from the same fields immediately before fixation. C, PI fluorescence produced by 60 min OGD is of neuronal origin. Cultures were exposed to 500 μm NMDA (n = 8 slices) or to 500 μm each of NMDA and kainate (n = 8 slices) for 24 hr, or to either 1 or 3 hr of OGD (n = 7 and 9 slices, respectively) followed by a 24 hr incubation in BSS. PI fluorescence was normalized to that obtained from the same slices after complete cell killing. Both the NMDA and the 1 hr OGD insults produced only neuronal cell loss as gauged histologically (as in Fig. 3). The combined NMDA and kainate insult, as well as the 3 hr OGD insult, killed an additional cell population (p < 0.05, one-tailed Student’st test) that was resistant to 500 μm NMDA or 60 min of OGD, and was most likely glial, although a contribution from a small resistant neuronal population outside the neuronal layers cannot be excluded. PI fluorescence values obtained by OGD and the EAA insults were ∼40% of the maximum. Thus, the value of 40% of maximal fluorescence (horizontal dotted line) was selected in subsequent analyses as equating 100% neuronal death. D, Method of calculating cell death at a given time t from PI fluorescence images. Images of the cultures were taken before (i) and during (ii andiii) the 24 hr observation period and normalized to the maximal PI fluorescence values obtained with complete cell killing (iv). The formula shown was applied to the pooled fluorescence from the entire image or to fluorescence values derived from regions of interest (ROIs; solid lines iniv) encompassing the CA1,CA3, or dentate granule layer (DG) of the culture. ROIs were selected on the final PI image (iv) and then applied to the previous images in the same series (i–iii). Gray-level scale iniv applies to all images.