FIGURE 1.

Global ischemia induces progressive neuronal injury and Zn²⁺ accumulation in CA1 pyramidal neurons. Rats were subjected to TGI followed by resuscitation as described. After 1- or 4-hour recovery, rats were killed, perfused, and brain tissue removed for histological examination as described. Injury was assessed on hippocampal slices subjected to a modified acid fuchsin labeling procedure (VAF; detailed in Materials and Methods) and counterstained with toluidine blue; Zn²⁺ accumulation was assessed via Timm’s labeling (see Materials and Methods). (A) Global ischemia induced injury to hippocampal CA1 pyramidal neurons. Left: Representative images. Hippocampal slices were photographed at low magnification (Bar = 500 µm), and CA1 and CA3 regions (indicated by blue and red rectangles, respectively) examined at higher magnification (Bar = 50 µm). While most neurons appear intact in control slices, note the increased numbers of VAF-stained neurons as well as of severely swollen neurons (with vacuolar changes and loss of distinct nuclear outlines) after ischemia. Further note that these changes are more prevalent in CA1 than in CA3 and appear to increase from 1 to 4 hours after ischemia (particularly in CA1). Examples of intact (arrow) and damaged (VAF+, triangle; swelling and vacuolar changes, circled) neurons are marked. Right: Quantitative assessment. All discernible neurons were rated as intact or injured, and the percentages of CA1 and CA3 neurons determined to be injured within each hippocampal section were calculated. Note that the extent of neuronal injury was far greater in CA1 that in CA3, and that in CA1 it was significantly greater after 4-hour than 1-hour recovery. Bars represent mean ± SEM from 4 to 7 independent animals (comprising ≥1000 cells from CA1; ≥500 from CA3; with ≥3 sections counted for each animal; ** indicates p < 0.01 by one-way ANOVA with Tukey post hoc). (B) Transient loss of mossy fiber Zn²⁺ and progressive Zn²⁺ accumulation in CA1 pyramidal neurons after transient global ischemia. Photomicrographs show low magnification images of Timm’s-labeled hippocampal sections (Bar = 500 µm), and greater magnification images of the CA1 regions from the same sections (indicated by rectangles; Bar = 50 µm). Note the robust stain in the mossy fibers (indicative of presynaptic vesicular Zn²⁺) in control, and the substantial loss of this Zn²⁺ at 1-hour recovery. Further note the appearance of some Zn²⁺ in the somata of CA1 pyramidal neurons that progresses from 1 hour to 4 hours after ischemia (a small amount of accumulation was also noted in some CA3 pyramidal neurons, not shown; Images are representative of ≥3 repetitions each condition).