Figure 5.
Early CaEDTA acts downstream of caspase-3 cleavage to block caspase-3 activity, p75NTR induction, and TUNEL. Representative Western blots and relative abundance of procaspase-3 (a) and caspase-3 (b) in protein samples isolated from control (Contl) and experimental animals subjected to ischemia or CaEDTA (300 mm, i.c.v.; 30 min before ischemia), followed by ischemia and killed at 24 hr after reperfusion. Westerns were probed with an anti-caspase-3 antibody. Global ischemia induced an upregulation of procaspase-3 (a) and promoted its proteolytic processing to generate cleaved (activated) caspase-3 (b). CaEDTA did not significantly alter the ischemia-induced upregulation of procaspase-3 (a) or the appearance of caspase-3 (b). Mean band densities for procaspase-3 and cleaved (activated) caspase-3 in protein samples from the CA1 of experimental animals were normalized to the corresponding values for samples from control animals. Representative brain sections at the level of the dorsal hippocampus from animals subjected to sham operation (control; c, d), global ischemia (e, f), or CaEDTA (300 mm, i.c.v.; 30 min before ischemia), followed by ischemia and killed at 24 hr (g, h), labeled with FAM-DEVD-FMK, a fluorescein-tagged analog of zDEVD-FMK, a potent inhibitor of activated caspases. c, d, Caspase-3 activity was low in control brain. e, f, Global ischemia induced caspase-3 activity in the CA1 pyramidal cell layer, evident at 24 hr (g, h). i, Summary of data for animals subjected to sham operation, ischemia, or CaEDTA, followed by ischemia. Scale bars, 400 μm (low magnification); 50 μm (high magnification). Error bars represent SEM.j-u, Triple labeling of representative brain sections with the nuclear stain Hoechst 33342 (j, m, p, s), an anti-p75NTR antibody (k, n, q, t), and TUNEL (l, o, r, u) in the CA1 pyramidal layer of control (j, l) and experimental gerbils subjected to global ischemia (m-r) or to CaEDTA (300 mm, i.c.v.; 30 min before ischemia), followed by ischemia and killed at 72 hr (s-u). p is the super imposition of m and n.q is a superimposition of m and o. r is a superimposition of n and o. Indicated boxes are enlarged in p-r. p75NTR immunolabeling visualized in red (Texas Red), TUNEL in green (fluorescein), and Hoechst in blue. so, Stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Global ischemia induced p75NTR expression and TUNEL positivity in CA1 pyramidal neurons; CaEDTA pretreatment reduced p75NTR immunolabeling and TUNEL. Scale bars: (in r) p-r, 10 μm; (in u) j-o and s-u, 60 μm. Statistical significance in a, b, i was assessed by ANOVA, followed by Newman-Keuls test (*p < 0.05; **p < 0.01).