Estrogen protects against global ischemia-induced neuronal death and prevents activation of apoptotic signaling cascades in the hippocampal CA1

Abstract

The importance of postmenopausal estrogen replacement therapy in affording protection against the selective and delayed neuronal death associated with cardiac arrest or cardiac surgery in women remains controversial. Here we report that exogenous estrogen at levels that are physiological for hormone replacement in postmenopausal women affords protection against global ischemia-induced neuronal death and prevents activation of apoptotic signaling cascades in the hippocampal CA1 of male gerbils. Global ischemia induced a marked increase in activated caspase-3 in CA1, evident at 6 hr after ischemia. Global ischemia induced a marked upregulation of the proapoptotic neurotrophin receptor p75(NTR) in CA1, evident at 48 hr. p75(NTR) expression was induced primarily in terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling-positive cells, indicating expression in neurons undergoing apoptosis. Global ischemia also induced a marked downregulation of mRNA encoding the AMPA receptor GluR2 subunit in CA1. Caspase-3, p75(NTR), and GluR2 were not significantly changed in CA3 and dentate gyrus, indicating that the ischemia-induced changes in gene expression were cell specific. Exogenous estrogen attenuated the ischemia-induced increases in activated caspase-3 and blocked the increase in p75(NTR) in post-ischemic CA1 neurons but did not prevent ischemia-induced downregulation of GluR2. These findings demonstrate that long-term estrogen at physiological levels ameliorates ischemia-induced hippocampal injury and indicate that estrogen intervenes at the level of apoptotic signaling cascades to prevent onset of death in neurons otherwise "destined to die."

Fig. 1.

Estrogen pretreatment affords neuroprotection against global ischemia-induced neuronal death in male gerbils. Toluidine blue staining of coronal brain sections at the level of the dorsal hippocampus at 7 d after reperfusion from control (n = 8; A, B) and experimental male gerbils subjected to global ischemia (BCCO, 5 min;n = 9; C, D) or to estradiol (0.36 mg pellet, s.c.; 60 d controlled time release), followed by global ischemia (BCCO, 5 min; n = 12;E, F). Global ischemia induced significant cell loss in the CA1 pyramidal cell layer; little or no cell loss was apparent in CA3 or dentate gyrus (C,D). Estradiol treatment for 14 d afforded nearly complete neuroprotection against ischemia-induced damage (E, F). Hippocampal injury was assessed quantitatively by the grading scale of Pulsinelli and Brierley: 0, no neurons damaged; 1, a few (<30%) neurons damaged; 2, many (30–70%) neurons damaged; and 3, the majority (>70%) of neurons damaged (Pulsinelli et al., 1982). Neuronal damage scores from a minimum of four microscopic sections per animal were analyzed; comparisons among group means were made using an ANOVA, followed by Newman–Keuls test and plotted as scatter graphs (G). Neuronal damage scores from two estrogen-treated gerbils with low plasma estradiol levels, indicating likely loss of the implanted pellet, are shown asdiamonds. Global ischemia was induced in adult male gerbils by BCCO as described in Materials and Methods. Scale bar: lower magnification, 400 μm; higher magnification, 40 μm.DG, Dentate gyrus; so,stratum oriens;sp,stratum pyramidale; sr, stratum radiatum.

Fig. 2.

Estrogen attenuates ischemia-induced increase of activated caspase-3. Activated caspase-3 immunolabeling in the CA1 pyramidal cell layer in sections of brain from control (sham-operated) (A) and experimental animals subjected to global ischemia (B–E); male, adult Mongolian gerbils were implanted with pellets containing placebo (B,D) or 17β-estradiol (E) 14 d before induction of global ischemia by BCCO (5 min). Data are typical of three animals per treatment group. Global ischemia induced a marked increase in activated caspase-3 (assessed by immunolabeling with an antibody to activated caspase-3 p20), evident at 6, 12, and 24 hr primarily in the hippocampal CA1 pyramidal cell layer (B). Estradiol pretreatment markedly reduced the ischemia-induced increase in activated caspase-3 in the CA1 pyramidal layer at 24 hr (E). so, Stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bar, 40 μm.

Fig. 3.

Estrogen attenuates ischemia-induced upregulation of p75^NTR protein expression in CA1. p75^NTR immunolabeling in the CA1 pyramidal cell layer in brain sections from control (A,B) and experimental animals at 48 hr (E,F), 72 hr (G,H), and 7 d (I,J) after global ischemia (BCCO, 5 min). Male, adult gerbils were implanted with pellets containing placebo (A, B, E–J) or 17β-estradiol 14 d before sham operation (C,D) or global ischemia (K–P). Data are typical of a minimum of three animals per time point and treatment group. Estradiol attenuated the ischemia-induced upregulation of p75^NTR in the CA1 pyramidal layer. p75^NTR immunolabeling was not altered in the CA3 pyramidal or dentate gyrus granule cell layer. so, Stratum oriens; sp, stratum pyramidale;sr, stratum radiatum. Scale bars: lower magnification, 400 μm; higher magnification, 40 μm.

Fig. 4.

p75^NTR colocalizes with GAD and β-tubulin in post-ischemic brain. Immunolabeling and merged images of p75^NTR and GAD (C, H,M) or β-tubulin (E,J, O) in the CA1 pyramidal cell layer in brain sections from control (A–E) and experimental animals at 72 hr (F–J) and 7 d (K–O) after global ischemia. Data are typical of three control and three ischemic animals at each time point. At 72 hr, p75^NTR immunolabeling was punctate over cell bodies within the pyramidal cell layer, suggestive of synaptic sites (G), and colocalized with GAD-positive (H) and a subpopulation of β-tubulin-positive (J) neurons. At 7 d, punctate p75^NTR immunolabeling was reduced in GAD-positive (M) and β-tubulin-positive (O) cells; increased diffuse labeling was observed over the pyramidal cell layer, which was primarily devoid of neurons (L). p75^NTRimmunolabeling was visualized in green and GAD or β-tubulin in red. so, Stratum oriens;sp, stratum pyramidale; sr, stratum radiatum. Scale bar, 40 μm.

Fig. 5.

Estrogen attenuates ischemia-induced increase in p75^NTR expression in CA1 neurons undergoing apoptosis. Triple-labeling of p75^NTR(B, E, H,K), TUNEL (C, F,I, L), and Hoechst 33342 (A, D, G,J) in the CA1 pyramidal cell layer in sections of control (A–C) and experimental male gerbils pretreated with estradiol (J–L) or placebo (D–I), followed by global ischemia.G is the merge of an inset ofD and E at high magnification,H is the merge of an inset ofE and F at high magnification, andI is the merge of an inset ofD and F at high magnification. p75^NTR immunolabeling was visualized inred (Texas Red), TUNEL reaction in green(fluorescein), and Hoechst-stained nuclei in blue. A high coincidence of p75^NTR immunolabeling and TUNEL was observed. so, Stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bars:first, second, and fourth rows, 40 μm; third row, 10 μm.

Fig. 6.

Estrogen attenuates ischemia-induced increase in p75^NTR abundance in the hippocampal CA1. Film autoradiograms of representative Western blots probed with an antibody to p75^NTR (top) and mean band densities (bottom) for protein samples extracted from the microdissected hippocampal CA1 of placebo- and estradiol-pretreated male gerbils subjected to sham operation (control) or at 24, 48, and 72 hr after global ischemia. Band densities are normalized to that of control gerbils. Data are means of four control and four experimental animals at each time point. Global ischemia induced an upregulation in p75^NTR protein abundance at 48 hr (p < 0.05) and 72 hr (p < 0.01) relative to control. Estrogen pretreatment attenuated ischemia-induced upregulation of p75^NTR protein levels in the CA1 pyramidal layer.

Fig. 7.

Estradiol does not attenuate ischemia-induced decrease in GluR2 mRNA expression. Representative in situ hybridization for GluR2 mRNA expression (top) and mean optical density values (bottom) for the CA1 pyramidal cell layer of control (placebo-treated, sham-operated) animals (far left) and experimental animals subjected to global ischemia (middle left), estradiol treatment, followed by global ischemia (middle right), or to estradiol treatment, followed by sham operation (far right) at 72 hr after reperfusion. Mean optical density values for experimental animals were normalized to the mean value for control gerbils. The relative abundance of GluR2 mRNA did not differ significantly in placebo-treated (decline to 49.46 ± 12.17% of control) versus estradiol-treated (decline to 35.93 ± 8.43% of control) ischemic animals at 72 hr. Estradiol did not detectably alter GluR2 mRNA expression in CA1 pyramidal neurons of control animals. n.s., Not significant.