Neuroprotective action of acute estrogens: animal models of brain ischemia and clinical implications

Abstract

The ovarian hormone 17β-estradiol (E2) exerts profound neuroprotective actions against ischemia-induced brain damage in rodent models of global and focal ischemia. This review focuses on the neuroprotective efficacy of post-ischemic administration of E2 and non-feminizing estrogen analogs in the aging brain, with an emphasis on studies in animals subjected to a long-term loss of circulating E2. Clinical findings from the Women's Health Initiative study as well as data from animal studies that used long-term, physiological levels of E2 treatment are discussed in this context. We summarize major published findings that highlight the effective doses and timing of E2 treatment relative to onset of ischemia. We then discuss recent findings from our laboratory showing that under some conditions the aging hippocampus remains responsive to E2 and some neuroprotective non-feminizing estrogen analogs even after prolonged periods of hormone withdrawal. Possible membrane-initiated signaling mechanisms that may underlie the neuroprotective actions of acutely administered E2 are also discussed. Based on these findings, we suggest that post-ischemic treatment with high doses of E2 or certain non-feminizing estrogen analogs may have great therapeutic potential for treatment of brain damage and neurodegeneration associated with ischemia.

Figure 1. Neuroprotective effects of acute post-ischemic E2 treatment are time and dose dependent

Therapeutic time window for 100 μg/kg of E2 administered after ischemia and dose-response relationships at 6 h after MCAO treatment were examined in adult female rats. Two weeks after bilateral ovariectomy (OVX), rats in the experimental groups were subjected to MCAO. Panel A: Therapeutic time window for post-ischemic treatment with 100 μg/kg of E2. E2 was administered to OVX females at 0.5 (n=8), 1 (n=6), 2 (n=7), 3 (n=6), or 4 (n=9) h after MCAO. Rats in the control groups, OVX (n=12) and intact females (INT, n=6), received saline and empty pellets at the same time points following MCAO. Data are means ± SEM. *, p<0.05 versus OVX and INT. Data are adapted from Yang et al. (2003) [40] with permission. Panel B: Dose-response relationships for 6 h delay treatment. E2 was administered subcutaneously 6 h after MCAO at doses of 100 (n=9), 500 (n=10), 1000 (n=10), 2000 (n=9), or 5000 ug/kg (n=9). Data are means ± SEM. INT, intact rats; *, p <0.05 versus OVX and intact rats (INT). Data are adapted from Yang et al. (2003) [40] with permission.

Figure 2. E2, G1 and STX afford neuroprotection in middle-aged females 8 weeks after hormone deprivation

Middle-aged female rats (11–13 mo) were subjected to sham surgery or global ischemia (Isch) 8 weeks after OVX. Top panel: Representative photomicrographs of hippocampal neurons in the dorsal CA1 subfield 7 days after sham surgery or global ischemia in animals injected ICV immediately after ischemia with either E2 (2.25 μg), G1 (50 μg), STX (50 μg) or vehicle (Veh). A separate group of animals was injected sc with E2 (100 μg/kg). Scale bars: low magnification, 400 μm; higher magnification, 60 μm. Bottom panel: Surviving CA1 neurons 7days after ischemia were counted in 3 sectors (lateral, middle and medial, 250 μm × 250 μm each) of 4 sections of the dorsal hippocampus. Data are means ± SEM. *, p<0.001 versus sham; **, p<0.001 versus ischemia/vehicle. Data are adapted from Lebesque et al. (2010) [23] with permission.

Figure 3. E2 affords neuroprotection in aged females 6 months after hormone deprivation

Female rats aged 15–18 mo were subjected to sham surgery or global ischemia 6 months after OVX. Left panels: Representative photomicrographs of neurons in dorsal CA1 7 d after sham or ischemia (Isch) surgery in rats infused immediately upon reperfusion with vehicle (veh) or 2.25 μg of E2. Scale bars, 60 μm. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Right panel: Quantification of surviving CA1 pyramidal neurons was performed in 3 sectors (lateral, middle and medial, 250 μm × 250 μm each) of 4 sections of the dorsal hippocampus 7 d after ischemia. Because there were no significant differences in cell counts in vehicle (black circles) and E2 (gray circles) treated sham rats, data were combined and shown as a single sham group. Data are means ± SEM. ***, p<0.001 versus ischemia; **, p<0.01 versus ischemia/vehicle. Data are adapted from Inagaki et al. (2012) [17] with permission.

Figure 4. CA1 neurons in the aging hippocampus retain responsiveness to E2 and high frequency stimulation after a long period of hormone deprivation

Evoked field excitatory postsynaptic potentials (fEPSPs) were recorded in hippocampal slices from aged (15–18 mo old, 6 mo after OVX, white circles, n=7 slices from 6 rats) and young (2 mo old, 7–10 days after OVX, black circles, n=5 slices from 4 rats) females. Panel A left: E2 (1 nM) was bath applied after 15 min stable baseline. Panel A right: the averaged responses for 5 min baseline recording (last 5 min before E2 infusion), and the averaged responses during 25–30 min after the start of E2 application, respectively, were compared by ANOVA. Panel B left:High frequency stimulati on (HFS, 2 trains of 100 stimulations at 100 Hz) was applied in acute slices from young (n=7 slices from 6 rats) and aged (n=4 slices from 4 rats) OVX rats after 20 min baseline recording in the presence of E2. Panel B right: the averaged responses for 5 min baseline (last 5 min before LTP induction), and the averaged responses during 25–30 min after delivery of tetanus, respectively, were compared by ANOVA. Data are means ± SEM. *, p<0.05; **, p<0.01. Data are adapted from Inagaki et al. (2012) [17] with permission.

Figure 5. G1 mimics E2 potentiation of synaptic transmission in CA1

Changes in fEPSPs recorded in CA1 in the presence of E2 or G1 were examined in acute hippocampal slices prepared from OVX young adult female rats. Evoked fEPSPs were recorded in response to stimulation of the Schaffer collaterals. After a stable 15 min baseline was established, vehicle solution containing 0.01% DMSO was introduced. Fifteen min later, 10 nM E2 (A, n=9 slices from 6 rats) or 10 nM G1 (B, n=14 slices from 9 rats) was bath applied. Data are adapted from Lebesque et al. (2010) [23] with permission.

Figure 6. PI3K inhibitor LY294002 attenuates E2 neuroprotection

Young OVX rats were subjected to global ischemia (white and black bars) or sham surgery (grey bars). E2 (50 μg) or vehicle was administered immediately after ischemia. Some rats also received the PI3K inhibitor LY294002 or vehicle at 0 and 12 h after sham or ischemia surgery (n=3–12). Surviving CA1 neurons were counted 7 days later (a–p). SO, stratum oriens; SP, stratum pyramidale; Sr, stratum radiatum. ***, p <0.001 vs. all sham groups; ##, p<0.01 ischemia+estradiol vs. ischemia and ###, p<0.001 vs. ischemia+estradiol+LY294002. Data are means ± SEM and are adapted from Jover-Mengual et al. (2010) [19] with permission.