Acute administration of non-classical estrogen receptor agonists attenuates ischemia-induced hippocampal neuron loss in middle-aged female rats

Abstract

Background: Pretreatment with 17beta-estradiol (E2) is profoundly neuroprotective in young animals subjected to focal and global ischemia. However, whether E2 retains its neuroprotective efficacy in aging animals, especially when administered after brain insult, is largely unknown.

Methodology/principal findings: We examined the neuroprotective effects of E2 and two agonists that bind to non-classical estrogen receptors, G1 and STX, when administered after ischemia in middle-aged rats after prolonged ovarian hormone withdrawal. Eight weeks after ovariectomy, middle-aged female rats underwent 10 minutes of global ischemia by four vessel occlusion. Immediately after reperfusion, animals received a single infusion of either E2 (2.25 microg), G1 (50 microg) or STX (50 microg) into the lateral ventricle (ICV) or a single systemic injection of E2 (100 microg/kg). Surviving pyramidal neurons in the hippocampal CA1 were quantified 1 week later. E2 and both agonists that target non-classical estrogen receptors (G1 and STX) administered ICV at the time of reperfusion provided significant levels of neuroprotection, with 55-60% of CA1 neurons surviving vs 15% survival in controls. A single systemic injection of a pharmacological dose of E2 also rescued approximately 50% of CA1 pyramidal neurons destined to die. To determine if E2 and G1 have similar mechanisms of action in hippocampal neurons, we compared the ability of E2 and G1 to modify CA1 pyramidal neuron responses to excitatory inputs from the Schaffer collaterals recorded in hippocampal slices derived from female rats not subjected to global ischemia. E2 and G1 (10 nM) significantly potentiated pyramidal neuron responses to excitatory inputs when applied to hippocampal slices.

Conclusions/significance: These findings suggest (1) that middle-aged female rats retain their responsiveness to E2 even after a long period of hormone withdrawal, (2) that non-classical estrogen receptors may mediate the neuroprotective actions of E2 when given after ischemia, and (3) that the neuroprotective efficacy of estrogens may be related to their modulation of synaptic activity in hippocampal slices.

Figure 1. Chemical structure of the natural estrogen 17β-estradiol and non-classical estrogen receptor agonists.

G1: GPR30 agonist; STX: diphenylacrylamide compound (SERM) that does not bind ER-α or ER-β.

Figure 2. G1 and E2 afford similar levels of neuroprotection when administered to young rats immediately after ischemia.

Panel A shows representative photomicrographs of hippocampal neurons in the dorsal CA1 region 7 days after sham surgery or global ischemia (Isch) in young adult female rats that were OVX for 1 week and injected ICV with either E2 (2.25 µg), G1 (50 µg) or vehicle (DMSO) immediately after ischemia. Scale bars: low magnification, 400 µm; higher magnification, 60 µm. Panel B: Quantification of living pyramidal neurons was performed in the CA1 region of the hippocampus 7 days after ischemia. Neurons were counted in 3 sectors (lateral, middle and medial) of the dorsal hippocampus. ANOVA followed by Newman Keuls, * p<0.001 versus sham, ** p<0.001 versus ischemia/vehicle.

Figure 3. STX affords neuroprotection in short-term OVX, middle-aged rats when administered after ischemia.

Middle-aged female rats were subjected to sham surgery or global ischemia (Isch) 1 week after OVX. Panel A shows representative photomicrographs of hippocampal neurons in the dorsal CA1 region 7 days after sham surgery or global ischemia in animals injected ICV immediately after reperfusion with either vehicle (DMSO) or STX (50 µg). Scale bars: low magnification, 400 µm; higher magnification, 60 µm. Panel B: Quantification of living pyramidal neurons was performed in the CA1 region of the hippocampus 7 days after ischemia. Neurons were counted in 3 sectors (lateral, middle and medial) of the dorsal hippocampus. ANOVA followed by Newman Keuls, * p<0.001 versus sham, ** p<0.001 versus ischemia/vehicle.

Figure 4. E2, G1 and STX afford neuroprotection in middle-aged females 8 weeks after hormone deprivation.

Middle-aged female rats were subjected to sham surgery or global ischemia (Isch) 8 weeks after OVX. Panel A shows representative photomicrographs of hippocampal neurons in the dorsal CA1 region 7 days after sham surgery or global ischemia in animals injected ICV immediately after reperfusion with either vehicle, E2 (2.25 µg), G1 (50 µg), STX (50 µg) or vehicle. Additional animals were injected sc with E2 (100 µg/kg). Scale bars: low magnification, 400 µm; higher magnification, 60 µm. Panel B: Quantification of living pyramidal neurons was performed in the CA1 region of the hippocampus 7 days after ischemia. Neurons were counted in 3 sectors (lateral, middle and medial) of the dorsal hippocampus. ANOVA followed by Newman Keuls, * p<0.001 versus sham, ** p<0.001 versus ischemia/vehicle.

Figure 5. The GPR30 agonist G1 mimics short latency E2 potentiation of hippocampal CA1 neuronal excitability.

Changes in field EPSPs in response to E2 or G1 were measured on hippocampal slices prepared from OVX young adult female rats. Schaffer collaterals were activated by monopolar stimulation. Slices were perfused with Ringer's solution until a stable baseline response was obtained. They were then perfused for 15 min with Ringer's solution containing the vehicle and then with 10 nM E2 (A) or G1 (B). Bins of 1 min were plotted. It was frequently possible to record synaptic activity from more than one slice from the same animal in a recording session. Therefore, to test G1, we recorded from 14 slices originating from 9 different OVX females; of these, 9 exhibited an increase in the fEPSP. Similarly, 8 out of 9 slices from 6 different animals responded to E2. Only responding slices are illustrated in the figures.