Progesterone exacerbates striatal stroke injury in progesterone-deficient female animals

BACKGROUND AND PURPOSE: We have previously shown that female animals experience substantial protection from brain injury after reversible middle cerebral artery occlusion (MCAO) compared with their male or ovariectomized female counterparts. The reproductive steroid estrogen has been shown to provide neuroprotection from a variety of experimental insults, but the importance of progesterone as an anti-ischemic treatment has not been well explored. We evaluated histological outcomes after MCAO in ovariectomized female rats with or without acute or chronic progesterone replacement therapy. METHODS: Age-matched, adult female Wistar rats were ovariectomized and treated with 0, 30, or 60 mg/kg progesterone IP 30 minutes before ischemia (n=12 to 14 per group) or with 30 mg/kg progesterone IP daily for 7 to 10 days before ischemia (n=16). Each animal subsequently underwent 2 hours of MCAO with the intraluminal filament technique, followed by 22 hours of reperfusion. Ipsilateral parietal cortex perfusion was monitored with laser Doppler flowmetry throughout ischemia. Cortical, caudate-putamen, and hemispheric infarction volumes were determined with 2,3,5-triphenyltetrazolium chloride staining and digital image analysis. RESULTS: Intraischemic plasma progesterone levels were 5+/-3, 102+/-20,* 181+/-28,* and 133+/-25* ng/mL in the 0, 30, and 60 mg/kg acute progesterone group and the 30 mg/kg chronic progesterone group, respectively (*P<0.05 compared with 0 mg/kg). Caudate-putamen infarction volume (percent contralateral structure) was significantly increased by chronic progesterone treatment: 45.6+/-5.1%* in the 30 mg/kg chronic progesterone group and 29.2+/-5.3%, 35.8+/-5.1%, and 42.0+/-5.0% in the 0, 30, and 60 mg/kg acute progesterone groups, respectively (*P<0.05 compared with 0 mg/kg). Cortical and total hemispheric infarction volumes (percent contralateral structure) were unchanged by progesterone treatment. CONCLUSIONS: Exogenous progesterone therapy does not ameliorate histological injury after MCAO in previously ovariectomized, adult female rats. Furthermore, chronic progesterone administration can exacerbate infarction in subcortical regions.     

Progesterone administration during reperfusion, but not preischemia alone, reduces injury in ovariectomized rats.

Although progesterone is neuroprotective in traumatic brain injury, its efficacy in stroke is unclear. The authors determined whether there are infarction differences after middle cerebral artery occlusion (MCAO) in ovariectomized rats treated acutely with progesterone before MCAO or both pre- and postischemia. Rats received vehicle, 5 (P5), 10 (P10), or 20 (P20) mg/kg progesterone intraperitoneally 30 minutes before MCAO. In another cohort, animals received vehicle or 5 (P5R) mg/kg progesterone intraperitoneally 30 minutes before MCAO, at reperfusion initiation, and at 6-hour reperfusion. Animals underwent 2-hour MCAO by the intraluminal filament technique, followed by 22-hour reperfusion. Cortical (CTX) and caudate-putamen (CP) infarctions were determined by 2,3,5-triphenyltetrazolium chloride staining and digital image analysis. End-ischemic and early reperfusion regional cerebral blood flow (CBF) was measured by [ C]-iodoantipyrine quantitative autoradiography in vehicle- or progesterone (5 mg/kg)-treated rats. Cortical infarction (% contralateral CTX) was 31 +/- 30% (vehicle), 39 +/- 23% (P5), 41 +/- 14% (P10), and 28 +/- 20% (P20). Caudate-putamen infarction (% contralateral CP) was 45 +/- 37% (vehicle), 62 +/- 34% (P5), 75 +/- 17% (P10), and 52 +/- 30% (P20). In vehicle and P5R groups, CTX infarction was 37 +/- 20% and *20 +/- 17%, respectively (* < 0.05 from vehicle). In vehicle and P5R groups, CP infarction was 63 +/- 26% and 43 +/- 29%, respectively. End-ischemic regional CBF and CBF recovery during initial reperfusion was unaffected by progesterone treatment. These data suggest that progesterone administration both before MCAO and during reperfusion decreases ischemic brain injury.     

Neuroprotective effects of female gonadal steroids in reproductively senescent female rats

BACKGROUND AND PURPOSE: Young adult female rats sustain smaller infarcts after experimental stroke than age-matched males. This sex difference in ischemic brain injury in young animals disappears after surgical ovariectomy and can be restored by estrogen replacement. We sought to determine whether ischemic brain injury continues to be smaller in middle-aged, reproductively senescent female rats compared with age-matched males and to test the effect of ovarian steroids on brain injury after experimental stroke in females. METHODS: Four groups of 16-month old Wistar rats (males [n=9], untreated females [n=9], and females pretreated with 17beta-estradiol [25-microgram pellets administered subcutaneously for 7 days; n=9] or progesterone [10-mg pellets administered subcutaneously for 7 days; n=9] were subjected to 2 hours of middle cerebral artery occlusion with the intraluminal filament technique, followed by 22 hours of reperfusion. Physiological variables and laser-Doppler cerebral cortical perfusion were monitored throughout ischemia and early reperfusion. In a separate cohort of males (n=3), untreated females (n=3), females pretreated with 17beta-estradiol (n=3), and females pretreated with progesterone (n=3), end-ischemic regional cerebral blood flow was measured by [(14)C]iodoantipyrine autoradiography. RESULTS: As predicted, infarct size was not different between middle-aged male and female rats. Cortical infarcts were 21+/-5% and 31+/-6% of ipsilateral cerebral cortex, and striatal infarcts were 44+/-7% and 43+/-5% of ipsilateral striatum in males and females, respectively. Both estrogen and progesterone reduced cortical infarct in reproductively senescent females (5+/-2% and 16+/-4% in estrogen- and progesterone-treated groups, respectively, compared with 31+/-6% in untreated group). Striatal infarct was smaller in the estrogen- but not in the progesterone-treated group. Relative change in laser-Doppler cerebral cortical perfusion from preischemic baseline and absolute end-ischemic regional cerebral blood flow were not affected by hormonal treatments. CONCLUSIONS: We conclude that the protection against ischemic brain injury found in young adult female rats disappears after reproductive senescence in middle-aged females and that ovarian hormones alleviate stroke injury in reproductively senescent female rats by a blood flow-independent mechanism. These findings support a role for hormone replacement therapy in stroke injury prevention in postmenopausal women.     

Differential effects of 17beta-estradiol upon stroke damage in stroke prone and normotensive rats.

We previously reported that during pro-estrus (high endogenous estrogen levels), brain damage after middle cerebral artery occlusion (MCAO) was reduced in stroke-prone spontaneously hypertensive rats (SHRSP) but not in normotensive Wistar Kyoto rat (WKY). In the present study, we examined the effect of exogenous estrogen on brain damage after MCAO in SHRSP and WKY. A 17beta-estradiol (0.025 mg or 0.25 mg, 21 day release) or matching placebo pellet was implanted into ovariectomized WKY and SHRSP (3 to 4 months old) who then underwent distal diathermy-induced MCAO 2 weeks later. Plasma 17beta-estradiol levels for placebo and 17beta-estradiol groups were as follows: WKY 0.025 mg 16.4 +/- 8.5 (pg/mL, mean +/- SD) and 25.85 +/- 12.6; WKY 0.25 mg 18.2 +/- 9.0 and 69.8 +/- 27.4; SHRSP 0.25 mg 20.7 +/- 8.8 and 81.0 +/- 16.9. In SHRSP, infarct volumes at 24 hours after MCAO were similar in placebo and 17beta-estradiol groups: SHRSP 0.025 mg 126.7 +/- 15.3 mm (n = 6) and 114.0 +/- 14.1 mm (n = 8) (not significant); SHRSP 0.25 mg 113.5 +/- 22.3 mm (n = 8) and 129.7 +/- 26.2 mm (n = 7) (not significant), respectively. In WKY, 17beta-estradiol significantly increased infarct volume by 65% with 0.025 mg dose [36.1 +/- 20.7 mm (n = 8) and 59.7 +/- 19.3 mm (n = 8) (P = 0.033, unpaired t-test)] and by 96% with 0.25 mg dose [55.9 +/- 36.4 mm (n = 8) and 109.7 +/- 6.7 mm (n = 4) (P = 0.017)]. Thus, 17beta-estradiol increased stroke damage in normotensive rats with no significant effect in stroke-prone rats. Despite being contrary to our hypothesis, our findings add substance to the recently reported negative effects of 17beta-estradiol in clinical studies.     

Estrogen receptor antagonist ICI182,780 exacerbates ischemic injury in female mouse.

Recent findings in animals emphasize that experimental ischemic brain damage can be strikingly reduced by estrogen: however, the neuroprotective mechanisms are not well understood. It was hypothesized that estrogen signaling via cognate estrogen receptors (ERs) within the vasculature is an important aspect of cerebral ischemic protection in the female brain, in part by amplifying intraischemic cerebral blood flow (CBF). In the present study, the hypothesis that chronic treatment with the pure ER antagonist ICI182,780 (ICI) would increase ischemic brain damage by a blood flow-mediated mechanism was investigated. Adult C57B1/6J mice were pretreated with either subcutaneous ICI (100 microg/day) or oil/ethanol vehicle for 1 week before 2 hours of middle cerebral artery occlusion (MCAO) and 22 hours of reperfusion. End-ischemic regional CBF was evaluated in additional cohorts using [14C]iodoantipyrine autoradiography. Infarction volume as measured by cresyl violet histology was greater in the striatum of ICI-treated females (70 +/- 3% of contralateral striatum vs. 40 +/- 12% in vehicle-treated females). Cortical injury was not enhanced relative to control animals (39 +/- 6% of contralateral cortex in ICI group vs. 27 +/- 8% in vehicle-treated group). Physiologic variables and ischemic reduction of the ipsilateral cortical laser-Doppler flow signal were similar between groups. Further, ICI treatment did not alter end-ischemic cortical or striatal CBF. The deleterious effect of ICI was limited to females, as there were no differences in stroke damage or CBF between male treatment groups. These data suggest that estrogen inhibits ischemic brain injury in striatum of the female by receptor-mediated mechanisms that are not linked to preservation of intraischemic CBF.     

Ovariectomy exacerbates and estrogen replacement attenuates photothrombotic focal ischemic brain injury in rats

BACKGROUND AND PURPOSE: We previously reported the infarct volumes in female spontaneously hypertensive rats (SHR) to be significantly smaller than those in male SHR. The purpose of the present study was to determine whether estrogen is responsible for the sex difference in ischemic vulnerability in SHR. METHODS: In experiment 1, 1 week (short-term) or 4 weeks (long-term) after the ovariectomy (OVX), female SHR (5 months old) were randomly subjected to photothrombotic occlusion of the middle cerebral artery, and the infarct volumes were determined. In experiment 2, the rats were randomly assigned to 3 groups (ie, the sham-ovariectomized, ovariectomized, and estrogen replacement groups). In the replacement group, estradiol valerate (200 microgram/kg) was subcutaneously injected once a week after the OVX. Four weeks after the OVX or sham-OVX, all rats were subjected to middle cerebral artery occlusion. Changes in regional cerebral blood flow were determined by laser-Doppler flowmetry. RESULTS: In experiment 1, the infarct volume produced 1 week after the OVX was not different from that of the sham-ovariectomized group. In contrast, the infarct volume produced 4 weeks after the OVX was significantly larger than that of the sham-ovariectomized group (82.4+/-11.6 versus 54.5+/-16.0 mm(3), P=0.0058). In experiment 2, estradiol replacement after the OVX was observed to attenuate the infarct volume compared with the ovariectomized group (55.6+/-18.8 versus 78.5+/-21.0 mm(3), P=0.0321). The degrees of regional cerebral blood flow reduction did not differ among the sham-ovariectomized, ovariectomized, and estrogen replacement groups. CONCLUSIONS: Chronic estrogen depletion was thus found to increase the infarct size, which was attenuated by estradiol replacement. These findings indicate that estrogen contributes to the sex difference in ischemic vulnerability and that endogenous estrogen also has a neuroprotective effect against ischemic brain damage.     

雌激素对脑缺血病理及超微结构的影响

目的：通过观察雌激素对去卵巢雌性大鼠局灶性脑缺血大脑皮质区病理及超微结构的影响，以探讨雌激素对缺血性脑损害的神经保护作用。方法：采用大鼠大脑中动脉闭塞制成局灶性脑缺血模型，在缺血２小时，再灌注２２小时后立即断头取脑，切片，用光镜（定性和定量）和电镜观察大脑皮质区的病理学变化及超微结构改变。结果：雌激素用药组较对照组正常神经细胞的密度显著增加（Ｐ〈０．０５），血管内皮细胞受损程度减轻。结论：雌激素可     

Estrogen is neuroprotective via an apolipoprotein E-dependent mechanism in a mouse model of global ischemia.

Estrogen can ameliorate brain damage in experimental models of focal cerebral ischemia., estrogen increases levels of apolipoprotein E (apoE), which also has neuroprotective effects in brain injury. The authors tested the hypotheses that physiologically relevant levels of 17beta-estradiol are neuroprotective in global cerebral ischemia and that neuroprotection is mediated via apoE. In the first study, subcutaneous implants of 17beta-estradiol were tested in female C57Bl/6J mice (ovariectomized and nonovariectomized) and plasma levels measured by radioimmunoassay to validate that physiologically relevant levels could be achieved. In the second study, female C57Bl/6J and apoE-deficient mice were ovariectomized and implanted with 17beta-estradiol or placebo pellet. Two weeks later, transient global ischemia was induced by bilateral carotid artery occlusion and the mice killed after 72 hours. Ischemic and normal neurons were counted in the caudate nucleus and CA1 pyramidal cell layer and the percentage of neuronal damage was compared between the treated groups. In C57Bl/6J mice, there was less neuronal damage in the 17beta-estradiol-treated group compared with placebo group in the caudate nucleus (15 +/- 20% versus 39 +/- 27%, = 0.02) and in the CA1 pyramidal cell layer (1.8 +/- 2% versus 10 +/- 14%, = 0.08). In contrast, neuronal damage was not significantly different between the 17beta-estradiol and placebo groups in apoE-deficient mice in the caudate nucleus (47 +/- 35% versus 53 +/- 29%, = 0.7) or in the CA1 pyramidal cell layer (24 +/- 19% versus 24 +/- 19%, = 1.0). The data indicate a neuroprotective role for estrogen in global ischemia, the mechanism of which is apoE-dependent.     

Gender differences and the effects of synthetic exogenous and non-synthetic estrogens in focal cerebral ischemia

The role of gender difference and estrogen in ischemic cerebrovascular events is controversial. Evidence is lacking as to whether or not there are significant gender differences in the incidence and outcome of stroke in the clinical setting. Recent clinical epidemiological studies have demonstrated that there is no significant association between the use of hormonal replacement therapy and the risk of stroke. However, several animal studies have shown that there are gender differences in stroke outcome and that exogenous administered estrogens are neuroprotective. In this study, the influence of gender differences and the effects of synthetic and non-synthetic estrogens were examined in a model of focal cerebral ischemia using 210 male, intact female, and ovariectomized female rats. All animals underwent 3 h of middle cerebral artery and bilateral common carotid artery occlusion. After 72 h, the rats were sacrificed and stained for histological assessment of infarction. There were no gender differences in infarction volume. Intravenous administration of either low or high dose 17 beta-estradiol or tibolone did not alter infarct volume. Subcutaneous administration of low and high dose 17beta-estradiol using 7-day release pellets did not alter infarct volume. Low dose tibolone using implanted 7-day release pellets did not alter infarct volume. However, high dose tibolone using implanted 7-day release pellets significantly (P<0.05) reduced infarct volume only in ovariectomized female rats. These results demonstrate that estrogen therapy has no effect on infarction volume following severe focal cerebral ischemia.     

Effects of combined estrogen and progesterone on brain infarction in reproductively senescent female rats.

Recent data from the Women's Health Initiative have highlighted many fundamental issues about the utility and safety of long-term estrogen use in women. Current hormone replacement therapy for postmenopausal women incorporates progestin with estrogen, but it is uncertain if combined therapy provides major cerebrovascular risks or benefits to these women. No experimental animal stroke studies have examined combined hormone administration. The authors tested the hypothesis that combined hormone treatment reduces ischemic injury in middle-aged female rat brain. Reproductively senescent female rats underwent 2-hour middle cerebral artery occlusion (MCAO) followed by 22 hours reperfusion. Estrogen implants were placed subcutaneously at least 7 days before MCAO, and progesterone intraperitoneal injections were given 30 minutes before MCAO, at initiation, and at 6 hours of reperfusion. Rats received no hormone, a 25-microg estrogen implant, a 25-microg estrogen implant plus 5 mg/kg intraperitoneal progesterone, or 5 mg/kg intraperitoneal progesterone. Cortical, caudoputamen, and total infarct volumes were assessed by 2,3,5-triphenyltetrazolium chloride staining and digital image analysis at 22 hours reperfusion. Cortical and total infarct volumes, except in the acute progesterone-treated group, were significantly attenuated in all estrogen-alone and combined hormone-treated groups. There were no significant differences in caudoputamen infarct volumes in all hormone-treated groups as compared with untreated rats. These data have potential clinical implications relative to stroke for postmenopausal women taking combined hormone replacement therapy.     

17beta-Estradiol extends ischemic thresholds and exerts neuroprotective effects in cerebral subcortex against transient focal cerebral ischemia in rats

Neuroprotective effects of estrogens are demonstrated consistently in the cerebral cortex, but not in subcortical areas. In the present study, transient middle cerebral artery occlusions (MCAO) were induced for various duration, and protective effects of estrogen treatment on the cerebral cortex and subcortex were evaluated. MCAO was induced for 30, 40 or 60 min in ovariectomized rats. Animals were treated with 17beta-estradiol (E2) or vehicle (OVX) 2 h before MCAO and sacrificed 24 h after the indicated duration of MCAO. Ischemic lesion was evaluated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin and eosin staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. E2 treatment reduced the magnitude and delayed the appearance of the total ischemic lesion area and largely prevented TUNEL staining in the cortex. In the subcortex, E2 treatment prevented the ischemic lesion in the 30-min group, reduced lesion area in the 40-min group, but had no effect on ischemic lesion area in the 60-min group. E2 treatment significantly decreased apoptotic cell number in the subcortical area at 30 and 40 min, but not at 60 min of MCAO. This study demonstrated that estrogen treatment can protect the cerebral subcortex in a severity-dependent manner, suggesting that the lack of protective effects of estrogen treatment in the subcortex is not due to the lack of estrogen receptors. Further, this study indicates that estrogens could be used as a neuroprotectant to prolong the therapeutic window of thrombolysis and prolong the time of cerebral circulation intervention for neurosurgical procedure.     

Estradiol alters only GAD67 mRNA levels in ischemic rat brain with no consequent effects on GABA.

The present study tested the hypothesis that estradiol reduces tissue infarction after middle cerebral artery occlusion (MCAO) in estradiol-deficient females by augmenting glutamic acid decarboxylase (GAD) expression and thus activity, leading to increases in gamma-amino-butyric acid (GABA) tissue levels. Glutamic acid decarboxylase is the principal enzyme for GABA synthesis and has two isoforms, GAD65 and GAD67, which differ in size and cellular distribution. Rats were ovariectomized 7 to 8 days before receiving no hormone, placebo, or 25 microg estradiol via subcutaneous implant 7 to 10 days before harvesting tissue in either ischemic cohorts after 2 h of MCAO (end-ischemia) or in nonischemic cohorts. Selected cortical and striatal regions were microdissected from harvested brains. GAD65/67 mRNA levels were determined by microlysate ribonuclease protection assay. End-ischemic GABA concentrations were determined by HPLC. Steroid treatment selectively decreased ischemic cortical GAD67 mRNA levels. In most brain regions evaluated, regional GABA concentrations increased with ischemia regardless of treatment. Estradiol blocked MCAO-induced increases in GABA concentration only in dorsomedial cortex. These data suggest that estradiol repletion in ischemic rat brain selectively decreases GAD67 mRNA levels but does not alter steady-state GABA concentrations. It may be that estradiol under ischemic conditions is attenuating GABA metabolism rather than enhancing synthesis or is augmenting other aspects of GABAergic transmission such as GABA transporters and receptors.     

Estrogen reduces leukocyte adhesion in the cerebral circulation of female rats.

In this study the authors addressed the hypothesis that estrogen (i.e., 17beta-estradiol) acts to repress leukocyte adhesion. The experiments involved comparing leukocyte adhesion in cerebral venules in vivo, in intact ovariectomized and 17beta-estradiol-treated (100 microg/kg/day for 1 week) ovariectomized female rats using topical applications of the adhesion-promoting drug, phorbol 12-myristate 13-acetate (PMA). Adherent Rhodamine-6G-labeled leukocytes were viewed through a closed cranial window using intravital microscopy/videometry. Leukocyte dynamics were recorded at baseline and after each dose of PMA. The PMA was suffused (1.0 mL/min) at increasing concentrations (0.01, 0.1, and 1.0 micromol/L, 15 minutes at each level). A videotape record of each experiment was made for subsequent analysis of leukocyte adhesion. The data showed that the percentage venular area occupied by adherent leukocytes at baseline was significantly greater in the ovariectomized compared to the intact and 17beta-estradiol-treated groups (12.2%, 3.4%, and 4.2% respectively). That relationship was maintained during PMA treatments to the extent that the percentage venular area occupied by adherent leukocytes increased to 26.4% in the untreated ovariectomized group compared to 14.4% and 11.3% in the intact and 17beta-estradiol-treated groups, respectively. In conclusion, the authors found chronic estrogen depletion enhances leukocyte adhesion in the rat cerebral circulation. Estrogen repletion in such animals is accompanied by a significant reduction in leukocyte adhesion. These findings could, at least in part, account for the ischemic brain damage seen in ovariectomized versus intact females, and the restored neuroprotection observed upon 17beta-estradiol treatment reported in earlier studies.     

Estrogen attenuates neuronal excitability in the insular cortex following middle cerebral artery occlusion

The current investigation examined the role of estrogen in the insular cortex (IC) under both normal and ischemic conditions. Experiments were done in anaesthetized male Sprague-Dawley rats. The effect of systemic 17beta-estradiol (estrogen) administration on levels of amino acids and of endogenous estrogen obtained by microdialysis and its effect on neuronal activity of cells located in the insular cortex were measured in the absence of, and following permanent occlusion of, the right middle cerebral artery (MCA). In normal rats, intravenous (i.v.) injection of estrogen resulted in a significant increase (greater than 25 spikes/bin) in the spontaneous activity of neurons located within the insular cortex, while there was a significant decrease in gamma-aminobutyric acid (GABA) levels measured in IC dialysate. Middle cerebral artery occlusion (MCAO) resulted in a biphasic response consisting of a transient increase in the extracellular concentration of glutamate, aspartate, and GABA, followed by sustained elevations in glutamate and aspartate, but reduced GABA levels 4 h post-MCAO. MCAO also resulted in a significant increase in neuronal activity in the IC (from 28 +/- 9 to 120 +/- 88 spikes/bin). This MCAO-induced excitation was completely blocked following the prior intravenous administration of estrogen. Systemic estrogen administration also resulted in a delay in the progression and decrease in the final infarct volume by approximately 56%. Taken together, these results suggest that under normal conditions, estrogen excites neurons in the insular cortex by decreasing GABA release (disinhibition) and it plays a role in attenuating the MCAO-induced excitability and death of these neurons.     

Genetic hypertension and increased susceptibility to cerebral ischemia.

A review of the sensitivity of genetically hypertensive rats to cerebral ischemia was presented together with original data describing the systematic comparison of the effects of focal ischemia (permanent and temporary with reperfusion) performed in hypertensive and normotensive rats (i.e., blood pressures verified in conscious instrumented rats). Microsurgical techniques were used to isolate and occlude the middle cerebral artery (MCAO) of spontaneously hypertensive (SHR), Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats at the level of the inferior cerebral vein. Following permanent (24 h) MCAO, persistent and similar decreases in local microvascular perfusion (i.e., to 15.6 +/- 1.7% of pre-MCAO levels) were verified in the primary ischemic zone of the cortex for all strains using Laser-Doppler flowmetry. A contralateral hemiplegia that occurred following MCAO, evidenced by forelimb flexion and muscle weakness, was greater in SHR (neurological grade = 2.0 +/- 0.1) than SD (1.0 +/- 0.4) or WKY (0.7 +/- 0.4) rats (N = 7-9, p less than 0.05). SHR also exhibited sensory motor deficits following MCAO compared to sham-operation, with decreased normal placement response of the hindlimb (% normal = 20 vs. 83, N = 23-30, p decreased rota-rod (41 +/- 7 vs. 126 +/- 19 on rod, N = 10-15, p less than 0.05) and balance beam (25 +/- 5 vs. 116 +/- 29 s on beam, N = 5-7, p less than 0.05) performance. However, an index of general motor activity was not affected by permanent MCAO. Triphenyltetrazolium-stained forebrain tissue analyzed by planimetry revealed a significantly larger and more consistent cortical infarction in SHR (hemispheric infarction = 27.9 +/- 1.5%) compared to SD (15.4 +/- 4.1%) and WKY (4.0 +/- 2.4%) rats (N = 7-9, p less than 0.05), occupying predominantly the frontal and parietal areas. Also, a significant degree of ipsilateral hemispheric swelling (4.6 +/- 0.9%, N = 7-9, p less than 0.05) and increased brain water content (78.4 +/- 0.3% to 80.4 +/- 0.2%, N = 8-9, p less than 0.05) was identified in SHR that was not observed in SD or WKY rats. A novel model of temporary MCAO also was evaluated in the hypertensive and normotensive rat strains. Initially, the effect of increasing MCAO-time followed by 24 h reperfusion in SHR was studied. During temporary MCAO (20 to 300 min), persistent and stable decreases in local microvascular perfusion (i.e., to 15-20% of pre-MCAO levels) were verified in the primary ischemic zones of the cortex.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of thrombin preconditioning on focal cerebral ischemia in rats

Our previous studies have shown that prior intracerebral infusion of a low dose of thrombin (thrombin preconditioning; TPC) reduces the brain edema that follows a subsequent intracerebral infusion of a high dose of thrombin or an intracerebral hemorrhage. In vitro studies have also demonstrated that low concentrations of thrombin protect neurons and astrocytes from hypoglycemia and oxidative stress-induced damage. This study, therefore, examines the hypothesis that TPC would offer protection from ischemic brain damage in vivo. This was a blinded design study. The rat brain was preconditioned with 1 U thrombin by direct infusion into the left caudate nucleus. Seven days after thrombin pretreatment, permanent middle cerebral artery occlusion (MCAO) was induced. Twenty-four hours post-ischemia, neurological deficit was evaluated and infarction volume, brain water and ion contents were measured. Compared to saline-treated rats, thrombin pretreatment significantly attenuated brain infarction in cortex (90+/-33 vs. 273+/-22 mm(3); P<0.05) and basal ganglia (56+/-17 vs. 119+/-12 mm(3); P<0.05) that followed 24 h of permanent MCAO. TPC also reduced the brain edema in cortex and basal ganglia by 50 and 53% (P<0.05). Neurological deficit was improved in thrombin pretreatment group (P<0.05). These effects of TPC were, in part, prevented by co-injection of hirudin, a thrombin inhibitor, indicating that the protection was indeed thrombin mediated. Cerebral TPC significantly reduces ischemic brain damage, perhaps by activation of the thrombin receptor. This finding provides a new mechanism by which to study ischemic tolerance.     

Immediate and residual effects of tamoxifen and ethynylestradiol in the female rat hypothalamus

Very little is known about the impact of selective estrogen receptor modulators (SERMs) on the brain. We examined the effects of tamoxifen (TAMOX) and the synthetic estrogen 17alpha-ethynylestradiol (EE) on estrogen-dependent gene expression and receptor binding in the female rat brain. Both immediate and residual effects were examined in both the presence and absence of 17beta-estradiol. Two groups of adult, ovariectomized, female rats (n=30 per group) were injected with TAMOX (5 mg/kg), EE (0.1 mg/kg), or sesame oil daily for 14 days. Animals from the first group were implanted with blank or 17beta-estradiol Silastic capsules concurrently with the last three SERM injections (immediate, group 1). Animals from the second group received either blank or 17beta-estradiol implants 2 weeks after the last injection (residual, group 2). All animals were sacrificed 72 h after implantation. TAMOX increased uterine weight in the absence of estrogen, but inhibited uterine weight gain in the presence of estrogen in both groups 1 and 2. TAMOX and EE increased oxytocin receptor binding in the ventromedial nucleus of the hypothalamus (VMN) in the absence of estrogen in both groups 1 and 2. The estrogen-dependent induction of PR mRNA expression in the VMN was significantly attenuated by TAMOX in group 1. Finally, TAMOX and EE had opposite effects on ERbeta mRNA expression in the paraventricular nucleus in the absence of 17beta-estradiol in group 1. Neither had any effect in group 2 when 17beta-estradiol was present. These results suggest that TAMOX has mixed agonist/antagonist effects in the female rat brain, many of which persist at least 2 weeks after the administration ceases.     

Estrogen modulation of NMDA-induced seizures in ovariectomized and non-ovariectomized rats

The objective of this study was to examine the neuroprotective effects of estrogen in response to N-methyl-D-aspartate (NMDA)-induced seizures in both male and female rats. Thirty-eight Long-Evans rats were divided into five groups: ovariectomized females, non-ovariectomized females, ovariectomized females with estrogen replacement (10 microg 17beta-estradiol in 100 microl sesame oil), males given exogenous estrogen and males receiving no estrogen. Using stereotaxic surgery, a cannula was placed in the lateral ventricle for convulsant agent administration (20 microg of NMDA), while an electrode was placed into the hippocampus for seizure recording. Seizure activity was monitored for 20 min. Onset to first seizure, first seizure duration, seizure frequency and total duration of seizures were determined. Rats were pretreated with either sesame oil (vehicle) or estrogen given subcutaneously for 4 days prior to seizure induction on the fourth day. Rats were euthanized 72 h later and the brains removed for histological processing. Electrode and cannula placement were verified microscopically and neuronal integrity was assessed via hematoxylin and eosin staining. Total seizure number was significantly higher in the ovariectomized females compared to the non-ovariectomized females and the ovariectomized females receiving estrogen (P<0.05). Moreover, hippocampal neuronal damage following seizure induction was significant in the ovariectomized rats compared to the non-ovariectomized rats (P<0.05). Pretreatment with estrogen did not affect any of the seizure parameters measured in the male rats. We conclude that estrogen appears to be neuroprotective against NMDA-induced seizures in female ovariectomized rats.     

17Beta-estradiol blocks NMDA-induced increases in regional cerebral O(2) consumption

We tested the hypothesis that 17beta-estradiol would reduce the cerebral O(2) consumption response to stimulation of N-methyl-D-aspartate (NMDA) receptors. We determined NMDA receptor density in 10 ovariectomized Wistar female rats equally divided into a control group and 17beta-estradiol (500 microg/21 days) treated group. An autoradiographic assay using 125I-MK-801, an NMDA antagonist, was used to measure specific binding to NMDA receptors. Another 14 ovariectomized rats were separated into 17beta-estradiol and control groups to determine cerebral blood flow (14C-iodoantipyrine) and O(2) consumption (microspectrophotometry). 17Beta-estradiol caused a 20% decrease in specific binding to cortical NMDA receptors. After topical cortical stimulation with 10(-3)M and 10(-4)M NMDA, blood flow increased significantly in control from 73+/-5 in the saline treated cortex to 110+/-8 ml/min/100 g with 10(-3)M NMDA. In contrast, there was no significant change in blood flow in the 17beta-estradiol treated animals. Cerebral O(2) extraction increased significantly in the 10(-3)M NMDA treated cortex in both groups. Cerebral O(2) consumption in the control group significantly increased by 53%, from 3.7+/-0.2 to 5.7+/-0.5 with 10(-4)M NMDA and 72% to 6.4+/-2.4 ml O(2)/min/100 g with 10(-3)M NMDA. The 17beta-estradiol group demonstrated no significant difference between the saline treated and NMDA treated cortex. Thus, 17beta-estradiol blocked the effects of NMDA on cerebral O(2) consumption and this was associated with a slightly decreased number of NMDA receptors.     

Deleterious effect of beta-estradiol in a rat model of transient forebrain ischemia

Estrogen has demonstrated great potential as a therapeutic agent in focal ischemic brain injury, as exogenous beta-estradiol has proven beneficial in a variety of focal stroke models. In contrast, the relatively few studies of estrogen's efficacy in transient forebrain ischemia have produced inconsistent results. The present study was therefore designed to clarify estrogen's neuroprotective potential in selective hippocampal neuronal injury resulting from four-vessel occlusion in the rat. Female Wistar rats (normal, ovariectomized, or ovariectomized and estradiol-treated) received 5 or 10 min of ischemia. No differences in hippocampal cell loss were found amongst the groups with 10 min of ischemia. Amongst the groups with 5 min of ischemia, the mildest injury was found in the ovariectomized animals, which lost only 32% of their CA1 pyramidal cells. In comparison, mean cell losses were 54% and 49%, respectively, in intact females and in ovariectomized animals with estradiol replacement. Linear regression analysis demonstrated a highly significant relationship between cell loss and plasma estradiol levels. The mechanism by which exogenous and endogenous estrogen exacerbated the injury is unclear, as estrogen has many neuroprotective effects. On the other hand, many other reported effects of estrogen in hippocampal area CA1 might confer increased sensitivity to ischemia, either by modulating the excitatory effects of glutamate or by modifying the inhibitory effects of GABA. Determining how to modulate the various competing effects of estrogen is of both theoretical and practical importance, as it is now clear that one cannot assume that estrogen administration will always improve outcome in cerebral ischemia.