Chronic Estrogen Replacement Research Articles

Estradiol replacement improves high-fat diet-induced insulin resistance in ovariectomized rats.

The role of 17β‐estradiol (E2) in high‐fat diet (HFD)‐induced alteration of the protein kinase B (Akt) signaling pathway in ovariectomized (OVX) rats is unclear. Therefore, we examined whether chronic estrogen replacement restores HFD‐induced impairment in insulin sensitivity by its effects concomitant with alterations in the Akt isoform 2 (Akt2) and Akt substrate of 160 kDa (AS160) phosphorylation in muscles of OVX rats. Nine‐week‐old female Wistar rats underwent ovariectomy under anesthesia; after 4 weeks, subcutaneous implantation of either E2 or placebo (PL) pellets was performed, and HFD feeding was initiated. Intravenous glucose tolerance tests were performed to assess insulin sensitivity. Following insulin injection into rats’ portal vein, the liver and gastrocnemius muscle were dissected for insulin signaling analysis. We observed that HFD increased energy intake and body weight in the PL group; however, it was temporarily decreased in the E2 group. Adipose tissue accumulation was larger in HFD‐fed rats than in normal chow diet (NCD)‐fed rats in the PL group; however, this difference was not observed in the E2 group. HFD reduced insulin sensitivity in the PL group only. In vivo insulin stimulation increased Akt2 phosphorylation in the muscles of NCD‐fed rats in both groups. In contrast, HFD affected insulin‐stimulated phosphorylation of Akt2 and AS160 in the muscles of rats in the PL group but not in the E2 group. Our data suggest that E2 replacement improves HFD‐induced insulin resistance, and this effect is accompanied by the alterations in the Akt2 and AS160 phosphorylation in insulin‐stimulated muscles of OVX rats.

Fluoxetine Mimics the Anorectic Action of Estrogen and Its Regulation of Circadian Feeding in Ovariectomized Female Rats.

Our previous study demonstrated that chronic estrogen replacement in ovariectomized rats reduces food intake and augments c-Fos expression in the suprachiasmatic nucleus (SCN), specifically during the light phase. Here, we hypothesized that serotonergic neurons in the central nervous system (CNS), which have anorectic action and play a role in regulating circadian rhythm, mediate the light phase-specific anorectic action of estrogen, and that selective serotonin reuptake inhibitors (SSRIs) mimic the hypophagic action of estrogen. Female Wistar rats were ovariectomized and treated with estradiol (E2) or cholesterol by subcutaneously implanting a silicon capsule containing E2 or cholesterol. Then, half of the cholesterol-treated rats were injected with the SSRI fluoxetine (5 mg/kg) (FLX group), while the remaining rats in the cholesterol-treated group (CON group) and all those in the E2 group were injected with saline subcutaneously twice daily at the onsets of the light and dark phases. Both E2 and FLX reduced food intake during the light phase but not the dark phase, and reduced body weight gain. In addition, both E2 and FLX augmented the c-Fos expression in the SCN, specifically during the light phase. These data indicate that FLX exerts estrogen-like antiobesity and hypophagic actions by modifying circadian feeding patterns, and suggest that estrogen regulates circadian feeding rhythm via serotonergic neurons in the CNS.

Estrogen replacement enhances insulin-induced AS160 activation and improves insulin sensitivity in ovariectomized rats.

Menopause predisposes women to impaired glucose metabolism, but the role of estrogen remains unclear. In this study, we examined the effects of chronic estrogen replacement on whole body insulin sensitivity and insulin signaling in ovariectomized rats. Female Wistar rats aged 9 wk were ovariectomized under anesthesia. After 4 wk, pellets containing either 17β-estradiol (E2) or placebo (Pla) were subcutaneously implanted in the rats. After 4 wk of treatment, the intra-abdominal fat accumulation was greater in the Pla group than that in the E2 group. Hyperinsulinemic-euglycemic clamp analysis and intravenous glucose tolerance test revealed that insulin sensitivity was significantly lower in the Pla group than in the E2 group. In addition, Western blotting showed that in vivo insulin stimulation increased protein kinase B (Akt) phosphorylation to a similar degree in the gastrocnemius and liver of both groups, but phosphorylated Akt2 Ser474 was enhanced in the muscle of the E2 group compared with the Pla group. Moreover, insulin-stimulated phosphorylation of Akt substrate of 160 kDa (AS160) Thr642 was observed only in the E2 group, resulting in the difference between the two groups. Additionally, AS160 protein and mRNA levels were higher in muscle of the E2 group than the Pla group. In contrast, E2 replacement had no effect on glucose transporter 4 protein levels in muscle and glycogen synthase kinase-3β in muscle and liver. These results suggest that estrogen replacement improves insulin sensitivity by activating the Akt2/AS160 pathway in the insulin-stimulated muscle of ovariectomized rats.

S-equol Exerts Estradiol-Like Anorectic Action with Minimal Stimulation of Estrogen Receptor-α in Ovariectomized Rats.

Chronic estrogen replacement in ovariectomized rats attenuates food intake and enhances c-Fos expression in the suprachiasmatic nucleus (SCN), specifically during the light phase. S-equol, a metabolite of daidzein, has a strong affinity for estrogen receptor (ER)-β and exerts estrogenic activity. The purpose of the present study was to elucidate whether S-equol exerts an estrogen-like anorectic effect by modifying the regulation of the circadian feeding rhythm in ovariectomized rats. Ovariectomized female Wistar rats were divided into an estradiol (E2)-replaced group and cholesterol (vehicle; Veh)-treated group. These animals were fed either a standard diet or an S-equol-containing diet for 13 days. Then, the brain, uterus, and pituitary gland were collected along with blood samples. In the rats fed the standard diet, E2 replacement attenuated food intake (P < 0.001) and enhanced c-Fos expression in the SCN (P < 0.01) during the light phase. Dietary S-equol supplementation reduced food intake (P < 0.01) and increased c-Fos expression in the SCN (P < 0.01) in the Veh-treated rats but not in the E2-replaced rats during the light phase. Dietary S-equol did not alter ER-α expression in the medial preoptic area or the arcuate nucleus, nor did dietary S-equol affect pituitary gland weight or endometrial epithelial layer thickness. By contrast, E2 replacement not only markedly decreased ER-α expression in these brain areas (P < 0.001) but also increased both the pituitary gland weight (P < 0.001) and the endometrial epithelial layer thickness (P < 0.001). Thus, dietary S-equol acts as an anorectic by modifying the diurnal feeding pattern in a manner similar to E2 in ovariectomized rats; however, the mechanism of action is not likely to be mediated by ER-α. The data suggest a possibility that dietary S-equol could be an alternative to hormone replacement therapy for the prevention of hyperphagia and obesity with a lower risk of adverse effects induced by ER-α stimulation.

Estrogen replacement attenuates stress-induced pressor responses through vasorelaxation via β2-adrenoceptors in peripheral arteries of ovariectomized rats.

We examined whether chronic estrogen replacement has an inhibitory effect on stress-induced pressor responses via activation of β2-adrenoceptor (AR) in peripheral arteries of ovariectomized rats. Female Wistar rats aged 9 wk were ovariectomized. After 4 wk, pellets containing either 17β-estradiol (E2) or placebo (Pla) were subcutaneously implanted into the rats. After 4 wk of treatment, rats underwent cage-switch stress, and, in a separate experiment, a subset received an infusion of isoproterenol (ISO) with or without pretreatment with the β1-AR blocker atenolol or the β2-AR blocker butoxamine. In addition, the isolated mesenteric artery was used to assess the concentration-related relaxing responses to ISO and the β1- or β2-AR mRNA level. The cage-switch stress-induced pressor response was significantly attenuated in the E2-treated group compared with the Pla-treated group. Pretreatment with atenolol reduced blood pressure responses in both groups. However, butoxamine enhanced the pressor response only in the E2-treated group, resulting in no difference between the two groups. In addition, the intravenous ISO-induced depressor response was significantly enhanced in the E2-treated group compared with the Pla-treated group. Furthermore, the difference in the depressor response was abolished by pretreatment with butoxamine but not by atenolol. In the isolated mesenteric artery, butoxamine caused a rightward shift in ISO-induced concentration-related relaxation in the E2-treated group. The β2-AR mRNA level in the mesenteric artery was higher in the E2-treated group than in the Pla-treated group. These results suggest that estrogen replacement attenuated the stress-induced pressor response probably by suppressing vasoconstriction via activation of β2-ARs in peripheral arteries of ovariectomized rats. NEW & NOTEWORTHY In this study, we show, for the first time, that estrogen replacement has an inhibitory effect on the psychological stress-induced pressor response through vasorelaxation via β2-adrenoceptors, probably due to overexpression of β2-adrenoceptor mRNA, in peripheral arteries of ovariectomized rats.

Abstract 046: Chronic Estrogen Replacement Prevents the Increase in Blood Pressure in Female IUGR Offspring at 12 Months of Age

Low birth weight (LBW ) is associated with an earlier age at menopause and a greater prevalence of hypertension in women in later life. Using a rodent model of LBW induced by placental insufficiency, previous studies from our lab indicate that intrauterine growth restriction ( IUGR ) programs a significant increase in blood pressure ( BP ) by 12 months of age in female IUGR offspring. Female IUGR are also acyclic by 12 months of age, or 6 months prior to control indicative of early reproductive senescence. The mechanisms involved in menopause related cardiovascular risk are not clear. Furthermore, the link between birth weight and the greater prevalence for hypertension in LBW women in later life is not known. Female IUGR exhibit a shift in the testosterone to estradiol ratio at 12 months of age. Thus, this study tested the hypothesis that a loss of estradiol ( E2 ) contributes to the increase in BP in female IUGR offspring that develops by 12 months of age. Female rats received vehicle or 17β-Estradiol valerate minipellets (1.5mg for 60-day release) for 6 weeks starting at 12 months of age with BP measured in conscious, chronically catheterized rats following treatment (2way ANOVA; Tukey’s multiple comparison). BP was significantly increased in vehicle-treated IUGR relative to vehicle-treated Control (*, p&lt;0.05); an increase that was abolished by chronic E2 in IUGR (**, p&lt;0.05). Uterine weight, a crude marker of E2, was significantly increased by chronic E2 in both Control and IUGR E2-treated offspring (**, p&lt;0.05). Therefore, this study indicates that E2 withdrawal after early reproductive senescence contributes to the increase in BP that develops in later life in female IUGR offspring.

Effects of estrogen replacement on stress-induced cardiovascular responses via renin-angiotensin system in ovariectomized rats

The purpose of this study was to determine whether chronic estrogen replacement in ovariectomized rats inhibits the pressor response to psychological stress by attenuating the activation of the renin-angiotensin system. Female Wistar rats aged 9 wk were ovariectomized. After 4 wk, the rats were randomly assigned to be implanted subcutaneously with pellets containing either 17β-estradiol (E2) or placebo (Pla). After 4 wk of treatment, the rats underwent cage-switch stress and, in a separate experiment, a subset received an infusion of angiotensin II. The cage-switch stress rapidly elevated blood pressure (BP) and heart rate (HR) as measured by radiotelemetry in both groups. However, the BP and HR responses to the stress were significantly attenuated in the E2 group compared with the Pla group. An angiotensin II type 1 receptor blocker, losartan, given in drinking water, abolished the difference in the pressor response to stress between the two groups. Moreover, the stress-induced elevation in plasma renin activity and angiotensin II concentration was significant in the Pla group, but not in the E2 group. In addition, the expression of renin mRNA in the kidney was lower in the E2 group relative to the Pla group. Finally, we found that intravenous angiotensin II infusion increased BP and decreased HR to a similar degree in both groups. These results suggest that the inhibitory effects of estrogen on psychological stress-induced activation of the renin-angiotensin system could be at least partially responsible for the suppression of the pressor responses to psychological stress seen in estrogen-replaced ovariectomized rats.

Estradiol replacement attenuates stress‐induced pressor response by regulating renin‐angiotensin system in ovariectomized rats

We examined whether chronic estrogen replacement has suppressive effects on psychological stress‐induced pressor response by attenuating renin‐angiotensin system (RAS) in ovariectomized rats. Female Wistar rats aged 9 wk were ovariectomized. After 4 wk, the rats were assigned either to a placebo‐treated (Pla) group or a group treated with 17β‐estradiol (E2) subcutaneously implanted with either pellet. Two wk later, they were implanted with radiotelemetry devices for blood pressure (BP) and heart rate (HR) measurements, and the half of them were denervated renally. These rats underwent cage‐switch stress 2 wk after the operation. The stress elevated the BP and HR rapidly and continuously both in the Pla and E2 groups. However, these responses to the stress were attenuated significantly in the E2 group compared with the Pla group. An angiotensin II type 1 receptor blocker, losartan, abolished the difference in the BP response to the stress between the two groups. In addition, the stress induced elevation of plasma renin activity and angiotensin II concentration only in the Pla group, but not in the E2 group. Furthermore, the renal sympathetic nerve denervation attenuated the pressor response in the Pla group. These results suggest that estradiol replacement attenuates psychological stress‐induced pressor response by suppressing RAS activation in ovariectomized rats.

Nongenomic effects of estrogen mediate the dose‐related myocardial oxidative stress and dysfunction caused by acute ethanol in female rats (652.19)

Acute ethanol lowers blood pressure (BP) and cardiac output in proestrus and after chronic estrogen (E2) replacement in ovariectomized (OVX) female rats. However, whether rapid nongenomic effects of E2 mediate these hemodynamic effects of ethanol remained unanswered. To test this hypothesis, we investigated the effect of ethanol (0.5, 1 or 1.5 g/kg; i.v.) on left ventricular (LV) function and oxidative markers in OVX rats pretreated 30 min earlier with 1 μg/kg E2 (OVXE2) or vehicle (OVX) and in proestrus sham‐operated (SO) rats. In SO rats, ethanol caused significant and dose‐ related reductions in BP, rate of rise in LV pressure (LV dP/dtmax), and LV developed pressure (LVDP). The ethanol effects disappeared in OVX rats and were restored in OVXE2 rats suggesting rapid estrogen receptor signaling mediates the detrimental LV effects of ethanol. The E2‐dependent LV dysfunction caused by ethanol was associated with higher LV: (i) generation of reactive oxygen species, (ii) expression of the malondialdehyde and 4‐hydroxynonenal protein adducts, (iii) Akt and ERK1/2, and (iv) catalase activity. Collectively, it is concluded that rapid estrogen receptor signaling is implicated in the cellular events that lead to the generation of aldehyde protein adducts and Akt/ERK1/2 phosphorylation, which ultimately mediate the estrogen‐dependent LV oxidative stress and dysfunction caused by ethanol in female rats. Grant Funding Source: Supported by NIAAA [2R01 AA014441‐07]

Nongenomic effects of estrogen mediate the dose-related myocardial oxidative stress and dysfunction caused by acute ethanol in female rats

Acute ethanol lowers blood pressure (BP) and cardiac output in proestrus and after chronic estrogen (E2) replacement in ovariectomized (OVX) female rats. However, whether rapid nongenomic effects of estrogen mediate these hemodynamic effects of ethanol remains unanswered. To test this hypothesis, we investigated the effect of ethanol (0.5 or 1.5 g/kg iv) on left ventricular (LV) function and oxidative markers in OVX rats pretreated 30 min earlier with 1 μg/kg E2 (OVXE2) or vehicle (OVX) and in proestrus sham-operated (SO) rats. In SO rats, ethanol caused significant and dose-related reductions in BP, rate of rise in LV pressure (LV dP/dtmax), and LV developed pressure (LVDP). These effects of ethanol disappeared in OVX rats and were restored in OVXE2 rats, suggesting rapid estrogen receptor signaling mediates the detrimental effects of ethanol on LV function. Ex vivo studies revealed that the estrogen-dependent myocardial dysfunction caused by ethanol was coupled with higher LV 1) generation of reactive oxygen species (ROS), 2) expression of malondialdehyde and 4-hydroxynonenal protein adducts, 3) phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2), and 4) catalase activity. ERK1/2 inhibition by PD-98059 (1 mg/kg iv) abrogated the myocardial dysfunction, hypotension, and the elevation in myocardial ROS generation caused by ethanol. We conclude that rapid estrogen receptor signaling is implicated in cellular events that lead to the generation of aldehyde protein adducts and Akt/ERK1/2 phosphorylation, which ultimately mediate the estrogen-dependent LV oxidative stress and dysfunction caused by ethanol in female rats.

Abstract 203: Chronic and Acute Estrogen Replacement Therapy Has Different Effects in Aged Animals After Experimental Stroke

Introduction: The effect of estrogen replacement therapy (ERT) on stroke incidence and severity has been extensively debated. Clinical trials of ERT demonstrated an increased risk of stroke in treated women, but study participants were well past menopause when therapy was initiated. It has been suggested that detrimental effects of ERT may be unmasked after prolonged periods of hypoestrogenicity. To date, very few studies have examined the effect of ERT in aged animals although the timing of replacement may be critical to the neuroprotective effects of ERT. Hypothesis: We hypothesized that chronic estrogen replacement (CER) initiated in late middle age, would decrease infarct size after an induced stroke whereas acute estrogen replacement (AER) would have no beneficial effects in the aged female brain. Methods: CER was administered to aged C57BL6 mice from 17 to 20 months of age, and AER was initiated at 20 months of age. Both CER and AER treated mice were subjected to 90-minute middle cerebral artery occlusion (MCAO) at 20.5 months of age. Stroke outcomes at 24 hours of MCAO were measured. Protein levels of nuclear factor κB (NFκB), estrogen receptor α (ERα) and serum levels of pro-inflammatory cytokines were also examined. Results: Female mice that received CER showed improved stroke outcomes after MCAO (total infarct: CER vs. vehicle 34.7±2.4% vs. 58.2±2.6%; n=9/gp, p &lt;0.05); whereas females that had AER did not. CER females exhibited diminished levels of NFκB translocation compared to AER females after stroke. AER females demonstrated both an increase in nuclear NFκB and enhanced expression of pro-inflammatory cytokines. The differential NFκB translocation was related to corresponding changes in ERα expression. Aged males benefited from ERT regardless of the timing of initiation of ERT, and had significantly reduced expression of pro-inflammatory markers after stroke compared to age-matched females. Conclusions: AER worsened stroke outcomes whereas CER was protective in aged females. A pro-inflammatory milieu emerges with age in females which was attenuated by CER treatment. Interestingly both AER and CER reduced stroke injury in aged males, suggesting sexually dimorphic effects on inflammation.

Chronic oestrogen replacement in ovariectomised rats attenuates food intake and augments c-Fos expression in the suprachiasmatic nucleus specifically during the light phase

Oestrogen replacement in ovariectomised (OVX) rats has been reported to attenuate food intake, especially during the light phase. To gain better insight into the central mechanism of oestrogen-induced reduction of food intake, we examined the effect of chronic oestrogen replacement in OVX rats on c-Fos expression in the suprachiasmatic nucleus (SCN) and on food intake during the light and dark phases. Eight-week-old female rats were ovariectomised and implanted with either an oestradiol (E2) or a vehicle pellet (Veh) subcutaneously. The animals were housed in an environment with a 12h light-12h dark cycle with the lights on at 07.00 hours. The amount of spontaneous food intake relative to each animal's body weight was significantly less for the E2 group than for the Veh group during the light phase, but there were no differences shown between these groups during the dark phase. There were no differences shown in the number of c-Fos-immunoreactive cells in the SCN in the E2 group compared with the Veh group during the early dark phase (22.00 hours; Zeitgeber time 15.00 (ZT15)), but the number was significantly higher than in the Veh group during the early light phase (10.00hours; ZT3). This finding suggests that chronic oestrogen replacement chronically enhances SCN activity, specifically during the light phase. The oestrogen-induced enhancement of SCN activity during the light phase is possibly involved in the light phase-specific attenuation of food intake by oestrogen replacement in OVX rats.

Estrogen Receptor β Does Not Influence Ischemic Tolerance in the Aged Female Rat Heart

Ischemic heart disease remains the leading cause of morbidityand mortality in aged women, with a 2- to 3-fold increased incidencefollowing menopause. Clinical trials have failed to demonstrate cardioprotectivebenefit from chronic estrogen (E2) replacement therapy, yet protective effects of E2 occur in adult animal models and are in part mediated by non-genomic estrogen receptor α (ERα) signaling. The role of ERβ is less clear. The purpose of the present study was to determine the efficacy of acute ERβ activation on reducing cardiac ischemia/reperfusion (I/R) injury in aged E2-deficient female rats. Hearts were isolated from adult (6mo; n=9), aged (24mo; n=13), and aged ovariectomized (OVX; n=14) F344 rats. Hearts were perfused in Langendorff mode and subjected to 47 min of global I and 60 min of R. Rats were acutely treated with the ERβ-agonist diarylpropionitrile (DPN) 45 min prior to I/R (5μg/kg, s.c.), and mRNA isolated to determine ERβ expression. Here, for the first time, our data suggest that acute treatment with DPN does not affect functional recovery following I/R injury in adult, aged, or aged OVX female rats. Additionally, we were unable to detect ERβ mRNA in the adult or aged female rat myocardium. These data indicate that acute ERβ activation does not impact I/R injury in the female rat heart and bring into question the relevance of non-genomic ERβ signaling during I/R injury. Funding Source: NIH R01 HL091097

Effect of Estrogen on Bladder Nociception in Rats

We assessed the effect of ovariectomy and estrogen replacement on nociceptive responses to bladder distention in a rat model. Female Sprague-Dawley rats (Harlan) underwent ovariectomy or sham surgery. Visceromotor responses (abdominal contractions) to bladder distention were determined 3 to 4 weeks later under isoflurane anesthesia. In rat subsets estrogen was chronically replaced with a subcutaneous estrogen pellet vs a placebo pellet or acutely replaced by subcutaneous injection 24 hours before testing. Effects of estrogen withdrawal were examined in another group of rats by implanting a pellet and explanting the pellet 24 hours before testing. Uterine weight was measured to assess the estrogen dose. Visceromotor responses to bladder distention were significantly less vigorous in ovariectomized rats vs controls. Acute estrogen replacement increased visceromotor responses in these rats but chronic estrogen replacement did not. Sudden chronic estrogen withdrawal resulted in increased visceromotor responses. Uterine weight was consistent with the physiological estrogen dose. Estrogen alone was not sufficient to produce increased nociceptive responses but an acute decrease in estrogen resulted in increased visceromotor responses. These data suggest that the pronociceptive effects of estrogen may be due to a mismatch between peripheral vs central and/or genomic vs nongenomic effects of the hormone, which occur during rapidly decreasing estrogen levels.

Testosterone and estrogen have opposing actions on inflammation-induced plasma extravasation in the rat temporomandibular joint

The present study was designed to test the hypothesis that estrogen exacerbates inflammation of the temporomandibular joint (TMJ). Evans blue dye was used to quantify plasma extravasation (PE) around the rat TMJ. In an initial set of experiments, TMJ PE was compared in naïve intact male and female rats, as well as in both groups after complete Freund's adjuvant (CFA)-induced inflammation of the TMJ. In contrast to our hypothesis, TMJ PE was significantly greater in both naïve and CFA-inflamed male rats than in females. To determine whether these differences were due to gonadal hormones, four additional groups of rats were studied: gonadectomized (Gx) males and females, Gx males with chronic testosterone (T) replacement, and Gx females with chronic estrogen (E) replacement. The sex difference in baseline TMJ PE appeared to reflect the actions of T. However, in the presence of TMJ inflammation, T augmented TMJ PE in males, while E attenuated TMJ PE in females. Changes in PE were also assessed in the contralateral TMJ. Results from this analysis indicated that there is a transient contralateral increase in TMJ PE in females but not males. Given that there is an inverse relationship between PE and joint damage, our results suggest that testosterone may mitigate, but estrogen may exacerbate, TMJ damage, particularly in the presence of overt inflammation. Importantly, our results may help explain both the higher prevalence and severity of temporomandibular disorder pain in females than males.

Vascular Adhesion Protein-1 Plays an Important Role in Postischemic Inflammation and Neuropathology in Diabetic, Estrogen-Treated Ovariectomized Female Rats Subjected to Transient Forebrain Ischemia

Endothelial vascular adhesion protein-1 (VAP-1) facilitates leukocyte adhesion and infiltration. This relates partly to the function of VAP-1 as a semicarbazide-sensitive amine oxidase (SSAO). We examined the effects of VAP-1/SSAO inhibition [via LJP-1207 (N'-(2-phenyl-allyl)-hydrazine hydrochloride)] on pial venular leukocyte adhesion and infiltration (at 2-10 h of reperfusion) and neuropathology (at 72 h of reperfusion) after transient forebrain ischemia (TFI). A model associated with increased postischemic inflammation was used-i.e., diabetic ovariectomized (OVX) female rats given chronic estrogen replacement therapy (ERT). We compared rats treated, either at the onset or at 6 h of reperfusion, with saline or LJP-1207. Additional rats, rendered neutropenic 24 h before TFI, were studied. In saline-treated controls, intravascular accumulation of adherent leukocytes gradually increased, reaching 15 to 20% of the venular area, at which point neutrophil infiltration commenced (at approximately 6 h). In the rats given LJP-1207 at the onset of reperfusion, limited neutrophil adhesion ( approximately 5% maximum) and no infiltration were observed. These results generally paralleled those in neutropenic rats. In rats treated at 6 h of reperfusion, the pattern of neutrophil adhesion was similar to that of the saline-treated group up to 6 h, but further infiltration was essentially prevented. Neurologic outcomes and histopathology were similar to one another in the LJP-1207-treated and neutropenic groups and significantly improved over those in saline-treated controls. Thus, VAP-1-mediated post-TFI leukocyte adhesion/infiltration in diabetic OVX females given chronic ERT contributes substantially to neuropathology. One implication is that specifically preventing leukocyte infiltration provides a substantial measure of neuroprotection. This could explain the finding of LJP-1207 having at least a 6-h therapeutic window in this model.

Influence of Oestrogenic Compounds on Monoamine Transporters in Rat Striatum

Oestrogens have been reported to modulate rat membrane (DAT) and vesicular (VMAT(2)) dopamine transporters. A recent pilot study of postmenopausal women showed that chronic oestrogen replacement therapy increases striatal DAT. In the present study, we first investigated whether the oestrogen receptors alpha and beta mediate the effects of oestradiol on DAT and VMAT(2). Two days after ovariectomy, Sprague-Dawley rats were treated for 2 weeks with oestradiol or specific ligands for oestrogen receptor alpha, 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or oestrogen receptor beta, 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN). Ovariectomy caused a decrease in [(125)I]-3beta-(4-iodophenyl)-tropane-2beta-carboxylic acid isopropyl ester ([(125)I] RTI-121) specific binding to DAT transporters in the middle striatum compared to values for intact rats, and this was reversed by oestradiol replacement therapy. DPN, but not PPT, mimicked the effect of oestradiol. [(125)I] RTI-121 specific binding in the anterior and posterior striatum was not affected by ovariectomy or any of the drug treatments. Second, we investigated whether oestradiol increased DAT specific binding after a longer period of hormonal withdrawal (a model of hormonal withdrawal at menopause) and whether the selective oestrogen receptor modulators (SERMs), tamoxifen and raloxifene, could reproduce the oestradiol-induced increase of [(125)I] RTI-121 specific binding in long-term ovariectomised rats. Four months after ovariectomy, Sprague-Dawley rats were treated for 2 weeks with oestradiol, tamoxifen or raloxifene, and then killed. Ovariectomy decreased [(3)H] RTI-121 specific binding to DAT transporters in the middle striatum compared to values for intact rats. Treatment with oestradiol, tamoxifen and raloxifene reversed this effect. [(125)I] RTI-121 specific binding in anterior and posterior striatum was not affected by ovariectomy or treatment with oestrogen receptor ligands. In both experiments, neither ovariectomy nor the oestrogenic treatments modulated striatal [(3)H] tetrahydrobenazine specific binding to VMAT(2). Overall, these results suggest that oestrogen receptor beta mediates the oestradiol-induced increase of striatal DAT and that oestradiol can increase DAT density even after long-term steroid withdrawal. The results also support the premise that the SERMs tamoxifen and raloxifene exert oestrogenic agonist effects in the brain.

Prolonged therapeutic window associated with pharmacologic blockade of vascular adhesion protein-1 (VAP-1)-related post-ischemic leukocyte adhesion in diabetic, ovariectomized (OVX) female rats given chronic estrogen replacement

Endothelial VAP-1 facilitates post-ischemic leukocyte adhesion and infiltration in cerebral venules (abstract-this meeting). Evidence suggests that this role of VAP-1 relates, in part, to its function as an amine oxidase and the formation of H2O2 and aldehydes. In the present study, we examined whether treatment with a specific inhibitor of the enzymatic function of VAP-1 (LJP-1207) was capable of reducing the neuropathology accompanying transient forebrain ischemia (TFI). We used a model associated with a high level of post-ischemic leukocyte adhesion and infiltration—diabetic (6–8 wks post-streptozotocin) OVX female rats given 1 week of estrogen replacement therapy (ERT). The rats were allowed to recover over 3 days following 20 min TFI. We compared rats treated, either at the onset or at 6 h reperfusion, with saline or LJP-1207 (30 mg/kg iv). Neurologic function was scored each day over the 3-day reperfusion period. The daily scores could range from 0 (no dysfunction) to 18 (severe dysfunction). In controls (n=10), the 3-day score was 13 2; while in the VAP-1-inhibited rats, the scores were 2 1 (LJP-1207 treatment @0 h reperfusion [n=7]) and 6 1 (LJP-1207 treatment at 6 h reperfusion [n=5]) (see figure 1). Both treatment groups were statistically different from control. Findings from our laboratory have shown that leukocyte infiltration, in diabetic OVX females given ERT, commences at >6 h reperfusion (Stroke 35: 1974–8, 2004), and that this transformation is largely prevented by LJP-1207 (abstract-this meeting). These findings, therefore, indicate that VAP-1-mediated post-TFI leukocyte adhesion/infiltration, in diabetic OVX females given chronic ERT, contributes substantially to neuropathology. One intriguing implication of these findings is that preventing leukocyte infiltration may provide a substantial measure of neuroprotection. This could explain the finding of LJP-1207 having at least a 6-hour therapeutic window, in this model.

17β-estradiol potentiates ischemia–reperfusion injury in diabetic ovariectomized female rats

To investigate the effect of 17β-estradiol (E 2) on ischemia–reperfusion (I/R) injury in diabetic ovariectomized female rats. Streptozotocin(STZ)-induced diabetic female rats received E 2 treatment for 2 weeks after ovariectomy (OVX). A period of 90 min of temporary middle cerebral artery occlusion (tMCAO) was used for the study. Rats were evaluated for physiological data including plasma glucose, E 2, MAP, PaCO 2 and PaO 2 before and after tMCAO. P-selectin expression, myeloperoxidase (MPO) enzyme activity and the cerebral infarct volume were analyzed. Results: The infarct volume in the E 2-treated OVX rats is bigger than that in intact and OVX groups. However, there is not a significant different area of cerebral infarct between diabetic OVX and intact rats. Significant upregulation of P-selectin expression and MPO activity of the ischemia–reperfusion hemisphere were observed in E 2 + OVX, intact and OVX groups at 8, 24, 72 h in time manner after tMCAO compared with that of the contralateral hemisphere of cerebral ischemia–reperfusion. Both P-selectin expression and MPO activity in the E 2 + OVX and intact rats are significantly higher than that in the untreated OVX rats. Chronic estrogen replacement therapy (ERT) potentiates the I/R injury in diabetes female rats. This may be related to the increased expression of P-selectin and MPO activity.

Differential effects of estrogen on hippocampal- and striatal-dependent learning

Estrogen’s role in learning and memory may be to predispose animals to use specific cognitive strategies ( Korol & Kolo, 2002). Specifically, estrogen may facilitate hippocampal-dependent learning, while at the same time attenuate striatal-dependent learning. As a stringent test of this hypothesis, place or response learning on an eight-arm radial maze was compared between ovariectomized (OVX) female Long–Evans rats and rats with chronic estrogen replacement (OVX + E; 5 mg 17-β estradiol 60-day release tablet). Reference and working memory errors were monitored separately for both place and response learning tasks. OVX + E rats learned the place task significantly faster than the response task, and faster than OVX rats. OVX rats required fewer days to reach criterion on the response task than OVX + E rats. Estrogen selectively enhanced reference memory performance, but only during place learning. The specific pattern of estrogen effects on learning suggests that future studies include verification of cognitive strategies used by animals.