Stephanie J. Murphy

A G-Protein-Coupled Estrogen Receptor Is Involved in Hypothalamic Control of Energy Homeostasis

Estrogens are involved in the hypothalamic control of multiple homeostatic functions including reproduction, stress responses, energy metabolism, sleep cycles, temperature regulation, and motivated behaviors. The critical role of 17β-estradiol (E2) is evident in hypoestrogenic states (e.g., postmenopause) in which many of these functions go awry. The actions of E2 in the brain have been attributed to the activation of estrogen receptors α and β through nuclear, cytoplasmic, or membrane actions. However, we have identified a putative membrane-associated estrogen receptor that is coupled to desensitization of GABAB and μ-opioid receptors in guinea pig and mouse hypothalamic proopiomelanocortin neurons. We have synthesized a new nonsteroidal compound, STX, which selectively targets the Gαq-coupled phospholipase C–protein kinase C–protein kinase A pathway, and have established that STX is more potent than E2 in mediating this desensitization in an ICI 182, 780-sensitive manner in both guinea pig and mouse neurons. Both E2 and STX were fully efficacious in estrogen receptor α,β knock-out mice. Moreover, in vivo treatment with STX, similar to E2, attenuated the weight gain in hypoestrogenic female guinea pigs. Therefore, this membrane-delimited signaling pathway plays a critical role in the control of energy homeostasis and may provide a novel therapeutic target for treatment of postmenopausal symptoms and eating disorders in females.

Inhalational anesthetics as neuroprotectants or chemical preconditioning agents in ischemic brain

This review will focus on inhalational anesthetic neuroprotection during cerebral ischemia and inhalational anesthetic preconditioning before ischemic brain injury. The limitations and challenges of past and current research in this area will be addressed before reviewing experimental and clinical studies evaluating the effects of inhalational anesthetics before and during cerebral ischemia. Mechanisms underlying volatile anesthetic neuroprotection and preconditioning will also be examined. Lastly, future directions for inhalational anesthetics and ischemic brain injury will be briefly discussed.

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 [14C]-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 (*P < 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.

Effects of Combined Estrogen and Progesterone on Brain Infarction in Reproductively Senescent Female Rats

Recent data from the Womens 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-μg estrogen implant, a 25-μg 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.

Hypertonic Saline Worsens Infarct Volume After Transient Focal Ischemia in Rats

BACKGROUND AND PURPOSE Hypertonic saline (HS) has been advocated as a hyperosmolar agent for the treatment of cerebral edema, especially after traumatic brain injury. We tested the hypothesis that continuous intravenous HS administered during reperfusion from transient focal cerebral ischemia attenuates infarct volume. METHODS Halothane-anesthetized male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO) by the intraluminal occlusion technique. At the onset of reperfusion, rats received a 10-mL/kg intravenous bolus of 0.9% saline (SAL, n=8) or 7.5% SAL (chloride:acetate 50:50, n=8) followed by a continuous infusion for 22 hours. In a second series of experiments, ischemic damage was determined in cohorts treated with equivolumetric 3% saline (n=8) or 20% mannitol (n=8). In a third series, regional cerebral blood flow was measured ([(14)C]iodoantipyrine autoradiography) at 6 hours of reperfusion in 7.5%-SAL-treated (n=5) or SAL-treated (n=5) animals. RESULTS In SAL rats, serum Na(+) was 137+/-3 and 138+/-2 mEq/L (mean+/-SEM) at baseline and 22 hours of reperfusion, respectively. In 7.5% SAL, serum Na(+) was 136+/-2 and 154+/-2 mEq/L at baseline and reperfusion, respectively. Physiological variables and reduction in laser-Doppler signal during MCAO and early reperfusion were not different between the 2 treatment groups. Cortical infarct volume was larger in 7.5%-SAL-treated rats (121+/-14 mm(3); 30+/-3% of contralateral cortex; P<0.05) than in SAL (64+/-15 mm(3); 16+/-4% of contralateral cortex). Striatal infarct volume was unchanged by HS therapy. Ipsilateral cortical tissue volume was increased relative to the contralateral side (by 26+/-5% with SAL; by 41+/-5% with 7.5% SAL). In contrast, ischemic damage was unaffected by 3%-SAL or 20%-mannitol treatment compared with SAL. Regional cerebral blood flow during reperfusion was heterogeneous in all animals, but there was no evidence of postischemic hypoperfusion or blood flow maldistribution in 7.5%-SAL-treated animals. CONCLUSIONS These data demonstrate that hypernatremia resulting from postischemic HS infusion worsens cortical infarct volume in transient focal cerebral ischemia. The deleterious effect is not linked to exacerbation of delayed hypoperfusion during early reperfusion (6 hours); however, blood flow defects at later recovery time points remain to be excluded. These results may have implications for HS therapy in clinical ischemic stroke.

Gender-specific response to isoflurane preconditioning in focal cerebral ischemia

Inhalation anesthetics are effective chemical preconditioning agents in experimental cerebral ischemia. However, previous work has been performed exclusively in male animals. We determined if there is a gender difference in ischemic outcome after isoflurane preconditioning (IsoPC), and if this sex-specific response is linked to differences in Akt phosphorylation or expression of neuronal inducible cell-death putative kinase (NIPK), a negative modulator of Akt activation. Young and middle-aged male and female mice were preconditioned for 4 h with air (sham PC) or 1.0% IsoPC and recovered for 24 h. Cortices were subdissected from preconditioned young male and female mice for measurement of Akt phosphorylation (Western blot) and NIPK mRNA (quantitative polymerase chain reaction). Additional cohorts underwent 2 h of reversible middle cerebral artery occlusion. Lastly, male and female Akt1+/+ and Akt1−/– mice were studied to determine if gender differences in ischemic outcome after IsoPC is Akt1-dependent. Infarction volume was determined at 22 h reperfusion (2,3,5-triphenyltetrazolium chloride). As expected, IsoPC decreased ischemic damage as compared with sham PC in young and middle-aged male mice. In contrast, IsoPC markedly increased infarction in young female mice and had no effect in middle-aged female mice. Cortical phospho-Akt was increased by IsoPC versus sham PC only in male mice. No increase was observed in IsoPC female mice. NIPK mRNA was higher in female mice than in male mice regardless of preconditioning status. Male IsoPC neuroprotection was lost in Akt1-deficient male mice. We conclude that IsoPC is beneficial only in ischemic male brain and that sex differences in IsoPC are mediated through Akt activation and basal NIPK expression.

A novel method for assessing sex-specific and genotype-specific response to injury in astrocyte culture

Female astrocytes sustain less cell death from oxygen-glucose deprivation (OGD) than male astrocytes. Arimidex, an aromatase inhibitor, abolishes these sex differences. To verify sex-dependent differences in P450 aromatase function in astrocyte cell death following OGD, we developed a novel method that uses sex-specific and genotype-specific single pup primary astrocyte cultures from wild-type (WT) and aromatase-knockout (ArKO) mice. After determining sex by external and internal examination as well as PCR and genotype by PCR amplification of tail cDNA, we established cultures from 1-3-day-old male and female WT and ArKO mice pups and grew them to confluence in estrogen-free media. Cell death was measured by lactate dehydrogenase (LDH) assay. Our study shows that, while WT female astrocytes are more resistant to OGD than WT male cells, sex differences disappear in ArKO cells. Cell death is significantly increased in ArKO compared to WT in female astrocytes but not male cells. Therefore, P450 aromatase appears to be essential in endogenous neuroprotection in females, and this finding may have clinical implications. This innovative technique may also be applied to other in vitro studies of sex-related functional differences.

Estrogen Is Renoprotective via a Nonreceptor-dependent Mechanism after Cardiac Arrest In Vivo

Background:Severe ischemia induces renal injury less frequently in women than men. In this study, cardiac arrest and cardiopulmonary resuscitation were used to assess whether estradiol is renoprotective via an estrogen receptor (ER)-dependent mechanism. Materials and Methods:Male and female C57BL/6 and ER gene-deleted mice underwent 10 min of cardiac arrest followed by cardiopulmonary resuscitation. Serum chemistries and renal stereology were measured 24 h after arrest. Results:Estrogen did not affect mean arterial pressure, regional renal cortical blood flow, and arterial blood gases. Hence, female kidneys were protected (mean ± SEM: blood urea nitrogen, 65 ± 21 vs.149 ± 27 mg/dl, P = 0.04; creatinine, 0.14 ± 0.05 vs. 0.73 ± 0.16 mg/dl, P = 0.01; volume of necrotic tubules, 7 ± 1% vs. 10 ± 0%, P = 0.04). Estrogen also reduced renal injury. In intact females (n = 5), ovariectomized/vehicle-treated (n = 8), and ovariectomized/estrogen-treated (n = 8) animals, blood urea nitrogen was 65 ± 21, 166 ± 28, and 50 ± 14 mg/dl (P = 0.002); creatinine was 0.14 ± 0.05, 0.74 ± 0.26, and 0.23 ± 0.27 mg/dl (P = 0.014); necrotic tubules were 2.5 ± 0.25%, 12.0 ± 1.9%, and 5.0 ± 1.6% (P = 0.004), respectively. In ER-α and ER-β gene-deleted mice and controls estradiol-reduced functional injury (blood urea nitrogen: estradiol 117 ± 71, vehicle 167 ± 56, P = 0.007; creatinine: estradiol 0.5 ± 0.5, vehicle 1.0 ± 0.4, P = 0.013), but the effect of estradiol was not different between ER-α or ER-β gene-deleted mice. Adding ICI 182,780 to estradiol did not alter injury. Conclusions:In women, kidneys were protected from cardiac arrest through estrogen. Estradiol-mediated renoprotection was not affected by ER deletion or blockade. Estradiol is renoprotective after cardiac arrest. The results indicate that estradiol renoprotection is ER-α and ER-β independent.

ISOFLURANE PRECONDITIONING NEUROPROTECTION IN EXPERIMENTAL FOCAL STROKE IS ANDROGEN-DEPENDENT IN MALE MICE

Isoflurane preconditioning neuroprotection in experimental stroke is male-specific. The role of androgens in the ischemic sensitivity of isoflurane preconditioned male brain and whether androgen effects are androgen receptor dependent were assessed. Male C57BL/6 mice were implanted with flutamide (androgen receptor antagonist), or castrated and implanted with testosterone, dihydrotestosterone, flutamide, letrozole (aromatase inhibitor), or vehicle 7-13 days before preconditioning. P450 estrogen aromatase wild-type and knockout mice were also evaluated. All mice were preconditioned for 4 h with 0% (sham preconditioning) or 1% isoflurane (isoflurane preconditioning) and recovered for 24 h. Mice then underwent 2 h of middle cerebral artery occlusion and were evaluated 22 h later for infarct volume. For neurobehavioral outcomes, sham and isoflurane preconditioned castrated male+/-dihydrotestosterone groups underwent 1 h of middle cerebral artery occlusion followed by 9 days of reperfusion. Isoflurane preconditioning neuroprotection relative to infarct volume outcomes were testosterone and dihydrotestosterone dose-specific and androgen receptor-dependent. Relative to long-term neurobehavioral outcomes, front paw sensorimotor function improved in isoflurane preconditioned mice regardless of androgen status while androgen replacement independently improved sensorimotor function. In contrast, isoflurane preconditioning improved cognitive function in castrates lacking endogenous androgens, but this improvement was absent in androgen replaced mice. Our findings suggest that androgen availability during isoflurane preconditioning may influence infarct volume and neurobehavioral outcomes in male mice following experimental stroke.

PD-L1 enhances CNS inflammation and infarct volume following experimental stroke in mice in opposition to PD-1

BackgroundStroke severity is worsened by recruitment of inflammatory immune cells into the brain. This process depends in part on T cell activation, in which the B7 family of co-stimulatory molecules plays a pivotal role. Previous studies demonstrated more severe infarcts in mice lacking programmed death-1 (PD-1), a member of the B7 family, thus implicating PD-1 as a key factor in limiting stroke severity. The purpose of this study was to determine if this protective effect of PD-1 involves either of its ligands, PD-L1 or PD-L2.MethodsCentral nervous system (CNS) inflammation and infarct volume were evaluated in male PD-L1 and PD-L2 knockout (-/-) mice undergoing 60 minutes of middle cerebral artery occlusion (MCAO) followed by 96 hours of reperfusion and compared to wild-type (WT) C57BL/6J mice.ResultsPD-L1-/- and PD-L2-/- mice had smaller total infarct volumes compared to WT mice. The PD-L1-/- and to a lesser extent PD-L2-/- mice had reduced levels of proinflammatory activated microglia and/or infiltrating monocytes and CD4+ T cells in the ischemic hemispheres. There was a reduction in ischemia-related splenic atrophy accompanied by lower activation status of splenic T cells and monocytes in the absence of PD-L1, suggesting a pathogenic rather than a regulatory role for both PD-1 ligands (PD-Ls). Suppressor T cells (IL-10-producing CD8+CD122+ T cells) trafficked to the brain in PD-L1-/- mice and there was decreased expression of CD80 on splenic antigen-presenting cells (APCs) as compared to the WT and PD-L2-/- mice.ConclusionsOur novel observations are the first to implicate PD-L1 involvement in worsening outcome of experimental stroke. The presence of suppressor T cells in the right MCAO-inflicted hemisphere in mice lacking PD-L1 implicates these cells as possible key contributors for controlling adverse effects of ischemia. Increased expression of CD80 on APCs in WT and PD-L2-/- mice suggests an overriding interaction leading to T cell activation. Conversely, low CD80 expression by APCs, along with increased PD-1 and PD-L2 expression in PD-L1-/- mice suggests alternative T cell signaling pathways, leading to a suppressor phenotype. These results suggest that agents (for example antibodies) that can target and neutralize PD-L1/2 may have therapeutic potential for treatment of human stroke.