Monique C. Saleh

Autonomic and cardiovascular reflex responses to central estrogen injection in ovariectomized female rats

The role of estrogen in central autonomic nuclei was examined in ovariectomized female Sprague-Dawley rats supplemented daily for 7 days with either estrogen (5 microg/kg; sc) or saline (0.9%; sc). Animals were subsequently anaesthetized with sodium thiobutabarbital (Inactin; 100 mg/kg; ip) and instrumented to record blood pressure and heart rate. Efferent vagal parasympathetic (VPNA) and renal sympathetic (RSNA) nerve activities were recorded and used to assess baseline and reflexive changes in autonomic tone. The cardiac baroreflex was evoked using a single bolus injection of phenylephrine (0.1 mg/kg) both before and following either intrathecal injection of estrogen (0.5 microM; 1 microl) or bilateral injection of estrogen (0.5 microM; 100 nl/side) into several central autonomic nuclei. In estrogen-replaced rats, both the baseline and PE-evoked values for mean arterial pressure and RSNA were significantly decreased following injection of estrogen into the nucleus tractus solitarius (NTS), rostral ventrolateral medulla (RVLM), parabrachial nucleus (PBN), central nucleus of the amygdala (CNA) and the intrathecal space. Baseline heart rate and VPNA were significantly decreased following injection of estrogen into NTS, nucleus ambiguous (Amb), PBN and the intrathecal space. PE-evoked changes in heart rate and VPNA were significantly enhanced following injection of estrogen into these same nuclei. Injection of estrogen into the insular cortex (IC) produced significant decreases in baseline and PE-evoked RSNA only. The cardiac baroreflex was significantly enhanced following injection of estrogen into all nuclei and the intrathecal space. In saline-replaced females, injection of estrogen into NTS, RVLM, Amb and the intrathecal space had similar effects on both baseline and PE-evoked parameters although of a reduced magnitude compared to estrogen-replaced rats. However, no significant changes in autonomic tone and baroreflex function were observed following the injection of estrogen into the PBN, CNA or IC of saline-replaced rats. These results demonstrate a role for estrogen in central autonomic nuclei in female rats and suggest a possible alteration of estrogen receptor distribution or efficacy within the central nervous system of estrogen-deficient female rats.

Uncoupling protein-2: evidence for its function as a metabolic regulator

Uncoupling protein-2, discovered in 1997, belongs to a family of inner mitochondrial membrane proteins that, in general, function as carriers. The function(s) of uncoupling protein-2 have not yet been definitively described. However, mounting evidence suggests that uncoupling protein-2 could act in multiple tissues as a regulator of lipid metabolism. A role as a modulator of reactive oxygen species as a defence against infection is also postulated. In this review, a brief overview of the general and specific properties of uncoupling protein-2 is given and evidence for metabolic and immune regulatory functions is summarized. Uncoupling protein-2 could have particular importance in the regulation of lipid metabolism in adipose tissue and skeletal muscle. In addition, its ability to inhibit insulin secretion could also promote fat utilization over storage. Inhibition by uncoupling protein-2 of reactive oxygen species formation in macrophages and other tissues could have implications for regulation of immune function. The possibility of functions of uncoupling protein-2 in other tissues such as the brain are beginning to emerge. [Diabetologia (2002) 45: 174–187]

Acute injection of 17β-estradiol enhances cardiovascular reflexes and autonomic tone in ovariectomized female rats

Abstract Among the many benefits of long-term hormone replacement therapy to postmenopausal women is a significant reduction in risk for and progression of cardiovascular disease. However, long-term estrogen replacement therapy has been associated with several undesirable, and likely dose-dependent, side-effects. There is some evidence to suggest that the dose of estrogen which confers optimal beneficial effects on the cardiovascular system is much lower than that which is currently prescribed for postmenopausal women. The following experiments were conducted to determine the dose–response relationship of acutely administered estrogen on autonomic tone and reflex control of heart rate in ovariectomized Sprague–Dawley female rats. Rats were anaesthetized with sodium thiobutabarbital (100 mg/kg) and instrumented to record blood pressure, heart rate and efferent parasympathetic and sympathetic nerve activities. The sensitivity of the cardiac baroreflex was tested using intravenous injection of either phenylephrine hydrochloride (0.025–0.1 mg/kg) or sodium nitroprusside (0.0025–0.01 mg/kg). Intravenous injection of estrogen produced dose-dependent increases in the magnitude of the baroreflex sensitivity and parasympathetic tone while reducing sympathetic tone with a maximal effect observed at 1×10−3 mg/kg. Prior administration of the selective estrogen receptor antagonist, ICI 182,780 blocked the estrogen-induced changes in baroreflex sensitivity and autonomic tone. These results demonstrate that acutely administered, low-dose estrogen has beneficial effects on autonomic tone and cardiovascular reflexes.

Medullary and intrathecal injections of 17β-estradiol in male rats

The following experiments were designed to investigate the role of estrogen in central autonomic nuclei on autonomic tone and reflex control of heart rate. Male Sprague-Dawley rats were anesthetized with sodium thiobutabarbital (100 mg/kg) and instrumented to record blood pressure and heart rate. Efferent vagal and renal nerve activities were recorded and used to assess changes in parasympathetic and sympathetic tone, respectively. The cardiac baroreflex was evoked using a single bolus injection of phenylephrine (0.1 mg/kg) both before and following either intrathecal injection of estrogen (0.5 microM; 1 microl) to influence sympathetic preganglionic neurons of the intermediolateral cell column or bilateral injection of estrogen (0.5 microM; 100 nl/side) into the nucleus tractus solitarius, rostral ventrolateral medulla or nucleus ambiguus. The cardiac baroreflex was significantly enhanced following both intrathecal and medullary injections of estrogen. Efferent vagal nerve activity was significantly increased following injection of estrogen into the nucleus tractus solitarius, nucleus ambiguus and the intrathecal space. Renal sympathetic nerve activity was significantly depressed following injection of estrogen into the nucleus tractus solitarius, rostral ventrolateral medulla and the intrathecal space. In all cases, simultaneous injection of estrogen with the selective estrogen receptor antagonist, ICI 182,780 (1 pM) blocked all previously observed changes in baroreflex function and autonomic tone. These results demonstrate a role for estrogen in the reflex control of heart rate and as a central modulator of autonomic tone in male rats.

Lipoic acid protects against reperfusion injury in the early stages of cerebral ischemia.

Lipoic acid (LA) is a known antioxidant currently used as a therapy in patients with vascular and metabolic disorders. We tested the hypothesis that lipoic acid is protective against the cell death observed following stroke. Lipoic acid was administered 30minutes prior to, or immediately following removal of sutures used to occlude the middle cerebral artery (MCA) in male Sprague-Dawley rats. Following removal of the sutures, the MCA territory was allowed to undergo 5.5hrs of reperfusion. This ischemia/reperfusion (I/R) resulted in a focal infarct restricted to the prefrontal cortex (24±3mm(3)). Pretreatment with LA 30minutes prior to occlusion resulted in a dose-dependent reduction in infarct volume. This reduction in infarct volume was not observed when the LA was administered immediately prior to reperfusion (30minutes post-occlusion). To investigate a potential hemodynamic mechanism for this LA-induced neuroprotection, blood pressure, heart rate and baroreceptor reflex sensitivity (BRS) were measured. Intravenous administration of LA did not result in any significant changes in any of these parameters compared to saline-treated rats. Similarly, there was no significant contribution of systemic nitric oxide or alteration in cerebral perfusion measured following pretreatment with lipoic acid or during the course of occlusion and reperfusion compared with saline-treated rats. Western blot analysis of tissue from the ischemic cortex showed an increase in protein expression of superoxide dismutase (SOD2), but not SOD1, in LA pretreated rats. This suggests a potential mechanism of action contributing to the LA-induced neuroprotection observed. Furthermore, the data in the present investigation suggest the potential use of LA pretreatment as a neuroprotectant in stroke patients.

Resveratrol induced neuroprotection is mediated via both estrogen receptor subtypes, ERα and ERβ

Resveratrol, a dietary polyphenol with antioxidant and anti-inflammatory activity, has been shown to provide neuroprotection in models of ischemia. However, the mechanism of action of resveratrol-induced neuroprotection remains unclear. Previous work in our laboratory has provided evidence that acute, systemic administration of resveratrol is neuroprotective in a permanent model of cerebral ischemia, an effect that was blocked when animals received the non-selective estrogen receptor antagonist, ICI, 182,780. The present study was designed to investigate whether the source of neuroprotection afforded by resveratrol action within the cerebral cortex itself is mediated preferentially via selective activation of either α or β estrogen receptor subtype. Intracortical injection of resveratrol (0.1 and 1.0 μM) 10 min prior to 30 min of ischemia followed by 5.5h of reperfusion significantly reduced infarct volume in the prefrontal cortex. This neuroprotective effect was significantly attenuated when resveratrol injection (1.0 μM) was preceded by injection of a selective estrogen receptor α antagonist, 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1N-pyrozole dihydrochloride (MPP) or a selective estrogen receptor beta (ERβ) antagonist, 4-[2-phenyo-5,7-bis(trifluoromrthyl)pyrazolo(1,5-a)pyrimidin-3-yl]phenol (PHTPP). These results provide evidence for rapidly induced neuroprotection mediated by resveratrol activation of either estrogen receptor subtype within the ischemic cortex of rats.

Resveratrol preconditioning induces cellular stress proteins and is mediated via NMDA and estrogen receptors

Resveratrol pretreatment has been shown to provide neuroprotection in models of cerebral ischemia. This phenomenon, commonly termed preconditioning, promotes ischemic tolerance and may involve mild activation of endoplasmic reticulum stress pathways in the affected tissue. Systemic injection of resveratrol (2 x 10(-3), 2 x 10(-4), 1 x 10(-4) mg/kg) 30 min prior to a 4 h period of right middle cerebral artery occlusion significantly reduced infarct area in the insular region of rat prefrontal cortex. This affect was blocked when resveratrol treatment was combined with a non-selective estrogen receptor antagonist, or preceded by intracortical injection of an NMDA receptor antagonist. The neuroprotective effect of resveratrol was associated with reduced renal sympathetic nerve activity as well as induction of resident endoplasmic reticulum chaperone proteins, glucose-regulated proteins 78 and 94. The calcium-sensitive chaperone heat shock protein 70 and the cysteine protease m calpain did not respond to resveratrol pretreatment. However, a significant induction of heat shock protein 70 was observed in the contralateral cortex of resveratrol pretreated rats following 4 h of right middle cerebral artery occlusion. These data suggest that resveratrol preconditioning promotes ischemic tolerance in the short term, in part via effects mediated through activation of estrogen and NMDA receptors, as well as through mild activation of cellular stress proteins.

Apocynin may limit total cell death following cerebral ischemia and reperfusion by enhancing apoptosis.

The present study was designed to determine a dose-response relationship between apocynin and infarct volume as well as to provide a possible molecular mechanism mediating this effect. We tested the hypothesis that apocynin protects against cell death following stroke and reperfusion injury. Apocynin was administered 30 min prior to, or immediately following removal of sutures used to occlude the middle cerebral artery (MCA) in male Sprague-Dawley rats. Following removal of the sutures, the MCA was allowed to undergo 5.5h of reperfusion. Pretreatment with apocynin 30 min prior to occlusion resulted in a dose-dependent reduction in infarct volume by ∼50 %. Analysis of tissue from the ischemic cortex of apocynin-treated rats showed an increase in the level of glutathione (GSH), protein adducts (HNE-His), hydrogen peroxide (H(2)O(2)) and DNA fragmentation (apoptotic cell death) was also observed. This suggests that apocynin may increase antioxidant defense systems (GSH) to limit the degree of ischemia-induced cellular stress. In addition, this moderate cell stress results in more apoptotic vs necrotic cell death, and thus may limit the spreading depression and total cell death that occurs following ischemia/reperfusion. These effects may serve as a potential novel mechanism of action contributing to the apocynin-induced neuroprotection observed.

Inhibitory effect of 17β-estradiol in the parabrachial nucleus is mediated by GABA

In the present investigation, electrophysiological recordings of thalamic relay neurons were used to investigate the role of estrogen as a modulator of visceral afferent information through the PBN to forebrain structures. Experiments were done in anaesthetized (sodium thiobutabarbitol; 100 mg/kg) male and ovariectomized female rats supplemented for 7 days prior with either 17beta-estradiol (OVX-E(2)) or saline (OVX-S). A portion of the right cervical vagus was isolated for the electrical activation (0.8 Hz, 2 ms duration) of visceral afferents. The evoked single and multi-unit activity was recorded via a recording electrode in the ventrobasal thalamus. Exogenous microinjection of 17beta-estradiol (0.1, 0.25 and 0.5 microM; 200 nl) into the parabrachial nucleus (PBN) produced a significant, dose-dependent attenuation in the magnitude of visceral afferent activation-evoked responses of neurons recorded in the thalamus in both male and OVX-E(2) groups. No effect on evoked thalamic activity was observed following injection of estrogen into the PBN of OVX-S animals. Co-injection of estrogen with the GABA(A) receptor antagonist, bicuculine (0.1 microM; 200 nl) but not phaclofen (GABA(B); 0.1, 0.5 or 1 microM; 200 nl) resulted in an increase in the evoked thalamic response in males (55+/-11%) and OVX-E(2) female (68+/-15%) rats. These studies suggest that estrogen inhibits neurotransmission in the PBN via an interaction with the GABA(A) receptor to modulate the flow of visceral information to the thalamus.

Guanosine protects against reperfusion injury in rat brains after ischemic stroke.

After ischemic stroke, early thrombolytic therapy to reestablish tissue perfusion improves outcome but triggers a cascade of deleterious cellular and molecular events. Using a collaborative approach, our groups examined the effects of guanosine (Guo) in response to ischemic reperfusion injury in vitro and in vivo. In a transient middle cerebral artery occlusion (MCAO) in rats, Guo significantly reduced infarct volume in a dose‐dependent manner when given systemically either immediately before or 30 min, but not 60 min, after the onset of the 5.5‐hr reperfusion period. In a separate experiment, Guo significantly reduced infarct volume after 24 hr of reperfusion when administered 5 min before reperfusion. Western blot analysis did not reveal any significant changes either in endoplasmic reticulum (ER) stress proteins (GRP 78 and 94) or HSP 70 or in levels of m‐calpain. In vitro oxygen and glucose deprivation (OGD) significantly increased production of both reactive oxygen species (ROS) and interleukin‐8 (IL‐8) in the primary astrocytes. Guo did not alter ROS or IL‐8 production when given to the astrocytes before OGD. However, Guo when added to the cells prior to or 30 min after reperfusion significantly reduced IL‐8 release but not ROS formation. Our study revealed a dose‐ and time‐dependent protective effect of Guo on reperfusion injury in vitro and vivo. The mechanisms by which Guo exerts its effect are independent of unfolded proteins in ER or the level of intracellular calcium or ROS formation. However, the effect may be induced, at least partially, by inhibiting IL‐8, a marker of reperfusion‐triggered proinflammatory events.