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 5.5 h 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

Role of estrogen in central nuclei mediating stroke-induced changes in autonomic tone

The current investigation examined the role of estrogen in central autonomic regulatory nuclei on the autonomic dysfunction resulting from middle cerebral artery occlusion (MCAO). Experiments were done in anaesthetized male Sprague-Dawley rats. The effect of MCAO on autonomic tone was assessed by monitoring vagal and renal efferent nerve activities before and following systemic administration of either estrogen or saline and the bilateral microinjection of the estrogen receptor antagonist, ICI 182, 780, into several autonomic nuclei (the intrathecal space of the spinal cord, nucleus tractus solitarius, nucleus ambiguus, rostral ventrolateral medulla, parabrachial nucleus, central nucleus of the amygdala or ventral posteromedial thalamus). Autonomic reflex function was evoked using intravenous injection of increasing doses of phenylephrine (0.025–0.1 mg/kg) and the peak changes in heart rate and blood pressure were plotted to obtain the baroreflex sensitivity. The presence of ICI 182, 780 in the intrathecal space of the spinal cord, nucleus ambiguous, nucleus tractus solitarius, rostral ventrolateral medulla, parabrachial nucleus, or central nucleus of the amygdala prior to the administration of estrogen resulted in a significant attenuation (ranging from 79% to 94 %) in the estrogen-induced recovery of autonomic function following MCAO. Blocking estrogen receptors in the ventral posteromedial thalamus had no effect on the ability of estrogen to prevent the MCAO-induced changes in autonomic function. These results suggest that the estrogen-mediated recovery of autonomic function following MCAO is dependent on the availability of estrogen receptors in several forebrain and brainstem autonomic nuclei

The role of dopaminergic neurodegeneration in equine pituitary pars intermedia dysfunction (equine Cushing's disease)

The objective of this study is to demonstrate the role of oxidative stress and dopaminergic neurodegeneration in the development of equine pituitary pars intermedia dysfunction (PPID). The results support the hypothesis that PPID is a dopaminergic neurodegenerative disease. Horses with PPID had 5 times less tyrosine hydroxylase staining, indicating five-fold reduction in the number of dopaminergic terminals in the pars intermedia.

Cocaine sensitization does not alter SP effects on locomotion or excitatory synaptic transmission in the NAc of rats

Substance P (SP) and cocaine employ similar mechanisms to modify excitatory synaptic transmission in the nucleus accumbens (NAc), a region implicated in substance abuse. Here we explored, using NAc slices, whether SP effects on these synaptic responses were altered in rats that have been sensitized to cocaine and whether SP could mimic cocaine in triggering increased locomotion in sensitized rats. Intraperitoneal (IP) injection of naïve rats with cocaine (15 mg/kg) caused increased locomotion by 408.5 ± 85.9% (n = 5) which further increased by 733.1 ± 157.8% (n = 5) following a week of cocaine sensitization. A similar challenge with 10 mg/kg of SP after cocaine sensitization did not produce significant changes in locomotion (170.6 ± 61.0%; n = 4). In contrast to cocaine, IP injection of rats with SP or SP5–11 (10–100 mg/kg) with or without phosphoramidon did not elicit changes in locomotion. In electrophysiological studies, both cocaine and SP depressed evoked NMDA and non-NMDA receptor-mediated excitatory synaptic currents (EPSCs) in slices obtained from naïve rats. In slices derived from cocaine-sensitized rats, cocaine but not SP produced a more profound decrease in non-NMDA compared to NMDA responses. Similar to that in naïve rats, cocaine’s effect on the EPSCs in these sensitized rats occluded those of SP. Thus, although SP and cocaine may employ similar mechanisms to depress EPSCs in the NAc, IP injection of SP does not mimic cocaine-induced hyperlocomotion indicating that not all of cocaine’s effects are mimicked by SP. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’

UPEI-300, A Conjugate Of Lipoic Acid And Edaravone, Mediates Neuroprotection In Ischemia/Reperfusion

Edaravone, an electron spin trapper with radical scavenging activity, has been shown to be effective in reducing infarct volume in humans following ischemic stroke. However, concerns of edaravone-induced renal toxicity have limited its clinical adoption. Previous work has demonstrated that edaravone produced significant neuroprotection when injected prior to a period of ischemia and/or reperfusion. The current investigation was designed to determine if a newly synthesized co-drug consisting of lipoic acid and edaravone, named UPEI-300, could produce neuroprotection in in vitro and/or an in vivo rodent model of stroke. UPEI-300 produced dose-dependent neuroprotection in vitro and was subsequently tested in vivo. Male rats were anaesthetized and the middle cerebral artery was occluded for 30 min followed by 5.5 hrs of reperfusion (ischemia/reperfusion; I/R). Pre-administration of UPEI-300 dose-dependently decreased infarct volume. Significant neuroprotection was also observed when UPEI-300 (1.0 mg/kg) was injected during the 30 min period of ischemia as well as up to 60 mins following the start of reperfusion. These results indicate that a co-drug consisting of edaravone and lipoic acid is a potent neuroprotectant, and clinically, the use of such a novel co-drug following an ischemic stroke might maintain neuroprotection while potentially decreasing edaravone associated renal toxicit

Guanosine protects against reperfusion injury in rat brain 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. © 2012 Wiley Periodicals, Inc. Nearly 80% of strokes are caused by occlusion of the middle cerebral artery (MCA) by a thrombus. Thus, effective stroke therapies require recanalization of occluded cerebral blood vessels. During cerebral ischemia, cerebral blood flow (CBF) is reduced by the occlusion of blood vessels that supply vital oxygen to brain tissues. Reperfusion strategies (i.e., early intravenous thrombolytic therapy) to reestablish tissue perfusion in order to reduce neurological deficits and improve functional outcome have been the most effective therapies (The National Institute of Neurological Disorders and Stroke, 1995; Hacke et al., 2008; Fisher, 2011). However, reperfusion of ischemic brain tissue can also have harmful consequences, including breakdown of the blood–brain barrier, which can lead to cerebral edema and/or brain hemorrhage as well as neurovascular injury and neuronal death. In the acute phase of reperfusion injury (within the first 6 hr), reperfusion after ischemia causes oxidative stress, which is the result of overproduction of reactive oxygen species (ROS) in mitochondria. This, in turn, triggers many cellular and molecular events, including protein oxidation, lipid peroxidation, and DNA damage, which can induce cell death (Sugawara and Chan, 2003; Saito et al., 2005; Jung et al., 2010). Reperfusion injury also recruits large numbers of inflammatory cells and causes other cellular and molecular cascades (Kidwell et al., 2000; Li et al., 2000; Warach and Latour, 2004). The complications resulting from reperfusion injury reduce the effectiveness of thrombolytic therapy, hence the narrow therapeutic window (within 4–6 hr following stroke onset) of many thrombolytics on the market today. This has led to a search for strategies to protect the brain against such injuries or at least to limit these effects. Ultimately, the development of neuroprotective drugs with multiple effects on the ischemic cascade will impede the tissue and cellular consequences of the vascular occlusion and its removal (Endres et al, 2008; Fisher, 2011). The purine nucleoside guanosine (Guo) has been shown to exist extracellularly and, like adenosine, to be an intercellular messenger demonstrating a plethora of both trophic and neuroprotective effects in vitro and in vivo (Rathbone et al., 1999, 2008; Ciccarelli et al., 2001; Traversa et al., 2002; Di Iorio et al., 2002, 2004; Pettifer et al., 2004, 2007; Moretto et al., 2005, 2009; Jiang et al., 2007, 2008a, b; Tavares et al., 2008; Su et al., 2009; Torres et al., 2010). Interestingly, after focal stroke in rats, Guo is elevated within 2 hr and remains high for 7 days (Uemura et al., 1991). This led to the investigation of the effects of exogenously administered Guo in stroke models, specifically, protection against combined oxygen and glucose deprivation (OGD) in vitro (Chang et al., 2008) and protection against stroke in an ex vivo model (Moretto et al., 2005, 2009), as well as in an in vivo rodent model of permanent ischemic stroke by MCA occlusion (MCAO; Rathbone et al., 2011). In the present study, our groups collaborated in examining the neuroprotective effects of Guo in response to reperfusion injury using two different in vivo rat models of transient MCAO. We examined the effect of Guo not only on infarct size but also on molecular chaperones such as glucose-regulated proteins (GRP 78 and GRP 94) and the cytosolic heat-inducible protein HSP 70. Their induction has been shown to increase when protein synthesis is interrupted following ischemia, and unfolded proteins accumulate in the lumen of the endoplasmic reticulum (ER) as a result of changes in oxidative status and calcium homeostasis. In addition, the widespread overactivation of glutamate receptors in response to focal ischemia can produce a sudden increase in intracellular calcium concentrations, resulting in activation of the cysteine protease m-calpain (Siman and Noszek, 1988; Choi, 1995). Sustained calpain activation can result in cellular apoptosis (Sareen et al., 2007), and inhibition of calpain has been shown to provide neuroprotection in a model of focal ischemia (Hong et al., 1994). Therefore, we investigated the effect of Guo on m-calpain after transient cerebral ischemia. We also examined whether Guo has any effect on ROS formation and the inflammatory events in primary cultured astrocytes after induction of OGD in a model of in vitro reperfusion–ischemia

Relaxin peptide hormones are protective during the early stages of ischemic stroke in male rats

The pregnancy hormone relaxin protects tissue from ischemic damage. The ability of relaxin-3, a relaxin paralog, to do so has not been explored. The cerebral expression of these peptides and their receptors make them logical targets for study in the ischemic brain. We assessed relaxin peptide-mediated protection, relative relaxin family peptide receptor (RXFP) involvement and protective mechanisms. Sprague-Dawley rats receiving permanent or transient middle cerebral artery occlusions (pMCAO and tMCAO, respectively), were treated with relaxin peptides and brains collected for infarct analysis. Activation of the endothelial nitric oxide synthase (eNOS) pathway was evaluated as a potential protective mechanism. Primary cortical rat astrocytes were exposed to oxygen glucose deprivation (OGD), treated with relaxin peptides, and viability examined. Receptor involvement was explored using RXFP3 antagonist or agonist treatment and PCR. Relaxin and relaxin-3 reduced infarct size after pMCAO. Both peptides activated eNOS. As relaxin-3 has not previously been associated with this pathway and displays promiscuous RXFP binding, we explored receptor contribution. Expression of rxfp1 was greater than rxfp3 in rat brain, although peptide binding at either receptor resulted in similar overall protection after pMCAO. Only RXFP3 activation reduced infarct size after tMCAO. In astrocytes, rxfp3 was greater than rxfp1 gene expression. Selective activation of RXFP3 maintained astrocyte viability after OGD. Relaxin peptides are protective during early stages of ischemic stroke. Differential responses among treatments and models suggest that RXFP1 and RXFP3 initiate different protective mechanisms. This preliminary work is a pivotal first step in identifying clinical implications of relaxin peptides in ischemic stroke