Poly(ADP-ribose) polymerase-2 (PARP-2) is a member of the PARP enzyme family, and, similarly to PARP-1, catalyzes the formation of ADP-ribose polymers in response to DNA damage. While PARP-1 overactivation contributes to ischemic cell death, no information is available regarding the role of PARP-2. In this study, we evaluated the impact of PARP-2 deletion on histopathological outcome from two different experimental models of cerebral ischemia. Male PARP-2(-/-) mice and wild-type (WT) littermates were subjected to either 2 h of middle cerebral artery occlusion (MCAO) followed by 22 h reperfusion, or underwent 10 mins of KCl-induced cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR) and 3-day survival. After MCAO, infarct volume was reduced in PARP-2(-/-) mice (38% +/- 12% of contralateral hemisphere) compared with WT (64% +/- 16%). After CA/CPR, PARP-2 deletion significantly increased neuronal cell loss in the hippocampal CA1 field (65% +/- 36% ischemic neurons) when compared with WT mice (31% +/- 33%), with no effect in either striatum or cortex. We conclude that PARP-2 is a novel executioner of cell death pathways in focal cerebral ischemia, but might be a necessary survival factor after global ischemia to mitigate hippocampal delayed cell death.

Objective:Despite recent advances in the treatment of cardiac arrest, neurologic outcome remains poor. 17β-Estradiol (E2) has been widely shown to reduce damage after experimental brain injury. The present study determined whether E2 also improves neuronal survival after experimental cardiac arrest and cardiopulmonary resuscitation and if any protection is dose-dependent. Design:A randomized trial. Setting:A research laboratory. Subjects:Male C57Bl/6 mice weighing 20–25 g. Interventions:Mice were randomized into one of six groups, receiving treatment with 0.5, 2.5, 12.5, 25, or 50 &mgr;g of E2 or vehicle 1.5 mins after return of spontaneous circulation. Ten minutes after induction of cardiac arrest (by KCl injection), cardiopulmonary resuscitation was initiated (with chest compressions, intravenous epinephrine, and ventilation with 100% O2). Additional animals of each E2-treated group were used for plasma estradiol-level analysis. Brains were removed for quantification of injury in the hippocampus and caudoputamen on day 3. Measurements and Main Results:The E2 0.5 group had physiologic estrogen levels 60 min after injection (mean ± se, 28 ± 5 pg/mL), whereas the E2 50 group still showed supraphysiologic levels 360 min after administration (245 ± 32 pg/mL). Hippocampal damage was not altered with E2 treatment. Only posttreatment with the lowest E2 dose (E2 0.5) resulted in attenuated neuronal injury in the rostral and caudal caudoputamen (34 ± 11% and 27 ± 11%), in comparison with vehicle (68 ± 5, p < .05; 63 ± 4%, p < .001). Higher E2 doses did not affect brain injury. Conclusions:We conclude that E2 has a critical dosing effect on neuronal survival, physiologic levels of E2 are neuroprotective after cardiac arrest/cardiopulmonary resuscitation, and acute exposure is sufficient for brain resuscitation.

Inhibition of glutamine synthesis reduces astrocyte swelling and associated physiological abnormalities during acute ammonium acetate infusion in anesthetized rats. We tested the hypothesis that inhibition of glutamine accumulation during more prolonged ammonium acetate infusion in unanesthetized rats reduces cortical astrocyte swelling and immunohistochemical changes in astrocytic proteins. Rats received a continuous i.v. infusion of either sodium acetate or ammonium acetate for 24 h to increase plasma ammonia from about 30–400 μmol/l. Cohorts were pretreated with vehicle or l-methionine-S-sulfoximine (MSO; 0.83mmol/kg). MSO reduced glutamine synthetase activity by 57% and glutamine synthetase immunopositive cell number by 69%, and attenuated cortical glutamine accumulation by 71%. Hyperammonemia increased the number of swollen astrocytes in cortex and MSO reduced this increase to control values. The number of glial fibrillary acidic protein immunopositive cells in cortex was greater in hyperammonemic rats and the increase in superficial cortical layers was attenuated by MSO. Immunoreactivity for the gap junction protein connexin-43 in the neuropil, assessed by optical density, was greater in the hyperammonemic group compared with controls, but this increase was not attenuated by MSO. No changes in the optical density of GLT1 glutamate transporter immunoreactivity in cortex were detected in any group. We conclude that glutamine synthetase inhibition reduces astrocyte swelling and ameliorates some of the reactive astroglial cytoskeletal alterations seen at 24 h of hyperammonemia, but that gap junction changes in astrocytes occur independently of glutamine accumulation and swelling.

Glutamine has been shown to influence endothelial-dependent relaxation and nitric oxide production in vitro, possibly by limiting arginine availability, but its effects in vivo have not been well studied. Hyperammonemia is a pathophysiological condition in which glutamine is elevated and contributes to depressed CO(2) reactivity of cerebral arterioles. We tested the hypothesis that acute hyperammonemia decreases pial arteriolar dilation to acetylcholine in vivo and that this decrease could be prevented by inhibiting glutamine synthetase with L-methionine-S-sulfoximine (MSO) or by intravenous infusion of L-arginine. Pial arteriolar diameter responses to topical superfusion of acetylcholine were measured in anesthetized rats before and at 6 h of infusion of either sodium or ammonium acetate. Ammonium acetate infusion increased plasma ammonia concentration from approximately 30 to approximately 600 microM and increased cerebral glutamine concentration fourfold. Arteriolar dilation to acetylcholine was intact after infusion of sodium acetate in groups pretreated with vehicle or with MSO plus methionine, which was coadministered to prevent MSO-induced seizures. In contrast, dilation in response to acetylcholine was completely blocked in hyperammonemic groups pretreated with vehicle or methionine alone. However, MSO plus methionine administration before hyperammonemia, which maintained cerebral glutamine concentration at control values, preserved acetylcholine dilation. Intravenous infusion of L-arginine during the last 2 h of the ammonium acetate infusion partially restored dilation to acetylcholine without reducing cerebral glutamine accumulation. Superfusion of 1 or 2 mM L-glutamine through the cranial window for 1 h in the absence of hyperammonemia attenuated acetylcholine dilation but had no effect on endothelial-independent dilation to nitroprusside. We conclude that 1) hyperammonemia reduces acetylcholine-evoked dilation in cerebral arterioles, 2) this reduction depends on increased glutamine rather than ammonium ions, and 3) increasing arginine partially overcomes the inhibitory effect of glutamine.

Cocaine- and amphetamine-regulated transcript (CART) peptides are known to be involved in the stress response and have been implicated in the regulation of the cardiovascular system. We evaluated the direct vasoactive properties of CART in the cerebral circulation and its potential mechanisms of action. Penetrating cerebral arterioles, isolated from male Sprague—Dawley rats, were cannulated using a concentric micropipette setup, pressurized and perfused. The vascular response to intraluminal and extraluminal CART peptide was characterized. The endothelium dependence of this response was assessed by means of the endothelial light—dye injury model. The nonspecific endothelin receptor antagonist PD-145065, the ETA-specific antagonist BQ-123, the ETB-specific antagonist BQ-788, and the inhibitor of endothelin-converting enzyme phosphoramidon were used to characterize the involvement of the endothelin pathway in the vascular response to CART peptide. Extraluminal and intraluminal application of CART peptide (0.1 nm to 1 μmol/L) evoked a long-lasting dose-dependent constriction of isolated penetrating cerebral arterioles to ~80% of resting myogenic tone. Disruption of the endothelium by the endothelial light/dye injury model resulted in the abolition of this response ( P<0.05). Extraluminal administration of PD-145065, BQ-123, and phosphoramidon blocked the constriction response to CART peptide ( P<0.01). The ETB antagonist, BQ-788, did not alter the constriction response to CART peptide. Cocaine- and amphetamine-regulated transcript peptide is a potent vasoconstrictor in the cerebral circulation. Its direct vasoactive properties are endothelium-dependent and are mediated by ETA, not ETB, endothelin receptors.

Immune system activation has implications for cerebrovascular health, but little is known about the function of the immune system after a major cerebrovascular event, such as cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Cardiac arrest and cardiopulmonary resuscitation damages the hippocampus, an important component of the hypothalamic—pituitary—adrenal (HPA) axis, and alterations in HPA axis activity can affect immune function. We tested the hypothesis that CA/CPR (approximately 8mins) would cause HPA axis dysregulation and alter the delayed type hypersensitivity (DTH) response to antigenic challenge. We also assessed the primary and secondary antibody response of mice exposed to CA/CPR. Of the mice exposed to CA/CPR, half had brains protected by hypothermia to isolate the effects of the CA/CPR procedure from the effects of CA/CPR-induced neuronal damage. Cardiac arrest and cardiopulmonary resuscitation-induced neuronal damage resulted in a persistent elevation of blood corticosterone concentration and a concomitant augmentation of the DTH response to antigenic challenge. Furthermore, immune activation before CA/CPR decreased survival after global ischemia. These data highlight the potential impact of neuronal damage on cell-mediated immune function and the role of humoral immune activation in outcome after global ischemia.

Estrogen receptor-α (ERα) regulates transcription through a number of molecular mechanisms. Two mechanisms by which ERα acts directly in the nucleus have emerged: (1) in classical ERα action, estrogen-bound receptor binds estrogen response elements (ERE) and regulates promoters by recruiting coactivators or corepressors to DNA; (2) non-classical action is not dependent on ERα binding to EREs; its mechanism is not as clearly defined as classical action. In many instances, non-classical action is mediated by tethering of ERα to other DNA-binding proteins, facilitating recruitment of coregulators to transcription regulatory sequences. In some cell types, non-classical stimulation can be enhanced by antagonists and repressed by agonists of ERα. Here, we show that non-classical action of ERα in 293 cells occurs in a wide range of enhancers and enhancer binding proteins. ERα stimulates AP-1 elements, cyclic AMP response elements (CRE), and serum response elements (SRE) in the presence of the antiestrogen ICI182,780. Further, in the presence of ICI182,780, ERα stimulates activation domains of Jun, ATF-2, Elk, and CRE-binding protein (CREB). Non-classical ERα regulation described here does not appear to be sensitive to point mutations which affect classical and tethered ERα action; moreover, in our experiments, non-classical action is uniquely sensitive to nuclear transport inhibition by leptomycin B. Because ICI182,780 appears to affect multiple and diverse transcriptional systems, our results are likely explained by ERα-dependent modulation of common components of the transcriptional machinery and may not be completely explained by tethering of ERα to specific transcription factors.

Estrogen receptor (ER) alpha localizes to both the nucleus and the plasma membrane, mediating estrogen-dependent genomic and nongenomic signaling, respectively. In some cells, ERalpha appears to be excluded from the nucleus, and it is unclear whether genomic signaling takes place. The purpose of this study was to determine whether membrane-associated ERalpha is capable of genomic signaling, or whether this pool of receptors strictly serves membrane-mediated signaling. ERalpha fused to the C-terminal cytoplasmic tail of bovine rhodopsin (Rh-ERalpha) activates ER response element-dependent transcription only in the presence of estrogen; the activity is antagonized by the estrogen antagonist ICI 182,780 and by the dominant-negative mutant of ERalpha and is unaffected by inhibitors of MAPKs and Akt signaling, indicating that this was due to direct genomic action. The activity of Rh-ERalpha containing the activating Y537S mutation was also estrogen dependent, suggesting that estrogen gated the entry of Rh-ERalpha into the nucleus. Indeed, cell fractionation studies demonstrated that Rh-ERalpha protein, in contrast to ERalpha that was nuclear at baseline, was excluded from the nucleus in the absence of hormone, and localized to the inner nuclear membrane on incubation with estrogen. These data demonstrate that membrane tethered ERalpha is capable of nuclear function and that its transcriptional activity is regulated by hormone-dependent entry into the inner nuclear membrane. Furthermore, these experiments provide evidence that under certain circumstances, membrane proteins are capable of nuclear function without detectable nucleoplasmic localization.

Stroke and cardiac arrest are major causes of death and disability in the United States and have been heavily investigated over the past 50 years. Stroke and cardiac arrest strike more than 1,000,000 individuals per year and while most strokes and cardiac arrests occur in older patients, there has been an alarming increase in stroke and cardiac arrest incidence in patients between 45 and 65 years of age. Despite much animal research concerning brain tissue injury and the mechanisms by which stroke and cardiac arrest produce injury to brain cells, little of this work has translated into effective treatment modalities for stroke and cardiac arrest in humans. Multiple mechanisms of injury to the brain from stroke and cardiac arrest have been identified: eg, production of oxygen free radicals, lipid peroxidation, release of excitatory amino acids such as glutamate and aspartate, involvement of mediators of inflammation, involvement of ionic pumps for Ca++ and Na+, acidosis, alterations in brain blood flow and metabolism, hyperthermia, involvement of genes and gene products, and others. As a result of injury, several of these mechanisms may be overlapping, making the issue of mechanisms extremely complex. The idea of protecting brain tissue from injury (neuroprotection) is not a new concept. Many neuroprotective agents, such as anesthetics, free radical scavengers, excitatory amino acid antagonists, calcium channel blockers, ionic pump modulators, anti neutrophil and platelet factors, growth factors, hypothermia, and, more recently, manipulation of genes and gene products and sex steroids, have been investigated for years. What remains curious is that while many of these agents appear quite effective neuroprotectants in cell culture systems or in small animal models (mice, rats, gerbils) of focal or global cerebral ischemia, they are much less effective in large animal models (cats, dogs, monkeys), and at this time, none of these potential neuroprotectants are effective in humans, with the possible exception of hypothermia.

Cardiac arrest is associated with high mortality and poor neurological outcome. We characterized functional and histological outcome in a novel mouse model of cardiac arrest and cardiopulmonary resuscitation (CPR) in order to study neuroprotective mechanisms. Cardiac arrest was induced in male C57Bl/6 and 129SVEV mice by i.v. injection of KCl. After 10 min cardiac standstill, CPR was initiated by administration of epinephrine, ventilation with 100% oxygen and chest compressions. Twenty-four hours before and 3 or 7 days after CPR, mice were subjected to behavioral testing using a passive avoidance task, locomotor activity in an open field, and spontaneous alternation in a T-maze. Hippocampal and caudoputamen injury was quantified 3 or 7 days after CPR. At both time points, caudoputamen injury was worse in 129SVEV mice. Post-ischemic mice of both strains showed a reduced number of correct choices in the T-maze up to 7 days after CPR, and were temporarily impaired in learning the passive avoidance task with a retention deficit on day 3 but not on day 7. Locomotor activity showed strain differences with C57Bl/6 mice being more active, but little ischemia-related effects. A dissociation between functional and histological outcome was found emphasizing the importance of combining both outcome measures for evaluation of neuroprotective strategies.

The hippocampus is highly sensitive to ischemia and is one of the most extensively damaged regions of brain during cardiac arrest. Damage to hippocampus can subsequently lead to learning and memory deficits. The current study used the Morris water maze to characterize spatial learning and memory deficits elicited by 8 min of cardiac arrest with cardiopulmonary resuscitation (CA/CPR) in mice, which is associated with a 25-50% decrease in CA1 neurons. Mice were trained to navigate the water maze prior to CA/CPR or sham surgery (SHAM). They were retested in the water maze on days 7 and 8 postsurgery; both CA/CPR and SHAM groups were able to perform the task at presurgical levels. However, when the hidden platform was moved to a new location, the SHAM mice were able to adapt more quickly to the change and swam a shorter distance in search of the platform than did CA/CPR mice. Thus, CA/CPR did not affect the ability of mice to retain a previously learned platform location, but it did affect their ability to learn a new platform location. This behavioural impairment was correlated with dendritic spine density in the CA1 region of the hippocampus. Data presented here suggest that morphological changes, such as spine density, that occur in neurons that survive CA/CPR may be associated with cognitive impairments.

s: 32nd Annual Meeting of the Society of Neurosurgical Anesthesia and Critical Care, Las Vegas, Nevada, October 22, 2004: CEREBRAL ISCHEMIA/MOLECULAR BIOLOGY

s: 32nd Annual Meeting of the Society of Neurosurgical Anesthesia and Critical Care, Las Vegas, Nevada, October 22, 2004: CEREBRAL BLOOD FLOW/PHARMACOLOGY

Cardiac arrest is often associated with poor neurologic outcome since therapeutic options are limited. We tested the hypothesis that overexpression of CuZn superoxide dismutase (SOD+/-) is neuroprotective in a new murine model of cardiac arrest and cardiopulmonary resuscitation (CPR). Second, we investigated if female and male mice sustain similar injury and if sex-specific outcomes are altered by SOD overexpression. Neuronal injury was quantified 3 days after 8 mins of KCl-induced cardiac arrest by calculating the percentage of ischemic neurons for caudoputamen and hippocampal CA1 region. In rostral caudoputamen, less neuronal cell loss was found for SOD+/- mice (31%+/-22%) when compared with wild-type (WT) mice (47%+/-31%, P<0.05). Superoxide dismutase overexpression did not reduce injury in the caudal caudoputamen. No sex-linked protection was evident in either genotype in the caudoputamen. Female WT mice had less CA1 injury than male WT mice (26%+/-31% versus 54%+/-30%, P<0.05), whereas no sex difference was found in SOD+/- mice (female: 42%+/-29%; male: 37%+/-37%). Comparison of hippocampal injury between genotypes revealed no differences for either males or females. In conclusion, SOD1 overexpression and female sex were associated with significant neuroprotection in this murine cardiac arrest model. However, no additive neuroprotection was observed, and these beneficial effects were restricted to specific brain regions.

kappa-Opioid receptors (KOR) have been implicated in neuroprotection from ischemic neuronal injury, but less work has been performed with transient focal cerebral ischemia to determine the role of KOR during reperfusion. We tested the effects of a selective and specific KOR agonist, BRL 52537 hydrochloride [(+/-)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine], and the standard KOR antagonist, nor-binaltorphimine dihydrochloride [nor-BNI; 17,17'-(dicyclopropylmethyl)6,6',7,7'-6,6'-imino-7,7'-binorphinan-3,4',14,14'-tetrol], on functional and histological outcome after transient focal ischemia in the rat. By use of the intraluminal filament technique, halothane-anesthetized adult male Wistar rats were subjected to 2 h of middle cerebral artery occlusion confirmed by laser Doppler flowmetry. In a blinded, randomized fashion, rats were treated with 1) saline (vehicle) 15 min before reperfusion followed by saline at reperfusion for 22 h, 2) saline 15 min before reperfusion followed by BRL 52537 (1 mg . kg(-1) h(-1)) at reperfusion for 22 h, 3) saline 15 min before reperfusion followed by nor-BNI (1 mg . kg(-1) . h(-1)) at reperfusion for 22 h, or 4) nor-BNI (1 mg/kg) 15 min before reperfusion followed by BRL 52537 (1 mg . kg(-1) . h(-1)) and nor-BNI (1 mg . kg(-1) . h(-1)) at reperfusion for 22 h. Infarct volume (percentage of ipsilateral structure) analyzed at 4 days of reperfusion was significantly attenuated in saline/BRL 52537 rats (n = 8; cortex, 10.2% +/- 4.3%; caudoputamen [CP], 23.8% +/- 6.7%) (mean +/- SEM) compared with saline/saline treatment (n = 8; cortex, 28.6% +/- 4.9%; CP, 53.3% +/- 5.8%). Addition of the specific KOR antagonist nor-BNI to BRL 52537 completely prevented the neuroprotection (n = 7, cortex, 28.6% +/- 5.3%; CP, 40.9% +/- 6.2%) conferred by BRL 52537. BRL 52537 did not produce postischemic hypothermia. These data demonstrate that KORs may provide a therapeutic target during early reperfusion after ischemic stroke.

OBJECT:Ibuprofen is an antiinflammatory drug that disrupts leukocyte-endothelial cell interactions by limiting expression of endothelial adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), also known as CD54. The authors hypothesized that ibuprofen could reduce the size of the infarct associated with transient focal ischemia by inhibition of ICAM-1 expression, and they evaluated its effects in rats treated with middle cerebral artery (MCA) occlusion. Ibuprofen treatment was compared with mild systemic hypothermia, which is known to be neuroprotective and is commonly used during neurosurgical procedures.METHODS:The maximum ibuprofen dose (240 mg/kg/day) that could be tolerated with no systemic toxicity was established in the initial experiments. In the efficacy experiment, rats were pretreated with vehicle, ibuprofen, or hypothermia (33 degrees C) prior to 2 hours of MCA occlusion; then their brains were harvested at 24 hours of reperfusion for histological studies. End-ischemic cerebral blood flow (CBF) was evaluated using [14C]iodoantipyrine autoradiography in additional cohorts. Expression of ICAM-1 within ischemic compared with nonischemic caudate nucleus and putamen (striatum) or cortex was evaluated using immunohistochemical studies. Compared with vehicle treatment, ibuprofen produced a 46.2% reduction (p = 0.01) in striatal infarcts, which was comparable to hypothermia (48.7% reduction, p = 0.02). Ibuprofen did not alter end-ischemic CBF in any region studied, and the ibuprofen treatment group had the lowest proportion of animals with marked ICAM-1 staining.CONCLUSIONS:Ibuprofen given in maximum tolerated doses reduces the striatal infarct size after focal cerebral ischemia. The neuroprotective mechanism does not work through preservation of intraischemic CBF and is consistent with inhibition of ICAM-1 expression; however, at the doses used in this study, other effects of ibuprofen on platelet and endothelial function are possible.