Richard J. Traystman

Differential effect of PARP-2 deletion on brain injury after focal and global cerebral ischemia

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.

Cardiac arrest with cardiopulmonary resuscitation reduces dendritic spine density in CA1 pyramidal cells and selectively alters acquisition of spatial memory

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 8u2003min 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 daysu20037 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.

Dose-dependent neuroprotection by 17β-estradiol after cardiac arrest and cardiopulmonary resuscitation

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.

Kappa-Opioid Receptor Selectivity for Ischemic Neuroprotection with BRL 52537 in Rats

&kgr;-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.

Ischemic insult to cerebellar Purkinje cells causes diminished GABAA receptor function and Allopregnanolone neuroprotection is associated with GABAA receptor stabilization

Cerebellar Purkinje cells (PC) are particularly vulnerable to ischemic injury and excitotoxicity, although the molecular basis of this sensitivity remains unclear. We tested the hypothesis that ischemia causes rapid down‐regulation of GABAA receptors in cerebellar PC, thereby increasing susceptibility to excitotoxicity. Oxygen‐glucose deprivation (OGD) caused a decline in functional GABAA receptors, within the first hour of re‐oxygenation. Decreased amplitude of miniature inhibitory post‐synaptic potentials confirmed that OGD caused a significant decrease in functional synaptic GABAA receptors and quantitative Western blot analysis demonstrated the loss of GABAA receptor current was associated with a decline in total receptor protein. Interestingly, the potent neuroprotectant allopregnanolone (ALLO) prevented the decline in GABAA receptor current and protein. Consistent with our in vitro data, global ischemia in mice caused a significant decline in total cerebellar GABAA receptor protein and PC specific immunoreactivity. Moreover, ALLO provided strong protection of PC and prevented ischemia‐induced decline in GABAA receptor protein. Our findings indicate that ischemia causes a rapid and sustained loss of GABAA receptors in PC, whereas ALLO prevents the decline in GABAA receptors and protects against ischemia‐induced damage. Thus, interventions which prevent ischemia‐induced decline in GABAA receptors may represent a novel neuroprotective strategy.

Recommendations of the National Heart, Lung, and Blood Institute Working Group on Cerebrovascular Biology and Disease

The National Heart, Lung, and Blood Institute (NHLBI) convened a conference call working group, consisting of experts in stroke and cerebrovascular biology on January 28, 2005. The purpose of this working group was to develop a prioritized set of recommendations for NHLBI to establish a focused and comprehensive set of research activities in cerebrovascular biology and disease. Three thematic areas of research emerged: (1) molecular and cellular neurobiology of cerebral blood vessels, focusing on genomics and proteomics, neurovascular signaling and cerebrovascular embryogenesis, development and plasticity; (2) resource development, involving the development of new methodological approaches for normal and altered function of the neurovascular unit, collaborative research, and training in cerebrovascular pathobiology; and (3) cerebrovascular diseases and translational approaches, addressing vascular mechanisms of disease, the role of risk factors, importance of biomarkers with the ultimate goal of developing new treatments.

Cardiac Arrest/Cardiopulmonary Resuscitation Increases Anxiety-Like Behavior and Decreases Social Interaction

Advances in medical technology have increased the number of individuals who survive cardiac arrest/cardiopulmonary resuscitation (CPR). This increased incidence of survival has created a population of patients with behavioral and physiologic impairments. We used temperature manipulations to characterize the contribution of central nervous system damage to behavioral deficits elicited by 8 minutes of cardiac arrest/CPR in a mouse model. Once sensorimotor deficits were resolved, we examined anxiety-like behavior with the elevated plus maze and social interaction with an ovariectomized female. We hypothesized that anxiety-like behavior would increase and social interaction would decrease in mice subjected to cardiac arrest/CPR and that these changes would be attributable to central nervous system damage rather than damage to peripheral organs or changes orchestrated by the administration of epinephrine. Mice that were subjected to cardiac arrest/CPR while the peripheral organs, but not the brain, were protected by hypothermia exhibited increased anxiety-like behavior and decreased social interaction, whereas mice with hypothermic brains and peripheral organs during cardiac arrest/CPR did not exhibit behavioral impairments. The present study demonstrates that central nervous system damage from cardiac arrest/CPR results in increased anxiety and decreased social interaction and that these behavioral changes are not attributed to underlying sensorimotor deficits, dynamics of arrest and CPR, or peripheral organ damage.

Non-classical regulation of estrogen receptor-α by ICI182,780

Estrogen receptor-alpha (ER alpha) regulates transcription through a number of molecular mechanisms. Two mechanisms by which ER alpha acts directly in the nucleus have emerged: (1) in classical ER alpha 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 ER alpha binding to EREs; its mechanism is not as clearly defined as classical action. In many instances, non-classical action is mediated by tethering of ER alpha 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 ER alpha. Here, we show that non-classical action of ER alpha in 293 cells occurs in a wide range of enhancers and enhancer binding proteins. ER alpha 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, ER alpha stimulates activation domains of Jun, ATF-2, Elk, and CRE-binding protein (CREB). Non-classical ER alpha regulation described here does not appear to be sensitive to point mutations which affect classical and tethered ER alpha 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 ER alpha-dependent modulation of common components of the transcriptional machinery and may not be completely explained by tethering of ER alpha to specific transcription factors.

Cocaine- and Amphetamine-Regulated Transcript (CART) Peptide: A Vasoactive Role in the Cerebral Circulation

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.

Experimental spinal cord ischemia: Model characterization and improved outcome with arterial hypertension

Objective:Paraplegia from spinal cord ischemia is a devastating complication of thoracoabdominal aortic aneurysm repair. Perioperative hypoperfusion of the spinal cord is a critical determinant of residual neurologic deficits. We determined if functional and histologic outcome is dependent on systemic blood pressure in a rat model of spinal cord ischemia. Design:Randomized, controlled, prospective study. Setting:Research laboratory at a university teaching hospital. Subjects:Adult male Wistar rats. Interventions:Endotracheally intubated adult male Wistar rats (300–450 g) anesthetized with halothane underwent a thoracotomy and placement of a clip across the descending aorta for 27 mins. Mean proximal arterial blood pressure (MPABP) was monitored with a cannula placed in the left common carotid artery. Halothane was adjusted (1.25–1.5%) to maintain MPABP between 70 and 90 mm Hg (n = 20) or 140 and 150 mm Hg (n = 20). Shamoperated rats (n = 10) had a thoracotomy without aortic clamping at an MPABP of 70–90 mm Hg. Following 1, 24, 48, and 72 hrs of recovery from anesthesia, motor function of the hind paws was scored as follows: 0, no evidence of deficit; 1, toes flat under body when walking but with ataxia; 2, knuckle walks; 3, movements in hind limbs but unable to knuckle walk; 4, no movement, drags hind limbs. Body temperature was maintained between 37 and 38°C throughout the experiment. Measurements and Main Results:All sham operated rats with MPABP 70–90 mm Hg recovered without neurologic deficits, whereas those that underwent aortic occlusion with MPABP between 70 and 90 mm Hg emerged from anesthesia with grade 3 and 4 deficits and remained in this condition without improvement at 72 hrs. Histopathology at 72 hrs demonstrated moderate to severe neuronal loss with involvement of dorsal, intermediate, and ventral horns. Only eight of 20 rats that underwent aortic occlusion with MPABP between 140 and 150 mm Hg had grade 1 and 2 deficits on emergence but had no neurologic deficit after 1 hr. Most of the surviving neurons in these animals appeared normal histologically, particularly motor neurons around the periphery of the ventral horn. Conclusions:Systemic blood pressure is a critical determinant of outcome following spinal cord ischemia, and controlled peri-operative blood pressure augmentation may ameliorate neurologic deficits in patients who undergo thoracoabdominal vascular procedures and are at risk for spinal cord hypoperfusion.