Ischemic nitric oxide and poly (ADP-ribose) polymerase-1 in cerebral ischemia: male toxicity, female protection.

It is well established that tissue damage and functional outcome after experimental or clinical stroke are shaped by biologic sex. We investigated the novel hypothesis that ischemic cell death from neuronally derived nitric oxide (NO) or poly-ADP ribose polymerase (PARP-1) activation is sexually dimorphic and that interruption of these molecular death pathways benefits only the male brain. Female neuronal nitric oxide synthase (nNOS) knockout (nNOS-/-) mice exhibited exacerbated histological injury after middle cerebral artery occlusion (MCAO) relative to wild-type (WT) females, unlike the protection observed in male nNOS-/- littermates. Similarly, treatment with the nNOS inhibitor (7-nitroindozole, 25 mg/kg) increased infarction in female C57Bl6 WT mice, but protected male mice. The mechanism for this sexually specific response is not mediated through changes in protein expression of endothelial NOS or inducible NOS, or differences in intraischemic cerebral blood flow. Unlike male PARP-1 knockouts (PARP1-/-), female PARP1-/- littermates sustained grossly increased ischemic damage relative to sex-matched WT mice. Treatment with a PARP inhibitor (PJ-34, 10 mg/kg) resulted in identical results. Loss of PARP-1 resulted in reversal of the neuroprotective activity by the female sex steroid, 17beta estradiol. These data suggest that the previously described cell death pathways involving NO and PARP ischemic neurotoxicity may be operant solely in male brain and that the integrity of nNO/PARP-1 signaling is paradoxically protective in the female.

Chronic behavioral testing after focal ischemia in the mouse: functional recovery and the effects of gender.

Several useful behavioral tests exist for measuring behavioral recovery after ischemia in higher-order animals and rats. With the increasing use of mice in focal stroke research, simple, reliable, and reproducible behavioral testing has become a priority. As neuroprotective agents are tested, long-term outcome must be assessed, especially in studies focused on neuronal plasticity and regeneration after ischemia. Our laboratory and others have previously shown that estrogen (E2) is neuroprotective in rodent stroke paradigms. We examined a battery of behavioral tests in male and female mice subjected to 90 min of middle cerebral artery occlusion (MCAO) to determine the most sensitive tests for detecting sensorimotor dysfunction after stroke, and to determine the functional significance of E2-mediated neuroprotection. Only two tests, the corner test and the cylinder test, were able to differentiate between groups (sham and stroke) after several days of repeated testing. The cylinder test was sensitive to the neuroprotective/neurorestorative effects of E2, but 2 weeks after stroke, the cylinder test was unable to distinguish between sham and stroke animals treated with E2. In contrast, the corner test was able to differentiate stroke and sham animals even 6 weeks after stroke, but did not distinguish animals treated with E2 vs. vehicle. These tests provide a simple, rapid, reliable assessment of sensorimotor dysfunction in the mouse after focal ischemia. Hormonal status influences speed of recovery on cylinder testing in animals of both genders. This suggests that a short battery of tests including the neurological score, cylinder, and corner test may be adequate to rapidly and repeatedly assess sensorimotor dysfunction in mice of both genders.

Pain evaluation and response to buprenorphine in rats subjected to sham middle cerebral artery occlusion.

Appropriate and efficacious use of analgesics in rodents must be balanced judiciously between animal needs and research objectives. A concern in many studies is that analgesia will confound experimental outcome or interpretation. Accordingly, determining whether rats subjected to surgical protocols show evidence of pain is important if we are to provide rational postoperative analgesia without compromising experimental objectives. The goal of this study was to evaluate both subjective and objective measures for pain evaluation in male Wistar rats subjected to sham middle cerebral artery occlusion (MCAO) surgery and to determine whether buprenorphine would be an appropriate analgesic in this surgical model. Male Wistar rats underwent a sham 2-h MCAO by the intraluminal filament technique followed by 22-h of recovery. Animals were randomly assigned to one of three postoperative treatment groups: vehicle only (VH; vehicle subcutaneously [s.c.] + plain jello orally), low-dose buprenorphine (LB; 0.05 mg/kg s.c. _ 0.25 mg/kg drug-in-jello orally), and high-dose buprenorphine (HB; 0.5 mg/kg s.c. + 0.25 mg/kg drug-in-jello orally). Animals received subcutaneous treatments prior to anesthetic recovery and approximately 18-h later. Jello treatments were given once at the end of the surgery day. We modified a previously published behavioral scoring system which is based on subjective and objective measures for pain evaluation. All animals were evaluated for pain before sham surgery (baseline) and at 3-, 18-, and 22-h postoperatively by observers who were blinded to treatment group. Brains were removed at 22-h after surgery and evaluated by 2,3,5-triphenyltetrazolium chloride staining to confirm lack of brain injury from the sham procedure. Sham intraoperative physiological variables were equivalent among treatment groups. Baseline assessment scores were zero for all groups. Postoperatively, all treatment groups showed elevated assessment scores relative to baseline values. Buprenorphine at the tested doses did not markedly reduce total assessment scores postoperatively relative to those in vehicle-treated animals. Further evaluation of rodent postoperative pain and response to analgesia is needed if we are to formulate a sound scientific approach to animal management in protocols requiring surgical manipulations.

Poly(ADP-ribose) polymerase impairs early and long-term experimental stroke recovery.

BACKGROUND AND PURPOSE: Poly(ADP-ribose) polymerase (PARP-1; Enzyme Commission 2.4.30) is a nuclear DNA repair enzyme that mediates early neuronal ischemic injury. Using novel 3-dimensional, fast spin-echo-based diffusion-weighted imaging, we compared acute (21 hours) and long-term (3 days) ischemic volume after middle cerebral artery (MCA) occlusion in PARP-1-null mutants (PARP-/-) versus genetically matched wild-type mice (WT mice). PARP-/- mice were also treated with viral transfection of wild-type PARP-1 to determine whether protection from MCA occlusion is lost with restoration of the gene product. METHODS: Halothane-anesthetized mice were treated with reversible MCA occlusion via intraluminal suture technique. Ischemic volumes were delineated by diffusion-weighted imaging with high spatial and temporal resolution during MCA occlusion and reperfusion. Recombinant Sindbis virus carrying beta-galactosidase (lacZ) or PARP-1 was injected into ipsilateral striatum, then animals underwent MCA occlusion 3 days later. Infarction volume was measured at 22 hours of reperfusion (2,3,5-triphenyltetrazolium chloride histology). RESULTS: Reduction in regional water apparent diffusion coefficient (ADC) during occlusion or secondary ADC decline during reperfusion was not different between groups. Ischemic volume was smaller early in occlusion in PARP-/- versus WT mice and remained less at 21 hours of reperfusion. Ischemic volume then increased from 1 to 2 days in all mice, then stabilized without further change. Ischemic damage was smaller in PARP-/- than in WT mice at 3 days. Transfection of PARP-1 into PARP-/- mice increased stroke damage relative to lacZ-injected PARP-/- and increased damage to that of the WT mice. Intraischemic laser-Doppler flowmetry and physiological variables were not different among groups. CONCLUSIONS: PARP-1 deficiency provides both early and prolonged protection from experimental focal stroke. The mechanism is not linked to preservation of ADC and mitigation of secondary energy depletion during early reperfusion.