The reason that estrogen is strongly protective in various estrogen-deficient animal models while seemingly detrimental in postmenopausal women remains unclear. It hypothesized that prolonged oral medroxyprogesterone (MPA) plus oral conjugated equine estrogens (CEE) diminishes estrogen ability to reduce stroke damage in the rodent stroke model. To test the hypothesis, we fed ovariectomized rats CEE or MPA, or a combination of CEE and MPA (CEP), before inducing 120 min of reversible focal stroke, using the intraluminal filament model. After 22 h reperfusion, the brains were harvested and infarction volumes were quantified. Treatment with CEE alone or with CEP reduced cortical infarction volume. However, CEP failed to provide ischemic protection in subcortical regions. It was concluded that CEE alone, or with CEP, is neuroprotective in the cortex, but interactive effects between the hormones may counteract CEE beneficial effects in subcortical brain regions.

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.

Spontaneously hypertensive stroke-prone rats (SHRSP), a model for genetic stroke susceptibility, suffer spontaneous stroke and enhanced injury after experimental stroke, in part due to abnormal cerebrovascular development. We hypothesized that angiopoietin system genes in SHRSP may follow unique patterns of expression after experimentally induced stroke. SHRSP, hypertensive control rats (SHR), and normotensive controls (WKY) were subjected to experimental middle cerebral artery occlusion, and brain RNA was analyzed for expression of angiogenic genes. Expression of angiopoietin-2 increased after stroke in all rat strains and was significantly enhanced in SHRSP compared with control strains. In addition, expression of angiopoietin-1 and the angiopoietin receptor dropped markedly after stroke in SHRSP animals, but was not different after ischemia in SHR and WKY strains. Thus, the SHRSP brain elaborates a unique and specific pattern of angiopoietin system gene expression after stroke which may underlie stroke susceptibility of these rats.

Background and Purpose— Recent results suggest that selective inhibitors of presynaptic neuronal ion channels can diminish glutamate release during cerebral ischemia and modulate excitotoxic cell death. The aim of the present study was to evaluate lamotrigine (LTG), an antiepileptic that inhibits presynaptic sodium and voltage-sensitive calcium channels, as a potential stroke resuscitation agent in the rat. Three dosages of LTG were examined for effect on infarction volume and sensorimotor behavioral recovery after middle cerebral artery (MCA) occlusion. Methods— Halothane-anesthetized male Wistar rats were subjected to 2 hours of MCA occlusion by the intraluminal occlusion technique. Physiological variables were controlled, and ipsilateral cortical perfusion was monitored by laser Doppler flowmetry throughout ischemia. At onset of reperfusion, rats received intravenous LTG 5, 10, or 20 mg/kg or PBS (n=9 to 11 per group) during 15 minutes. Behavioral assessment was completed at 3 and 7 days after stroke, and the brain was harvested for histology (triphenyltetrazolium chloride staining). Results— Values are mean±SE. Cortical infarction volumes were unchanged in LTG-treated animals: 14±6% of contralateral cortex at 5 mg/kg LTG, 17±7% at 10 mg/kg, and 30±6% at 20 mg/kg, versus saline-treated cohorts (12±3%;P =0.19; n=9). Caudate-putamen infarction injury was also unchanged (37±11% of contralateral caudate-putamen at 5 mg/kg LTG, 44±8% at 10 mg/kg, and 65±9% at 20 mg/kg versus saline (38±11%;P =0.18). Total infarction was not different among groups (P =0.15). Consistent with histology, behavioral outcomes were unimproved by treatment. Conclusions— Histological damage and behavioral recovery at 7 days after MCA occlusion was not altered by LTG treatment over the dosage range used in the present study.

P66 Angiogenic growth factors play an essential role in the development and structural integrity of the cerebral vasculature, but their importance in stroke is poorly defined. Stroke prone hypertensive rats (SHRSP), a model for genetic stroke susceptibility, suffer spontaneous stroke and enhanced injury after experimental stroke, at least in part due to abnormal cerebrovascular development. We hypothesized that angiogenic genes in SHRSP may follow unique patterns of expression after experimentally induced stroke. Ribonuclease protection assays were used to determine expression levels of the angiogenic receptor Tie-2 and its ligands in the brain after reversible middle cerebral artery occlusion (2 hours, followed by 22 hours of reperfusion) in SHRSP, and in control SHR and WKY rats. The expression of Tie-2 and Ang-1, an agonist for Tie-2, dropped markedly after stroke in SHRSP animals (70% and 45%; p<0.05), but not in controls. In addition, enhanced expression of Ang-2, a Tie-2 antagonist, is significantly higher in SHRSP animals (50% versus WKY; p<0.05). Together, these data demonstrate that SHRSP may dramatically downregulate Tie-2 signaling after stroke. This suggests that in stroke prone animals, impairment of Tie-2 signaling contributes to stroke vulnerability.

BACKGROUND AND PURPOSE The importance of postmenopausal estrogen replacement therapy for stroke in females remains controversial. We previously showed that female rats sustain less infarction in reversible middle cerebral artery occlusion (MCAO) than their ovariectomized counterparts and that vascular mechanisms are partly responsible for improved tissue outcomes. Furthermore, exogenous estrogen strongly protects the male brain, even when administered in a single injection before MCAO injection. The present study examined the hypothesis that replacement of 17beta-estradiol to physiological levels improves stroke outcome in ovariectomized, estrogen-deficient female rats, acting through blood flow-mediated mechanisms. METHODS Age-matched, adult female Wistar rats were ovariectomized and treated with 0, 25, or 100 microgram of 17beta-estradiol administered through a subcutaneous implant or with a single Premarin (USP) injection (1 mg/kg) given immediately before ischemia was induced (n=10 per group). Each animal subsequently underwent 2 hours of MCAO by the intraluminal filament technique, followed by 22 hours of reperfusion. Ipsilateral parietal cortex perfusion was monitored by laser-Doppler flowmetry throughout ischemia. Cortical and caudate-putamen infarction volumes were determined by 2,3, 5-triphenyltetrazolium chloride staining and digital image analysis. End-ischemic regional cerebral blood flow was measured in ovariectomized females with 0- or 25-microgram implants (n=4 per group) by (14)C-iodoantipyrine quantitative autoradiography. RESULTS Plasma estradiol levels were 3.0+/-0.6, 20+/-8, and 46+/-10 pg/mL in the 0-, 25-, and 100-microgram groups, respectively. Caudate-putamen infarction (% of ipsilateral caudate-putamen) was reduced by long-term, 25-microgram estrogen treatment (13+/-4% versus 31+/-6% in the 0-microgram group, P<0.05, and 22+/-3% in the 100-microgram group). Similarly, cortical infarction (% of ipsilateral cortex) was reduced only in the 25-microgram group (3+/-2% versus 12+/-3% in the 0-microgram group, P<0.05, and 6+/-3% in the 100-microgram group. End-ischemic striatal or cortical blood flow was not altered by estrogen treatment at the neuroprotective dose. Infarction volume was unchanged by acute treatment before MCAO when estrogen-treated animals were compared with saline vehicle-treated animals. CONCLUSIONS Long-term estradiol replacement within a low physiological range ameliorates ischemic brain injury in previously ovariectomized female rats. The neuroprotective mechanism is flow-independent, not through preservation of residual ischemic regional cerebral blood flow. Furthermore, the therapeutic range is narrow, because the benefit of estrogen in transient vascular occlusion is diminished at larger doses, which yield high, but still physiologically relevant, plasma 17beta-estradiol levels. Lastly, unlike in the male brain, single-injection estrogen exposure does not salvage ischemic tissue in the female brain. Therefore, although exogenous steroid therapy protects both male and female estrogen-deficient brain, the mechanism may not be identical and depends on long-term hormone augmentation in the female.