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Introduction: Sex is a known predictor of outcome after acute ischemic stroke. However, the effect of sex on outcome after subarachnoid hemorrhage (SAH) is not well studied. Methods: Five studies from the SAH International Trialists repository were included (4 randomized trials and 1 prospective study). Patients were divided into groups based on sex. The primary outcome was favorable outcome which was defined as Glasgow Outcome Score (GOS) of 4 or 5 and the secondary outcome was delayed cerebral ischemia (DCI). Binary logistic regression was done to assess the association between sex and outcomes. Results: A total of 8015 patients (2186 males and 5829 females) were included. Female patients were older (mean age 53 vs 50 years, p Conclusion: Female patients had worse functional outcome and higher risk of DCI after subarachnoid hemorrhage when compared to men.

Introduction: The pathophysiology of subarachnoid hemorrhage (SAH) is complex and includes disruption of the blood-brain barrier (BBB). We have previously optimized methods to freshly isolate BBB endothelial cells (BECs) and performed genome-wide expression profiling to uncover new therapeutic targets in an unbiased manner. In this study we validated 2 of the most highly upregulated targets, cyclooxygenase-2 (Cox2) and angiopoietin-2 (Angpt2). Methods: The prechiasmatic blood injection mouse model of SAH was used with experimental end-points at 24h. BBB disruption was assessed using intraperitoneal-injected cadaverine-based fluorescent dye. Global neurobehavioral assessments utilized the modified Garcia score. BECs were purified either by magnetic-based sorting for CD45-CD31+ cells or by fluorescence-activated cell sorting for Tie2+Pdgfrb- cells. Microarray results from RNA extracted from BECs were validated with real-time PCR. Immunofluorescence of coronal brain sections and enzyme-linked immunosorbent assays of brain homogenates were performed to determine protein expression. Intraperitoneal injections of the Cox2 inhibitor celecoxib (10mg/kg) were administered 30min and 12h after SAH induction. Results: CD45-CD31+ BECs derived from SAH mice demonstrated 6.6-fold and 5.4-fold increase in Cox2 and Angpt2 gene expression, respectively (n=4 per group, p Conclusions: In summary, we identified Cox2 as a potential therapeutic target in SAH. The observed beneficial effects of celecoxib treatment suggest that Cox2 may be critical to BBB disruption after SAH and its mechanism appears to be independent of Angpt2.

Mutations in the gene encoding the gamma2 subunit of the gamma-aminobutyric acid type A receptor (GABRG2) have been reported to cause childhood absence epilepsy (CAE), febrile seizures (FS), and generalized epilepsy with FS plus (GEFS+). The authors analyzed GABRG2 in 47 unrelated patients with CAE, FS, and GEFS+ and identified a novel mutation that cosegregated with FS. Electrophysiologic studies demonstrated altered current desensitization and reduced benzodiazepine enhancement in mutant receptors.

Background and Purpose —We observed that the second application of cerebrospinal fluid (CSF) from subarachnoid hemorrhage (SAH) patients onto cerebral arterial segments in vitro produces a greater contraction than does the initial application. It was hypothesized that the difference between the first and second applications of SAH CSF was due to the activity of thrombin. Methods —Canine vertebrobasilar artery was removed under general anesthesia, cut into rings, and suspended in tissue culture baths so as to measure isometric tension. CSF was taken from patients 1 to 3 days after SAH via ventricular drains. CSF was administered in 10 − 5 to 10 − 1 dilutions. The thrombin antagonist hirudin (5 U) was administered before CSF in some experiments. The arterial tension response to pure oxyhemoglobin (10 − 4 to 3.2 g/dL) and thrombin (10 − 4 to 3.2 U/mL), administered alone or in combination, was also examined. Results —Hirudin increased arterial tension generated on the initial application of SAH CSF but had no effect on the tension generated by the second application of the SAH CSF, suggesting that thrombin limits the tension generated by vasoconstrictive agents in the CSF. Thrombin and pure oxyhemoglobin administered together produced less tension than that generated in response to oxyhemoglobin administered alone; no additive response was observed by coadministering the 2 vasoconstrictive agents. Conclusions —In the presence of oxyhemoglobin, thrombin acts to reduce cerebral arterial tension. This interaction between thrombin and hemoglobin may account for the observation that the second application of CSF from SAH patients onto cerebral arterial segments in vitro produces a greater contraction than does the initial application.

OBJECTIVE AND IMPORTANCE: We describe combined laminectomy and thoracoscopic surgery for removal of a dumbbell thoracic spinal tumor to demonstrate the feasibility of such an approach. CLINICAL PRESENTATION: We present the case of a 29-year-old man who developed chest pain and spinal cord compression from a thoracic dumbbell neurofibroma. TECHNIQUE: Surgical approaches for benign nerve sheath tumors that extend from the spinal cord into the thoracic cavity include combined laminectomy and thoracotomy either in one or two stages, or a lateral extracavitary approach involving laminectomy, facetectomy, and rib resection in a single stage. We performed a combination laminectomy and thoracoscopic tumor resection in a single stage with good results. CONCLUSION: This technique has not been reported previously in the literature. It has the advantage of avoiding the potential morbidity of a thoracotomy, as well as the extensive muscle dissection and pain associated with the lateral extracavitary approach.

OBJECTIVE: Oxygen-derived free radicals may contribute to vasospasm after the rupture of an intracranial aneurysm through direct vasoconstricting effects occurring within the arterial wall or, secondarily, by causing lipid peroxidation in the subarachnoid erythrocytes with secondary induction of vasoconstriction. U74389G is a potent inhibitor of lipid peroxidation and a scavenger of oxygen-derived free radicals. This study determined the relative contributions of oxygen-derived free radicals and lipid peroxidation to vasospasm in the double-hemorrhage dog model. METHODS: Sixteen dogs underwent baseline (Day 0) cerebral angiography and induction of subarachnoid hemorrhage by two injections of blood into the cisterna magna 2 days apart. They were randomized to receive drug vehicle (n = 8) or U74389G (n = 8, 3 mg/kg of body weight/d) intravenously. Drug administration and end point analysis were blinded. The end points were angiographic vasospasm, as assessed by comparison of angiograms obtained before and 7 days after subarachnoid hemorrhage, and the levels of malondialdehyde and salicylate hydroxylation products (dihydroxybenzoic acids) in cerebrospinal fluid and of malondialdehyde in subarachnoid blood clots and basilar arteries 7 days after hemorrhage. RESULTS: Comparisons within groups of Day 0 and Day 7 angiograms and between groups of angiograms obtained at Day 7, showed significant vasospasm in animals in the vehicle group (mean ± standard error, 51% ± 4) but not in the U74389G group (25% ± 11, P < 0.05, unpaired t test). High-pressure liquid chromatographic assays of malondialdehyde and dihydroxybenzoic acids in cerebrospinal fluid, subarachnoid blood clots, and basilar arteries showed no significant differences between groups. CONCLUSION: The significant prevention of vasospasm by U74389G without change in levels of indicators of free radical reactions suggests that the effect of the drug is related to other processes occurring in the arterial wall and that cerebrospinal fluid levels of oxygen radicals and lipid peroxides are not useful markers of vasospasm.

OBJECTIVE Adenosine 5'-triphosphate (ATP) causes vasoconstriction by activation of P2-purinoceptors on vascular smooth muscle cells. Erythrocytes contain ATP at a concentration (1.6 mmol/L) that contracts smooth muscle. Previous studies of hemoglobin solutions did not assess whether the vasoactivity was caused by ATP rather than or in addition to hemoglobin. It was hypothesized that the hemolysis of erythrocytes that occurs after subarachnoid hemorrhage releases ATP in concentrations that cause vasospasm. METHODS Thirty-eight rats were randomly assigned to undergo placement of one of the following compounds in a silastic elastomer cuff around each femoral artery: 1) agarose gel (n = 8); 2) dog erythrocyte hemolysate (n = 8); 3) purified human hemoglobin (Hemolink; Hemosol, Inc., Toronto, Canada; n = 8); 4) ATP (n = 8); or 5) clotted autologous blood (n = 6). The amounts of hemoglobins and adenine nucleotides in the compounds were measured by spectrophotometry and high pressure liquid chromatography. Hemolysate, purified hemoglobin, and ATP were mixed with agarose gel to create an artificial clot. Rats were killed and fixed by perfusion at physiological blood pressure 7 days after perivascular cuff and spasmogen placement. Vasospasm was assessed by image analysis of cross sections of fixed femoral arteries. Arteries were assessed for histopathological changes on 3-point scales. RESULTS There was significant variance in arterial diameters among groups (mean diameter +/- standard deviation: agarose gel, 0.29 +/- 0.06; purified hemoglobin, 0.28 +/- 0.04; hemolysate, 0.24 +/- 0.05; ATP, 0.25 +/- 0.05; clotted blood, 0.24 +/- 0.01; P < 0.05, analysis of variance, n = 11-20). Animals exposed to clotted blood, hemolysate that contained ATP, or ATP, developed vasospasm, whereas purified hemoglobin and agarose did not cause vasospasm. Endothelial proliferation and perivascular inflammation were more severe (P < 0.05) in arteries exposed to clotted blood, purified hemoglobin, and hemolysate. CONCLUSION These results suggest that ATP may be a vasospastic substance released by erythrocyte hemolysis. The concentration of ATP in impure solutions of hemoglobin is too low to account for the vasoactivity of these solutions. The discrepancy between arterial narrowing and histopathological changes suggests that either histopathological changes may not be an important correlate of arterial vasospasm or that other substances are important in vasospasm.

Many new antiepileptic drugs have been developed to treat seizure disorders. The established antiepileptic drugs reduce neuronal excitability by promoting sodium channel inactivation, inhibiting T-type calcium channels, or enhancing gamma-aminobutyric acid type A receptor-mediated inhibition. Several of the newer agents employ similar mechanisms, whereas others may enhance gamma-aminobutyric acid-ergic inhibitory systems or inhibit glutamatergic excitatory neurotransmission, and may be neuroprotective or antiepileptogenic.

Controversy has arisen over the results of, among other studies, clinical trials of clazosentan in patients with aneurysmal subarachnoid hemorrhage (SAH).1–3 In a double-blind, dose-finding study, where patients were randomized to placebo or 1 of 3 doses of clazosentan, the drug significantly reduced angiographic vasospasm (aVSP) as assessed by independent, blinded review of angiograms done on all patients. There was no effect on 90-day outcome on the extended Glasgow outcome scale.1 The study was not powered to detect a difference in 90-day clinical outcome. In 2 phase 3 studies, clazosentan was associated with a trend toward or with a significant reduction in aVSP-related morbidity but again no effect on clinical outcome at 90 days.2,3 This led to the suggestion that reducing aVSP does not improve outcome after aneurysmal SAH because aVSP is an epiphenomenon or marker of other processes that are the true causes of poor outcome, an idea that has been around for ≤35 years.4,5 The other processes that are postulated to cause poor outcome include early brain injury, microthromboemboli, and cortical spreading ischemia (Figure).6–8 Whether aVSP is an epiphenomenon, the clazosentan trials are consistent with meta-analysis of clinical trials of drugs to improve outcome after SAH, which found that drug treatment significantly reduced aVSP, but that there was only a statistically insignificant trend toward improvement in clinical outcome.5 Other hypotheses to explain this disconnection include (1) rescue therapy, which in many studies was used more frequently in the placebo groups, improves outcome in the placebo groups to the same extent as the drug treatment in the drug treatment groups, (2) drug side effects counterbalance any benefit, (3) the sample sizes of the trials were too small, and (4) the outcome measure was too crude to measure the …

Background: Patients who survive aneurysmal subarachnoid hemorrhage (SAH) often have deficits in learning, memory and executive function, although structural brain damage may not be detectable. We previously reported that rodents with SAH develop cognitive deficits and loss of long-term potentiation (LTP), a probable electrophysiological correlate of learning and memory. We hypothesize that loss of LTP may be caused by diminished synapses and/or dysfunction of synaptic molecules responsible for LTP, and that this occurs without neuronal death. Methods: SAH was created by injection of 300 μl of fresh, unheparinized arterial blood into the prechiasmatic cistern of Sprague-Dawley rats (300-350g). Controls were injected with the same amount of saline. Cell death was detected with Fluoro-jade B and TUNEL staining. The number of synapses in dendritic layer of CA1 was quantified by double immunohistochemical staining of MAP2 and synaptophysin, or directly by transmission electron microscopy. Glutamate receptor subunits (GluR1/2) and CaM kinase II were quantified by immunohistochemical staining. Superoxide and nitric oxide (NO) concentrations in freshly homogenized hippocampal tissues were detected by spectrophotometry with DAF-2DA and MCLA dyes. Results: In the dendritic area of CA1, the number of synapses was significantly decreased after SAH compared to controls (54±4/image for SAH, 74±3 for controls, p Conclusions: Loss of LTP after SAH in rats may be due to a synaptic plasticity rather than cell death. Decreased immunoreactivity to GluR1, GluR2 and CaM kinase II suggests reduction in key proteins that mediator LTP may also contribute. Decreased superoxide and increased NO suggest oxidative stress is involved in the loss of LTP.

Background: Subarachnoid hemorrhage (SAH) is characterized by an initial hemorrhagic and ischemic brain injury followed by delayed macro- and microvascular constriction. Large-artery vasospasm and enhanced microcirculatory myogenic tone may contribute to delayed cerebral ischemia. Although this implies that therapeutic interventions must specifically correct the SAH-induced myogenic tone enhancement, current therapeutic approaches non-selectively interfere with vasoconstriction and risk disrupting cerebral autoregulation. This may explain why most interventions do not improve clinical outcome. This study identifies the molecular basis for exacerbated cerebrovascular constriction and validates new targets for SAH treatment. Methods: Wild-type, tumor necrosis factor α (TNFα) knockout, sphingosine-1-kinase (Sphk1) knockout and inducible, smooth muscle cell-targeted TNFα knockout mice were used. SAH was created by injection of 80 μl of arterial blood into the prechiasmatic cistern. Myogenic tone in the olfactory artery was assessed with a myograph system. Standard procedures for fluorescent immunolocalization, Western blotting and assessment of apoptosis were used. Results: SAH increased myogenic tone and vascular wall TNFα expression, without enhancing overall vascular contractility in response to phenylephrine. Knockout of TNFα globally or smooth muscle cell-specifically prevented SAH-induced increased myogenic tone. Inhibition of TNFα-shedding (TAPI, 50 μmol/L) or receptor-binding (etanercept, 10 mg/ml) eliminated SAH-mediated myogenic tone augmentation. Cystic fibrosis transmembrane regulator (CFTR) protein expression was down-regulated in cerebral arteries after SAH, which was abolished by antagonism of TNFα. Genetic mouse models confirmed that S1P signaling mediates the myogenic tone augmentation in SAH. Finally, disrupting TNFα signaling attenuated neuronal apoptosis in SAH animals. Conclusion: We identify a novel smooth muscle cell autocrine/paracrine signaling network that augments myogenic tone in SAH. It links TNFα, CFTR and sphingosine-1-phosphate (S1P) signaling. Targeting TNFα and the S1P 2 receptor subtype are potential therapeutic options to improve clinical outcome in SAH.

Background: Glutamate toxicity (excitotoxicity) is well-studied in the pathogenesis of brain damage after cerebral ischemia and traumatic brain injury. Patients with subarachnoid hemorrhage (SAH) also have increased intracerebral glutamate as detected by microdialysis. While neurons and glia are potential sources of glutamate, platelets also release glutamate as part of their recruitment and might mediate neuronal damage. Studies have shown that intraluminal platelets escape into brain parenchyma after SAH. Therefore, we studied the hypothesis that formation of platelet microthrombi after SAH, and their subsequent extravasation releases glutamate that mediates excitotoxic brain injury and neuron dysfunction after SAH. Methods: We used two models, primary neuronal cultures exposed to activated platelets, and a model of SAH created by injection of 300 μl of fresh, unheparinized arterial blood into the prechiasmatic cistern of Sprague-Dawley rats (300-350 g). Glutamate was measured using amperometric microelectrode arrays. Propidium iodide was used to evaluate neuronal viability in neuronal cultures, and surface glutamate receptor immunohistochemical staining was used to evaluate the phenotype of platelet-exposed cultured neurons and brain after SAH. Microthrombi were stained with anti-fibrinogen antibodies. Results: We first demonstrated that thrombin-activated platelet-rich plasma releases glutamate in concentrations that exceed 300 μmol/l. When applied to neuronal cultures, this activated plasma was neurotoxic, and neurotoxicity was attenuated by glutamate receptor antagonism. Exposure of cultured neurons to thrombin-activated platelets induced a marked downregulation of the surface glutamate receptor GluR2, a marker of excitotoxicity and a possible mechanism of neuron dysfunction. Microthrombi were detected in rat cerebral cortex 7 days after SAH and linear regression demonstrated a strong correlation between proximity to microthrombi and reduction of surface glutamate GluR2 receptors. Conclusions: These correlative data support the novel hypothesis that platelet-mediated microthrombosis contributes to neuronal glutamate receptor dysfunction and might therefore influence clinical outcome following SAH.

Introduction: In CONSCIOUS-1, clazosentan, an endothelin receptor antagonist, significantly and dose-dependently reduced angiographic vasospasm (VSP) after aneurysmal subarachnoid hemorrhage (aSAH). CONSCIOUS-3 aimed to assess whether clazosentan improves VSP-related morbidity/all cause mortality after aSAH. Methods: This was a randomized, double-blind, placebo-controlled trial. Patients included in the study were 18-75 years old with SAH due to ruptured saccular aneurysm secured by endovascular coiling, any thick clot and WFNS grades I-IV prior to coiling procedure. Patients were randomized 1:1:1 to intravenous clazosentan (5 or 15 mg/h) or placebo for ≤2 weeks. The primary composite endpoint (all-cause mortality; VSP-related new cerebral infarcts; delayed ischemic neurological deficit [DIND] due to VSP; rescue therapy in the presence of confirmed angiographic VSP) was evaluated 6 weeks post-aSAH and assessed centrally by a blinded critical events committee, with significance determined using logistic regression adjusted for WFNS. The main secondary endpoint was the extended Glasgow Outcome Scale (GOSE; dichotomized) at week 12. Results: CONSCIOUS-3 was halted prematurely following nonsignificant results from the parallel CONSCIOUS-2 clipping study. There were 571 treated patients (placebo n=189, clazosentan 5 mg/h n=194, clazosentan 15 mg/h n=188). The primary endpoint occurred in 27% of the placebo group compared with 24% and 15% in the 5 and 15 mg/h clazosentan groups, respectively; significant improvement was seen with 15 mg/h clazosentan (odds ratio [OR] 0.474, 95% CI 28-82%; p=0.007) but not 5 mg/h (OR 0.786, 95% CI 48-129%; p=0.340). DIND decreased with increasing clazosentan dose (placebo 21%; clazosentan 5 mg/h 18%; clazosentan 15 mg/h 10%). VSP-related new cerebral infarct occurred in 13%, 16% and 7% in the placebo, clazosentan 5 and 15 mg/h groups, respectively. A 3-fold greater use of rescue therapy was seen in patients receiving placebo (21%) compared with 15 mg/h clazosentan (7%). Poor functional outcome (GOSE score ≤4) occurred in 24% of patients in the placebo group compared with 25% (OR 0.918, 95% CI 55-154%; p=0.748) and 28% (OR 1.337, 95% CI 80-223%; p=0.266) in the clazosentan 5 and 15 mg/h groups, respectively. At week 12, mortality rates were 6%, 4% and 6% with placebo, clazosentan 5 and 15 mg/h, respectively. Treatment-emergent adverse events of specific interest were lung complications (21%, 36%, 37%), anemia (10%, 13%, 13%) and hypotension (7%, 11%, 16%) in the placebo, clazosentan 5 and 15 mg/h groups, respectively. Conclusions: Clazosentan (15 mg/h) significantly reduced mortality/VSP-related morbidity; however, no significant effect on GOSE occurred, possibly due to greater use of rescue therapy with placebo. Pulmonary complications, anemia and hypotension were more common in patients receiving clazosentan.

Subarachnoid hemorrhage (SAH) accounts for about 6% of cases of stroke and afflicts more than 50,000 individuals annually in North America. Cerebral angiographic vasospasm has been recognized as being commonly associated with aneurysmal SAH for many years. However, recent studies showed that angiographic vasospasm does not always predict the location of infarction, and alleviation of vasospasm did not result in better outcome in SAH patients. One hypothesis gaining more attention is that separate or interacting pathways may cause delayed cerebral ischemia (DCI) after SAH, and that these, in combination with angiographic vasospasm, lead to poor outcome. Postulated mechanisms include acute brain injury, microcirculatory dysfunction, microthromboembolism, and cortical spreading ischemia. The focus of this study is to determine first, if experimental SAH is associated with microcirculatory constriction and thrombosis, second, to determine if any microthromboemboli are thrombi or emboli and third, to determine if these changes correlate with brain injury. We used a prechiasmatic cistern injection model of SAH. 15 mice (n = 5 for each group) underwent sham surgery or injection of 100 μl 0.9% NaCl or autologous blood into the prechiasmatic cistern. Mice were sacrificed 2 days later and brains were examined by scanning (SEM) and transmission electron microscopy (TEM) and by immunohistochemistry for fibrinogen and P-selectin. Neuronal apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling. Nitric oxide (NO) was measured with 4,5-diaminofluorescein-2-diacetate. TEM and SEM demonstrated that mice with SAH had significantly more arterioles filled with lesions consistent with microthrombi. Quantification of microvessel constriction showed significantly decreased microvessel lumen area but increased micrvessel wall thickness in animals with SAH compared to sham or saline-injected controls (P < 0.01, ANOVA). This was accompanied by decreased concentration of NO in mice with SAH compared to saline and sham mice. There are a significantly increased number of TUNEL positive cells in both cerebral cortex and hippocampus (p < 0.01). Marked increase of fibrinogen positive microvessels are found in both cerebral cortex and hippocampus (p < 0.05 in cortex, p < 0.001 in hippocampus). The number of microthrombi correlates with the number of apoptotic neurons in cerebral cortex (R 2 =0.98). Cell membrane P-selectin is increased in the endothelium of arterioles in mice with SAH. This correlates with decreased NO in the brain. In conclusion, SAH causes microthrombosis and constriction of arterioles, which correlates with neuronal death. Increased P-selectin and decreased NO suggest a mechanism for microthrombosis and arteriolar constriction.

P114 Nitric oxide (NO) may be important in vasospasm following subarachnoid hemorrhage (SAH). We evaluated the time course of changes in 3 isoforms of NO synthase (NOS) after SAH in monkeys. Right SAH was created and vasospasm was assessed on angiograms obtained at baseline and after 3, 7 and 14 days. Animals were euthanized at these times (n = 4 - 6 per time) and the right and left (control) middle cerebral arteries were removed. Levels of nNOS, eNOS and iNOS messenger ribonucleic acid (mRNA) and protein were measured by reverse transcriptase polymerase chain reaction and Western blotting. Angiography showed a 45 ± 13% (mean ± s.d., p < 0.05) decrease in middle cerebral artery diameter 3 days, a 41 ± 23% (p< 0.05) decrease 7 days and an insignificant 6 ± 14% decrease 14 days after SAH. The RNA for eNOS was significantly reduced (1.7 ± 0.5-fold) 7 days after SAH. There was a significant, 1.7 ± 0.2-fold reduction in eNOS protein on days 3 and 7 after SAH that returned to normal by day 14. There were no significant changes in nNOS mRNA or protein at any time after SAH. There were no significant changes in iNOS mRNA whereas iNOS protein increased on days 3 and 7 (7 ± 9 and 2.7 ± 2.8-fold, respectively, p > 0.05) and significantly decreased (2.7 ± 1.1-fold, p < 0.05) on day 14. Immunohistochemistry localized eNOS to endothelium, nNOS to brain and perivascular adventitia of the middle cerebral arteries and iNOS to inflammatory cells in the subarachnoid space. These results show a correlation between decreased eNOS and increased iNOS during vasospasm, suggesting a complex role for changes in NO in vasospasm.

78 This study tested the hypothesis that adenovirus expressing heme oxygenase-1 (HO-1), the enzyme that metabolises hemoglobin, would reduce contractions of cerebral arteries to hemoglobin and decrease vasospasm after subarachnoid hemorrhage (SAH). Rats underwent injection of vehicle or replication defective adenovirus expressing HO-1 (Ad5HO-1) or β-galactosidase (Ad-βGal) into the cisterna magna. Transgene expression was assessed by reverse transcriptase polymerase chain reaction for messenger ribonucleic acid (mRNA) levels, immunoblotting for protein levels, immunohistochemistry, response of the basilar artery to pure, ferrous hemoglobin and carboxyhemoglobin production 1 day after virus injection. Effects of Ad5HO-1 and Ad-βGal on vasospasm were assessed in a rat double hemorrhage model. Injection of Ad5HO-1 significantly increased HO-1 mRNA, protein and activity in basilar artery compared to Ad-βGal and vehicle. Injection of Ad-βGal result in low level expression and activity of HO-1 but this was significantly less than after injection of Ad5HO-1. HO-1 immunoreactivity was present in the basilar artery adventitia after injection of Ad5HO-1 and Ad-βGal. Injection of Ad5HO-1 and Ad-βGal increased basilar artery diameter and brainstem blood flow compared to control (P < 0.001, ANOVA). Ad5HO-1, however, significantly and selectively prevented contraction of the basilar artery and reduction in brainstem cerebral blood flow due to hemoglobin and significantly increased carboxyhemoglobin concentration compared to Ad-βGal and vehicle. Basilar artery diameter 7 days after SAH was significantly greater after injection of Ad5HO-1 (380 ± 36 μm) compared to Ad-βGal (282 ± 44 μm) and vehicle (287 ± 31μm, P < 0.001, ANOVA). In conclusion, injection of Ad5HO-1 into the cisterna magna of rats results in expression of functional HO-1 in the adventitia of the basilar artery. This is associated with an increase in basilar artery diameter. HO-1 expression with Ad5HO-1 inhibits arterial contractions to hemoglobin and after SAH.

P112 It has been suggested that changes in the arterial wall contribute to the maintenance and resolution of vasospasm after subarachnoid hemorrhage (SAH). This study determined if such changes contribute to the resolution of vasosapsm after SAH, if vasospasm resolves because of loss of subarachnoid blood clot or if a combination of these mechanisms is involved. 27 monkeys underwent baseline angiography and unilateral (n = 17) or bilateral (n = 8) SAH. Animals with bilateral SAH underwent angiography 1, 3, 5 and 7 days later followed by euthanasia. Animals with unilateral SAH had angiography 7 days later. The clot then was removed in these animals and replaced with fresh clot (n = 7) or removed and not replaced (n = 5). The removed clot was placed on the left side (n = 12). Controls did not have clot removal (n = 5) and/or had fresh clot placed on the left on day 7 (n = 5). Angiography was repeated every 2 days until euthanasia on day 14. SAH caused significant middle cerebral artery vasospasm on day 7 that slowly resolved by day 14. Removal of clot on day 7 resulted in more rapid reversal of vasospasm suggesting that vasospasm depended on continued presence of subarachnoid clot rather than on or in addition to structural changes in the artery. Placing fresh clot on the right side on day 7 produced vasospasm that persisted without resolving suggesting that there is no preconditioning response in the artery that prevents or conributes to reversal of vasospasm. On the other hand, placing 7-day-old clot from the right onto the left caused significantly more rapid onset of severe vasospasm than placing fresh clot from animals 7 days after SAH, suggesting that breaking up the clot to replace it on the left increases release of spasmogens or that there is a negative adaptive response that exacerbates vasospasm. In conclusion, vasospasm resolves because of loss of subarachnoid clot. There is no evidence for a preconditioning response in cerebral arteries that renders them less responsive to subarachnoid clot. Spasmogen release from clot 7 days after SAH may be reduced in this model so that breaking up the clot increases spasmogen release resulting in rapid onset of severe vasospasm and/or that there is a negative adaptive response 7 days after SAH that worsens vasospasm.

Mammalian spinal neurons grown in tissue culture were used to study the effects of the four convulsants-penicillin, pentylenetetrazol, picrotoxin, and bicuculline-on these neurons' responses to amino acids. Bath application of all four convulsants produced paroxysmal depolarizing events in the neurons; iontophoresis of the four convulsants selectively depressed responses produced by iontophoresis of the putative inhibitory transmitter GABA, and effected this depression without altering either inhibitory responses to beta-alanine or glycine, or excitation mediated by glutamate. These results support the hypothesis that the convulsant activity of these agents comes in part from selective antagonism of GABA-mediated postsynaptic inhibition.

The convulsant bicuculline (BICUC) had both synaptic and nonsynaptic actions on mouse spinal cord neurons in primary dissociated cell culture. BICUC antagonized postsynaptic responses to the inhibitory neurotransmitter GABA (a synaptic action) and produced direct membrane depolarization by blocking a membrane potassium conductance and directly prolonging calcium-dependent action potentials (nonsynaptic actions). In cultured spinal cord neurons, BICUC also produced paroxysmal depolarizing events (PDE), which might be equivalent to in vivo convulsant-induced bursting in spinal cord neurons or paroxysmal depolarizing shifts (PDS) in cortical neurons. Thus, the concentrations of BICUC producing both synaptic and nonsynaptic actions overlapped those producing paroxysmal activity. The results suggest that in addition to antagonism of GABA-mediated synaptic inhibition, direct nonsynaptic actions of BICUC may be required for some neurons to develop PDS.

Mammalian spinal cord neurons were grown in dissociated cell culture and used to study the effects of the anticonvulsant barbiturates phenobarbital and mephobarbital, and the anesthetic barbiturates pentobarbital, secobarbital, and 1,3-dimethyl-butylethyl barbituric acid on amino acid responses and neuronal membrane properties. All barbiturates augmented responses to GABA and diminished glutamate (GLU) responses, but the anesthetic barbiturates were more potent. The anesthetic barbiturates directly depressed excitability by increasing membrane conductance, an effect reversed by the GABA antagonists picrotoxin and penicillin. Anticonvulsant barbiturates, however, had only minimal GABA-mimetic inhibitory action at high doses. Modulation of synaptic events mediated by GABA and GLU might contribute to barbiturate anticonvulsant activity; and direct GABA-mimetic inhibition, combined with similar modulation of synaptic transmission, might underlie barbiturate anesthesia.