Christopher J. Winfree

Procedural and Strain-related Variables Significantly Affect Outcome in a Murine Model of Focal Cerebral Ischemia

The recent availability of transgenic mice has led to a burgeoning number of reports describing the effects of specific gene products on the pathophysiology of stroke. Although focal cerebral ischemia models in rats have been well described, descriptions of a murine model of middle cerebral artery occlusion are scant and sources of potential experimental variability remain undefined. We hypothesized that slight technical modifications would produce widely discrepant results in a murine model of stroke and that controlling surgical and procedural conditions could lead to reproducible physiological and anatomic stroke outcomes. To test this hypothesis, we established a murine model that would permit either permanent or transient focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery. This study provides a detailed description of the surgical technique and reveals important differences among strains commonly used in the production of transgenic mice. In addition to strain-related differences, infarct volume, neurological outcome, and cerebral blood flow appear to be importantly affected by temperature during the ischemic and postischemic periods, mouse size, and the size of the suture that obstructs the vascular lumen. When these variables were kept constant, there was remarkable uniformity of stroke outcome. These data emphasize the protective effects of hypothermia in stroke and might help to standardize techniques among different laboratories to provide a cohesive framework for evaluating the results of future studies in transgenic animals.

Exacerbation of Cerebral Injury in Mice That Express the P-Selectin Gene Identification of P-Selectin Blockade as a New Target for the Treatment of Stroke

There is currently a stark therapeutic void in the treatment of evolving stroke. Although P-selectin is rapidly expressed by hypoxic endothelial cells in vitro, the functional significance of P-selectin expression in stroke remains unexplored. In order to identify the pathophysiological consequences of P-selectin expression and to identify P-selectin blockade as a potential new approach for the treatment of stroke, experiments were performed using a murine model of focal cerebral ischemia and reperfusion. Early P-selectin expression in the postischemic cerebral cortex was demonstrated by the specific accumulation of radiolabeled anti-murine P-selectin IgG, with the increased P-selectin expression localized to the ipsilateral cerebral microvascular endothelial cells by immunohistochemistry. In experiments designed to test the functional significance of increased P-selectin expression in stroke, neutrophil accumulation in the ischemic cortex of mice expressing the P-selectin gene (PS +/+) was demonstrated to be significantly greater than that in homozygous P-selectin-null mice (PS -/-). Reduced neutrophil influx was accompanied by greater postischemic cerebral reflow (measured by laser Doppler) in the PS -/- mice. In addition, PS -/- mice demonstrated smaller infarct volumes (5-fold reduction, P<.05) and improved survival compared with PS +/+ mice (88% versus 44%, P<.05). Functional blockade of P-selectin in PS +/+ mice using a monoclonal antibody directed against murine P-selectin also improved early reflow and stroke outcome compared with control mice, with reduced cerebral infarction volumes noted even when the blocking antibody was administered after occlusion of the middle cerebral artery. These data are the first to demonstrate a pathophysiological role for P-selectin in stroke and suggest that P-selectin blockade may represent a new therapeutic target in the treatment of stroke.

Dehydroascorbic acid, a blood–brain barrier transportable form of vitamin C, mediates potent cerebroprotection in experimental stroke

Neuronal injury in ischemic stroke is partly mediated by cytotoxic reactive oxygen species. Although the antioxidant ascorbic acid (AA) or vitamin C does not penetrate the blood–brain barrier (BBB), its oxidized form, dehydroascorbic acid (DHA), enters the brain by means of facilitative transport. We hypothesized that i.v. DHA would improve outcome after stroke because of its ability to cross the BBB and augment brain antioxidant levels. Reversible or permanent focal cerebral ischemia was created by intraluminal middle cerebral artery occlusion in mice treated with vehicle, AA, or DHA (40, 250, or 500 mg/kg), either before or after ischemia. Given before ischemia, DHA caused dose-dependent increases in postreperfusion cerebral blood flow, with reductions in neurological deficit and mortality. In reperfused cerebral ischemia, mean infarct volume was reduced from 53% and 59% in vehicle- and AA-treated animals, respectively, to 15% in 250 mg/kg DHA-treated animals (P < 0.05). Similar significant reductions occurred in nonreperfused cerebral ischemia. Delayed postischemic DHA administration after 15 min or 3 h also mediated improved outcomes. DHA (250 mg/kg or 500 mg/kg) administered at 3 h postischemia reduced infarct volume by 6- to 9-fold, to only 5% with the highest DHA dose (P < 0.05). In contrast, AA had no effect on infarct volumes, mortality, or neurological deficits. No differences in the incidence of intracerebral hemorrhage occurred. Unlike exogenous AA, DHA confers in vivo, dose-dependent neuroprotection in reperfused and nonreperfused cerebral ischemia at clinically relevant times. As a naturally occurring interconvertible form of AA with BBB permeability, DHA represents a promising pharmacological therapy for stroke based on its effects in this model of cerebral ischemia.

Matrix metalloproteinase-9 in cerebral aneurysms

OBJECTIVE Generalized disruption of arterial wall morphological changes in patients harboring cerebral aneurysms has been documented; however, little is known regarding the pathogenesis of these changes. To explore the role of the elastolytic gelatinase, matrix metalloproteinase-9 (MMP-9), levels of this enzyme in the wall of intracranial aneurysms were compared with those in both intracranial and extracranial arteries. The tissue levels of its major inhibitor, tissue inhibitor of metalloproteinase (TIMP), were measured in these tissues as well. The activity of MMP-9 in plasma was also evaluated. METHODS The aneurysm wall was excised from three of six patients undergoing craniotomies for aneurysm clipping. A 1-cm segment of superficial temporal artery (STA) was obtained from each of six patients. Additional STAs were obtained from six patients in the control group who were undergoing craniotomies for nonvascular disease. An intracranial artery was also obtained from the anterior temporal neocortical resection of a patient undergoing a craniotomy for mesial temporal sclerosis. MMP-9 and TIMP levels were determined via Western blot analysis. Using substrate gel Zymography, MMP-9 plasma activity was determined for a separate cohort of patients with aneurysms (n = 6) and patients in the control group (n = 6). RESULTS MMP-9 and TIMP levels in the aneurysm wall were markedly increased beyond levels in both extracranial arteries (STAs from patients with aneurysms and patients in the control group) and the intracranial artery. There were no differences in the levels of MMP-9 in the STAs of patients harboring aneurysms when compared with patients in the control group. Also, no differences were noted in plasma MMP-9 activity. CONCLUSION Local rather than systemic perturbations in MMP-9 levels may contribute to the matrix disruption associated with cerebral aneurysms. This local up-regulation is not the result of TIMP down-regulation. The lack of increased systemic metalloproteinase activity precludes the use of plasma MMP-9 activity as a screening tool for presymptomatic aneurysms. However, local therapeutic modulation of MMP-9 activity may help arrest aneurysm progression.

Intramedullary spinal cord metastasis: Report of three cases and review of the literature

BACKGROUND Intramedullary spinal cord metastasis is rare; but it is being encountered with increasing frequency. Optimal treatment after diagnosis remains controversial. METHODS In the last 3 years, we have encountered three cases of intramedullary metastasis presenting as focal mass lesions with minimal systemic evidence of cancer. We present our results in these patients and review the literature in an effort to more optimally define both the natural course of this disease, as well as a potential subset of patients who might benefit from more aggressive treatment. RESULTS With the availability of more sensitive imaging techniques, these tumors are being diagnosed with increasing frequency. Magnetic resonance imaging is sensitive, but nonspecific, in distinguishing intramedullary spinal cord metastases from primary cord tumors. Urgent biopsy is often necessary prior to definitive treatment. Radiation with chemotherapy significantly prolongs survival. Radical subtotal resection may offer additional quality survival, especially in cases of metastatic melanoma with an occult primary. CONCLUSIONS Regardless of treatment, many patients survive less than 1 year. Intramedullary spinal cord metastasis is a devastating condition, but with appropriate diagnosis and aggressive treatment, selected patients may have substantially increased survival.

Postischemic cerebrovascular E-selectin expression mediates tissue injury in murine stroke.

Background and Purpose Although the deleterious role of several proinflammatory mediators, including P-selectin, in reperfused stroke is well established, the role of E-selectin has not been fully characterized. Methods E-selectin mRNA expression was studied at 4, 10, and 24 hours after reperfusion with reverse transcription and polymerase chain reaction in mice (n=18) subjected to transient intraluminal middle cerebral artery occlusion (MCAO). Mice received intravenous injection with anti–E-selectin monoclonal antibody (10, 35, or 50 &mgr;g), nonimmune IgG, or vehicle immediately before MCAO and 90 minutes later (n=85). Others received anti–E-selectin antibody 3 or 6 hours after MCAO (n=32). Myeloperoxidase activity was measured in sham-operated mice and after 10 hours of reperfusion in saline-, nonimmune IgG–, or anti–E-selectin IgG–treated cohorts (n=17). Serial cerebral blood flow was measured with laser-Doppler flowmetry, and outcomes were assessed by neurological deficits and infarct volumes with the use of planimetric analysis of triphenyltetrazolium chloride–stained sections. Results Upregulated E-selectin expression occurred in the ischemic cerebral vasculature within 4 hours of reperfusion and persisted for 24 hours. Anti–E-selectin antibody increased ischemic cortical cerebral blood flow up to 2.6-fold (P <0.05). In addition to dose-dependent reductions in neurological deficits (P <0.05), mortality, and infarct volumes (P <0.01 for 35 and 50 &mgr;g), anti–E-selectin treatment reduced cerebral neutrophil accumulation (P <0.05) and was neuroprotective even if delayed until 3 hours after ischemia (P <0.05). Conclusions These findings establish a functional role for E-selectin in the pathogenesis of tissue injury after cerebral ischemia and reperfusion and suggest that E-selectin blockade may be clinically useful in the treatment of reperfused stroke.

Mild Hypothermia Reduces Penumbral Glutamate Levels in the Rat Permanent Focal Cerebral Ischemia Model

Although the cerebroprotective effects of hypothermia in focal models of ischemia are undisputed, the underlying mechanisms of this protection are still subject to much controversy. To analyze whether mild hypothermia attenuates glutamate levels in the penumbra surrounding permanent focal infarcts, extracellular glutamate concentration was analyzed bilaterally by microdialysis 20 minutes before to 120 minutes after a middle cerebral artery occlusion (MCAO) in rats. Normothermic animals (n = 11) had a baseline glutamate concentration of 1.14 +/- 0.40 mumol/ml (standard error of the mean) before the MCAO. Extracellular glutamate levels increased gradually after vessel occlusion to peak at 10.1 +/- 1.45 mumol/ml 80 minutes after the MCAO. This level gradually decreased to 5.72 +/- 1.67 mumol/ml by 120 minutes. Hypothermic animals (n = 11) had a baseline glutamate concentration of 1.73 +/- 0.83 mumol/ml before the MCAO. Extracellular glutamate levels increased after vessel occlusion but stabilized at 3.47 +/- 1.37 mumol/ml 30 minutes after the MCAO and remained stable until completion of the experiment. There were no significant differences in cortical blood flow between the normothermic and hypothermic groups at any time during the experiment. Infarct volumes, expressed as a percentage of the volume of the right (ipsilateral) hemisphere, were 19.8 +/- 2.16% in the normothermic group and 13.0 +/- 1.42% in the hypothermic group (P < 0.02). Although the normothermic penumbral glutamate levels began to increase immediately after the MCAO, they did not peak until 80 minutes after occlusion. In contrast, the normothermic core glutamate levels peaked within 30 minutes after the MCAO. Glutamate diffusion from the core region to the penumbra might account for this delay. Hypothermic cerebroprotection might involve a reduction in the pool of potentially diffusable glutamate in the core region but have little direct effect on glutamate release in the penumbra.

Serum S100B protein levels are correlated with subclinical neurocognitive declines after carotid endarterectomy.

OBJECTIVECarotid endarterectomy (CEA) is an effective means of stroke prevention among appropriately selected patients; however, neuropsychometric testing has revealed subtle cognitive injuries in the early postoperative period. The purpose of this study was to establish whether serum levels of two biochemical markers of cerebral injury were correlated with postoperative declines in neuropsychometric test performance after CEA. METHODSFifty-five consecutive patients underwent a battery of neuropsychometric tests 24 hours before and 24 hours after elective CEA. Two patients were excluded because of postoperative strokes. The pre- and postoperative serum levels of S100B protein and neuron-specific enolase for injured patients, defined as those who exhibited significant declines in neuropsychometric test performance (n = 12), were compared with the levels for uninjured patients (n = 41). RESULTSThere were no significant differences in the baseline S100B levels for the two groups. Injured patients exhibited significantly higher S100B levels, compared with uninjured patients, at 24, 48, and 72 hours after surgery (P < 0.05). There were no significant differences in neuron-specific enolase levels for injured and uninjured patients at any time point. CONCLUSIONThese data suggest that subtle cerebral injuries after CEA, even in the absence of overt strokes, are associated with significant increases in serum S100B but not neuron-specific enolase levels. Analyses of earlier time points in future studies of subtle cognitive injuries and biochemical markers of cerebral injury after CEA may be revealing.

Ultrarapid, Convection-Enhanced Intravascular Hypothermia A Feasibility Study in Nonhuman Primate Stroke

Background and Purpose— Hypothermia has been shown to be neuroprotective in a variety of clinical settings. Unfortunately, poor delivery techniques and insufficient data in appropriate preclinical models have hampered its development in human stroke. To address these limitations, we have devised a 10F intravascular catheter capable of rapid systemic cooling of nonhuman primates. Methods— Placed in the inferior vena cava via a transfemoral approach, the catheter was used to induce mild systemic hypothermia 3 hours after the onset of hemispheric stroke in baboons. Results— Cooling was achieved at a rate of 6.3±0.8°C/h. Target brain temperatures (32.2±0.2°C) were reached at the same time (47.7±6.32 minutes) as target esophageal temperatures (32.0±0.0°C). Hypothermia was maintained for 6 hours in all animals. Animals did not experience the infections, coagulopathy, or cerebral edema commonly seen with surface cooling methods in human stroke. Conclusions— These data suggest that a brief episode of mild core hypothermia instituted at a clinically relevant time point can be achieved in primate stroke and that our intravascular cooling technique provides safe, rapid, and reproducible hypothermia.

Mild hypothermia and MK-801 have similar but not additive degrees of cerebroprotection in the rat permanent focal ischemia model

Although not the sole factor, glutamate-mediated excitotoxicity is accepted as a major mechanism of ischemic neuronal damage. MK-801 and mild hypothermia, two cerebroprotective modalities, which have been documented to alter glutamatergic action, were tested in the rat middle cerebral artery occlusion (MCAO) model simulating permanent focal ischemia. We administered normothermic (37 degrees C) animals with either MK-801 (1.0 mg/kg 30 min before MCAO or 2.5 mg/kg 30 min before, immediately after, 4 hours, and 8 hours after MCAO) or saline vehicle (30 min before MCAO). Mildly hypothermic (33 degrees C) animals were administered either MK-801 (1.0 mg/kg) or saline vehicle 30 minutes before MCAO. Mild hypothermia was induced over a 20-minute period before MCAO in hypothermic animals. All animals were killed 24 hours after MCAO; their brains were sectioned and stained with 2,3,5-triphenyltetrazolium chloride and their infarct volumes were calculated. In normothermica animals given 1.0 mg/kg and multidose 2.5-mg/kg intraperitoneal injections of MK-801, the infarct volumes (as a percentage of right hemispheric volume) were 16.8 +/- 3.5% and 16.3 +/- 3.0%, respectively. These infarct volumes were significantly different (P < 0.05; single-variable analysis of variance) from the normothermic, drug-free control (26.8 +/- 1.9%), but not significantly different from each other. Analysis of the data using a nonparametric test (Kruskal-Wallis; P = 0.02) confirmed the same significant differences in infarct size. The infarct volumes from the mildly hypothermic groups were not different (1 mg/kg of MK-801, 15.5 +/- 2.3% and saline control, 15.4 +/- 1.1%).(ABSTRACT TRUNCATED AT 250 WORDS)