Reduction of tissue plasminogen activator-induced hemorrhage and brain injury by free radical spin trapping after embolic focal cerebral ischemia in rats.

Thrombolytic stroke therapy with tissue plasminogen activator (tPA) remains complicated by serious risks of cerebral hemorrhage and brain injury. In this study, a novel model of tPA-induced hemorrhage was used in spontaneously hypertensive rats to examine the correlates of hemorrhage, and test methods of reducing hemorrhage and brain injury. Homologous blood clot emboli were used to occlude the middle cerebral artery in spontaneously hypertensive rats, and delayed administration of tPA (6 hours postischemia) resulted in high rates of cerebral hemorrhage 24 hours later. Compared with untreated rats, tPA significantly increased hemorrhage volumes by almost 85%. Concomitantly, infarction and neurological deficits were worsened by tPA. A parallel experiment in normotensive Wistar-Kyoto rats showed markedly reduced rates of hemorrhage, and tPA did not significantly increase hemorrhage volumes. To examine whether tPA-induced hemorrhage was caused by the delayed onset of reperfusion per se, another group of spontaneously hypertensive rats was subjected to focal ischemia using a mechanical method of arterial occlusion. Delayed (6 hours) reperfusion via mechanical means did not induce hemorrhage. However, administration of tPA plus delayed mechanical reperfusion significantly increased hemorrhage volumes. Since reperfusion injury was implicated, a final experiment compared outcomes in spontaneously hypertensive rats treated with tPA plus the free radical spin trap alpha-phenyl tert butyl nitrone (alpha-PBN) versus tPA alone. tPA-induced hemorrhage volumes were reduced by 40% with alpha-PBN, and infarction and neurological deficits were also decreased. These results indicate that (1) blood pressure is an important correlate of tPA-induced hemorrhage, (2) tPA interacts negatively with reperfusion injury to promote hemorrhage, and (3) combination therapies with anti-free radical treatments may reduce the severity of tPA-induced hemorrhage and brain injury after cerebral ischemia.     

Effects of tissue type plasminogen activator in embolic versus mechanical models of focal cerebral ischemia in rats.

Tissue type plasminogen activator (tPA) can be effective therapy for embolic stroke by restoring cerebral perfusion. However, a recent experimental study showed that tPA increased infarct size in a mouse model of transient focal ischemia, suggesting a possible adverse effect of tPA on ischemic tissue per se. In this report, the effects of tPA in two rat models of cerebral ischemia were compared. In experiment 1, rats were subjected to focal ischemia via injection of autologous clots into the middle cerebral artery territory. Two hours after clot injection, rats were treated with 10 mg/kg tPA or normal saline. Perfusion-sensitive computed tomography scanning showed that tPA restored cerebral perfusion in this thromboembolic model. Treatment with tPA significantly reduced ischemic lesion volumes measured at 24 hours by >60%. In experiment 2, three groups of rats were subjected to focal ischemia via a mechanical approach in which a silicon-coated filament was used intraluminally to occlude the origin of the middle cerebral artery. In two groups, the filament was withdrawn after 2 hours to allow for reperfusion, and then rats were randomly treated with 10 mg/kg tPA or normal saline. In the third group, rats were not treated and the filament was not withdrawn so that permanent focal ischemia was present. In this experiment, tPA did not significantly alter lesion volumes after 2 hours of transient focal ischemia. In contrast, permanent ischemia significantly increased lesion volumes by 55% compared with transient ischemia. These results indicate that in these rat models of focal cerebral ischemia, tPA did not have detectable negative effects. Other potentially negative effects of tPA may be dependent on choice of animal species and model systems.     

Intravenous tissue-type plasminogen activator therapy for ischemic stroke: Houston experience 1996 to 2000.

CONTEXT: Intravenous tissue-type plasminogen activator (tPA) therapy using the National Institute of Neurological Disorders and Stroke criteria has been given with variable safety to less than 5% of the patients who have ischemic strokes nationwide. Our center is experienced in treating large numbers of stroke patients with intravenous tPA. OBJECTIVE: To report our total 4-year experience in the treatment of consecutive patients who had an ischemic stroke. DESIGN: Prospective inception cohort registry of all patients seen by our stroke team and an additional retrospective medical record review of all patients treated between January 1, 1996, and June 1, 2000. SETTING: A veteran stroke team composed of fellows and stroke-specialty faculty servicing 1 university and 3 community hospitals in a large urban setting. PATIENTS: Consecutive patients with ischemic stroke treated within the first 3 hours of symptom onset. INTERVENTION: According to the National Institute of Neurological Disorders and Stroke protocol, 0.9 mg/kg of intravenous tissue-type plasminogen activator was administered. MAIN OUTCOME MEASURES: Number and proportion treated, patient demographics, time to treatment, hemorrhage rates, and clinical outcome. RESULTS: A total of 269 patients were treated between January 1, 1996, and June 1, 2000. Their mean age was 68 years (age range, 24-93 years); 48% were women. This represented 9% of all patients admitted with symptoms of cerebral ischemia at our most active hospital (over the final 6 months, 13% of all patients with symptoms of cerebral ischemia and 15% of all acute ischemic stroke patients). Before treatment the mean +/- SD National Institutes of Health Stroke Scale (NIHSS) score was 14.4 +/- 6.1 points (median, 14 points; range, 4-33 points). A tPA bolus was given at 137 minutes (range, 30-180 minutes); 28% of the patients were treated within 2 hours. The mean door-to-needle time was 70 minutes (range, 10-129 minutes). The symptomatic intracerebral hemorrhage rate was 5.6% of those patients with a second set of brain scans (4.5% of all patients), with a declining trend from 1996 to 2000. Protocol violations were found in 13% of all patients; the symptomatic intracerebral hemorrhage rate in these patients was 15%. At 24 hours, the NIHSS score was 10 +/- 8 points (median, 8 points; range, 0-36 points). In-hospital mortality was 15% and the patients' discharge NIHSS scores were 7 +/- 7 points (median, 3 points; range, 0-35 points). CONCLUSIONS: Intravenous tPA therapy can be given to up to 15% of the patients with acute ischemic stroke with a low risk of symptomatic intracerebral hemorrhage. Successful experience with intravenous tPA therapy depends on the experience and organization of the treating team and adherence to published guidelines.     

Matrix metalloproteinase activation and blood-brain barrier breakdown following thrombolysis

Thrombolysis with tissue plasminogen activator (tPA) is the only pharmacotherapy available for cerebral ischemia. However, the use of tPA can increase the risk of hemorrhage due to blood-brain barrier (BBB) breakdown. Recent evidence suggests that increased activation of matrix metalloproteinases (MMPs) may be involved in this breakdown. This study examines the temporal profile of MMP-2 and -9 following tPA administration to ischemic rats. Male Sprague-Dawley rats were randomly assigned to one of four groups (Sham-tPA; Sham-Saline; Ischemia-tPA; Ischemia-Saline; group n = 6, total N = 120). Focal embolic ischemia was induced by middle cerebral artery occlusion through injection of an autologous clot. One hour post-surgery, tPA (10 mg/kg) or saline was delivered intravenously and animals were euthanized at 3, 6, 12, or 24 h after onset of ischemia. Infarct volume was measured by TTC staining; BBB components examined immunohistochemically; and MMP activation measured by gelatin zymography. Our results show that tPA significantly reduced infarct volumes (overall infarct volume-Sham-tPA: 5.80 +/- 4.55 [mean +/- SE]; Sham-Saline: 5.00 +/- 4.23; Ischemia-tPA: 186.1 +/- 73.45; Ischemia-Saline: 284.8 +/- 88.74; all P < 0.05). Treatment with tPA was also associated with the activation of MMP-9 at 6, 12, and 24 h following ischemia. No temporal changes were observed in MMP-2 activation, although tPA administration increased its activity compared to saline treatment. Analyses of immunohistochemistry showed that destruction of components of the BBB followed MMP-9 activation. Thus, increased MMP-9 activation may, in part, be responsible for the increases in hemorrhagic transformation reported with use of tPA. Our study is the first to demonstrate the temporal profile of MMP activation following thrombolysis with tPA in a model of thrombotic focal cerebral ischemia.     

Progesterone attenuates hemorrhagic transformation after delayed tPA treatment in an experimental model of stroke in rats: involvement of the VEGF–MMP pathway

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for acute stroke, but its use remains limited. Progesterone (PROG) has shown neuroprotection in ischemia, but before clinical testing, we must determine how it affects hemorrhagic transformation in tPA-treated ischemic rats. Male Sprague–Dawley rats underwent middle cerebral artery occlusion with reperfusion at 4.5 hours and tPA treatment at 4.5 hours, or PROG treatment intraperitoneally at 2 hours followed by subcutaneous injection at 6 hours post occlusion. Rats were killed at 24 hours and brains evaluated for cerebral hemorrhage, swelling, blood–brain barrier (BBB) permeability, and levels of matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor level (VEGF), and tight junction (TJ) proteins. We also evaluated PROG''s efficacy in preventing tPA-induced impairment of transendothelial electrical resistance (TEER) and TJ proteins under hypoxia/reoxygenation in the endothelial cells. Delayed tPA treatment induced significant hemorrhagic conversion and brain swelling. Treatment with PROG plus tPA ameliorated hemorrhage, hemispheric swelling, BBB permeability, MMP-9 induction, and VEGF levels compared with controls. Progesterone treatment significantly prevented tPA-induced decrease in TEER and expression of occludin and claudin-5, and attenuated VEGF levels in culture media subjected to hypoxia. The study concluded that PROG may extend the time window for tPA administration in ischemic stroke and reduce hemorrhagic conversion.     

Acute enoxaparin treatment widens the therapeutic window for tPA in a mouse model of embolic stroke

OBJECTIVES: The purpose of this experiment was to determine if the low molecular weight heparin (LMWH) enoxaparin could extend the treatment window for thrombolysis in a mouse model of embolic stroke. METHODS: To establish the treatment window, mice were treated with tPA 2, 3 or 4 hours after clot insertion. Results showed that only the 2 hour treatment group exhibited infarct volumes significantly smaller than untreated controls. We attempted to widen this window by pre-treating mice with enoxaparin (10 mg/kg, s.c.; n=36) 1 hour before embolization. A control group (n=24) was given a saline injection. The enoxaparin-treated animals were subdivided and treated with tPA either 4 (n=12) or 6 hours (n=12) after clot insertion, while the third group (n=12) was given saline. The saline-pre-treated mice were dived into two groups: one group (n=12) received tPA and the other group (n=12) received saline 4 hours post-stroke. Embolization was confirmed by laser Doppler flowmetry and the effects of the resulting infarcts were evaluated by triphenyltetrazolium chloride staining and by behavioral testing. RESULTS: Results showed large infarcts and impaired sensorimotor coordination in the saline pre-treated animals confirming the narrow treatment window. Enoxaparin pre-treatment produced significantly smaller infarcts and improved motor behavior in groups treated with tPA both 4 and 6 hours after embolization. Neither the 4 nor the 6 hour tPA-treated groups showed evidence of intracerebral hemorrhage or external bleeding. CONCLUSION: These data indicate that the LMWH enoxaparin can significantly increase the therapeutic time window in a mouse model of embolic stroke.     

Tissue plasminogen activator extravasated through the cerebral vessels: evaluation using a rat thromboembolic stroke model

Neurotoxic effects of endogenous tissue plasminogen activator (tPA) have recently been reported. Employing a rat model of thromboembolic stroke, we evaluated the extent and degree of extravasation of exogenous tPA administered for the purpose of fibrinolysis. In a thromboembolic model using Sprague-Dawley rats, focal cerebral ischemia was induced at the territory of the middle cerebral artery (MCA). Early reperfusion was induced by administering tPA (10 mg/kg) intravenously at 30 minutes after the onset of ischemia. Extravasated tPA was evaluated by immunohistochemistry, and the concentration of tPA in the brain tissue was quantified by enzyme-linked immunosorbent assay methods. The integrity of the blood-brain barrier (BBB) was examined electronmicroscopically. In a thread model of transient ischemia, reperfusion was induced without tPA administration at 30 minutes or 2 hours after the onset of ischemia, and the tPA content of the brain was quantified. In the rats with thromboembolic stroke, extravasation of tPA was observed at the territory of the MCA. Both the endogenous and exogenous tPA contents were 3.5 +/- 1.6 ng/ml of homogenized brain in saline. Electronmicroscopically, mild ischemic changes were observed, although the integrity of the BBB was preserved. In the thread model rats, the endogenous tPA contents of the ischemic hemisphere were 0.9 +/- 0.1 and 1.0 +/- 0.2 ng/ml in the 30-minute and 2-hour ischemia groups, respectively, and were significantly lower than the tPA contents in the thromboembolic stroke rats (p<0.01). The present findings indicate that significant extravasation of exogenous tPA occurs through the cerebral vessels even though early reperfusion is induced.     

Tissue plasminogen activator does not increase neuronal damage in rat models of global and focal ischemia

BACKGROUND: Intravenous tissue plasminogen activator (tPA) is the only approved treatment for acute ischemic stroke. Recent results from both in vitro and in vivo animal model experiments suggest the possibility that tPA is neurotoxic. METHODS: The authors evaluated the putative neurotoxicity of tPA in both global and focal animal models of ischemic stroke. Global ischemia was induced in male Wistar rats using a modified four-vessel occlusion technique, with percentage neuronal injury assessed at 7 days through necrotic and normal cell count in the CA1 region. Transient focal ischemia was induced in male spontaneously hypertensive rats subjected to middle cerebral artery clipping, with measurement of cortical infarct volume at 24 hours. tPA was administered in 1, 5, or 10 mg/kg doses given intravenously as a 10% bolus, 90% over the following hour, analogous to current human treatment protocols. RESULTS: In the global model, percent hippocampal injury was 60%+/-23%, 66%+/-26%, 55%+/-26%, and 52%+/-12% in the saline control, 1, 5, and 10 mg/kg tPA groups, respectively. In the focal model, after 120 minutes of ischemia, the control infarct size was 151+/-39 mm3, and for the group given 10 mg/kg of tPA, it was 158+/-28 mm3. CONCLUSIONS: Despite sublethal insults, with moderate injury induced by ischemia, there was no evidence that increasing doses of tPA exacerbated ischemic injury.     

Partial Intra‐Aortic Occlusion Improves Perfusion Deficits and Infarct Size Following Focal Cerebral Ischemia

Reperfusion with intravenous tissue plasminogen activator (tPA) has been the goal of therapy for acute ischemic stroke; however, tPA is contraindicated in many patients, has low recanalization rates in major occlusions, and carries a substantial risk of symptomatic intracerebral hemorrhage. In the present study, we hypothesized that partial intra‐aortic occlusion of the abdominal aorta would increase salvage of ischemic penumbra and reduce infarct volume after focal embolic stroke in rats. We examined the effects of aortic occlusion on infarct volume, expression and activation of matrix metalloprotease‐9, and hemorrhagic transformation with or without treatment with tPA. We then examined the effects of aortic occlusion on perfusion deficits following embolic occlusion. Results showed that partial aortic occlusion significantly reduces brain infarction volume with or without treatment with tPA after focal ischemia, but does not increase risk for hemorrhagic transformation or matrix metalloprotease‐9 expression and activation. Partial intra‐aortic occlusion also reduces perfusion deficits after focal cerebral ischemia as compared to control. The present study shows that partial intra‐aortic occlusion significantly decreases infarction volume and perfusion deficits following ischemic injury in an embolic model of cerebral ischemia. Moreover, combination treatment with tPA and partial intra‐aortic occlusion further reduces infarction volume without any increase in hemorrhagic transformation.     

Treatment of embolic stroke in rats with bortezomib and recombinant human tissue plasminogen activator

Stroke elicits a progressive vascular dysfunction, which contributes to the evolution of brain injury. Thrombolysis with tissue plasminogen activator (tPA) promotes adverse vascular events that limit the therapeutic window of stroke to three hours. Proteasome inhibitors reduce vascular thrombotic and inflammatory events, and consequently protect vascular function. The present study evaluated the neuroprotective effect of bortezomib, a potent and selective inhibitor of the proteasome, alone and in combination with delayed thrombolytic therapy on a rat model of embolic focal cerebral ischemia. Treatment with bortezomib reduces adverse cerebrovascular events including secondary thrombosis, inflammatory responses, and blood brain barrier (BBB) disruption, and hence reduces infarct volume and neurological functional deficit when administrated within 4 h after stroke onset. Combination of bortezomib and tPA extends the thrombolytic window for stroke to 6 h, which is associated with the improvement of vascular patency and integrity. Real time RT-PCR of endothelial cells isolated by laser-capture microdissection from brain tissue and Western blot analysis showed that bortezomib upregulates endothelial nitric oxide synthase (eNOS) expression and blocks NF-kappaB activation. These results demonstrate that bortezomib promotes eNOS dependent vascular protection, and reduces NF-kappaB dependent vascular disruption, all of which may contribute to neuroprotection after stroke.     

Timing of recanalization after tissue plasminogen activator therapy determined by transcranial doppler correlates with clinical recovery from ischemic stroke

BACKGROUND: The duration of cerebral blood flow impairment correlates with irreversibility of brain damage in animal models of cerebral ischemia. Our aim was to correlate clinical recovery from stroke with the timing of arterial recanalization after therapy with intravenous tissue plasminogen activator (tPA). METHODS: Patients with symptoms of cerebral ischemia were treated with 0.9 mg/kg tPA IV within 3 hours after stroke onset (standard protocol) or with 0.6 mg/kg at 3 to 6 hours (an experimental institutional review board-approved protocol). National Institutes of Health Stroke Scale (NIHSS) scores were obtained before treatment, at the end of tPA infusion, and at 24 hours; Rankin Scores were obtained at long-term follow-up. Transcranial Doppler (TCD) was used to locate arterial occlusion before tPA and to monitor recanalization (Marc head frame, Spencer Technologies; Multigon 500M, DWL MultiDop-T). Recanalization on TCD was determined according to previously developed criteria. RESULTS: Forty patients were studied (age 70+/-16 years, baseline NIHSS score 18.6+/-6.2). A tPA bolus was administered at 132+/-54 minutes from symptom onset. Recanalization on TCD was found at the mean time of 251+/-171 minutes after stroke onset: complete recanalization occurred in 12 (30%) patients and partial recanalization occurred in 16 (40%) patients (maximum observation time 360 minutes). Recanalization occurred within 60 minutes of tPA bolus in 75% of patients who recanalized. The timing of recanalization inversely correlated with early improvement in the NIHSS scores within the next hour (polynomial curve, third order r(2)=0.429, P<0.01) as well as at 24 hours. Complete recanalization was common in patients who had follow-up Rankin Scores if 0 to 1 (P=0.006). No patients had early complete recovery if an occlusion persisted for >300 minutes. CONCLUSIONS: The timing of arterial recanalization after tPA therapy as determined with TCD correlates with clinical recovery from stroke and demonstrates a 300-minute window to achieve early complete recovery. These data parallel findings in animal models of cerebral ischemia and confirm the relevance of these models in the prediction of response to reperfusion therapy.     

Postischemic intracarotid treatment with TNK-tPA reduces infarct volume and improves neurological deficits in embolic stroke in the unanesthetized rat

BACKGROUND AND PURPOSE: To simulate human stroke, we developed a model of focal cerebral embolic ischemia in the unanesthetized rat. Using this model, we tested the hypothesis that intra-arterial administration of TNK-tPA, a fibrin specific second generation thrombolytic agent, is effective in reducing ischemic volume without increasing intra-cerebral hemorrhage. METHODS: Under anesthesia, a catheter was inserted to the origin of the MCA of male Wistar rats. Forty-five minutes after recovery from anesthesia, the MCA was occluded in the awake rat by a single fibrin rich clot placed via the catheter. TNK-tPA (1.5 mg/kg) was administered intraarterially via the catheter at either 2 h or 4 h after stroke. All rats were sacrificed at 48 h after ischemia. Neurological deficits, gross hemorrhage and ischemic lesion volume were measured. RESULTS: A clot was detected at the origin of the MCA 4 h after MCA occlusion in the awake rats (n=4). Rats (n=12) subjected to MCA occlusion showed immediate neurological deficits which persisted for 48 h of ischemia. Ischemic rats had a lesion volume of 38.2+/-3.8% and 25% of rats exhibited gross hemorrhage. Ischemic rats (n=10) treated with TNK-tPA at 2 h showed a significant (P<0.05) reduction of neurological deficits, body weight loss and infarct volume (22.8+/-2.1%) without an increase in gross hemorrhage (10%) compared with the non treated ischemic rats (25%). Although treatment with TNK-tPA of ischemic rats (n=12) at 4 h did not significantly (P=0.06) reduce infarct volume (28.6+/-3.0%), it also did not increase gross hemorrhage (25%) compared with the control group (25%). CONCLUSIONS: This study demonstrates that intraarterial administration of TNK-tPA at 2 h of ischemia in the unanesthesthetized rat is effective in reducing neurological deficits and ischemic lesion volume without increasing hemorrhagic transformation and that administration of TNK-tPA at 4 h of ischemia does not increase the incidence of hemorrhagic transformation.     

Interplay between the gamma isoform of PKC and calcineurin in regulation of vulnerability to focal cerebral ischemia.

Protein phosphorylation and dephosphorylation mediated by protein kinases and protein phosphatases, respectively, represent essential steps in a variety of vital neuronal processes that could affect susceptibility to ischemic stroke. In this study, the role of the neuron-specific gamma isoform of protein kinase C (gammaPKC) in reversible focal ischemia was examined using mutant mice in which the gene for gammaPKC was knocked-out (gammaPKC-KO). A period of 150 minutes of unilateral middle cerebral artery and common carotid artery (MCA/CCA) occlusion followed by 21.5 hours of reperfusion resulted in significantly larger (P < 0.005) infarct volumes (n = 10; 31.1+/-4.2 mm3) in gammaPKC-KO than in wild-type (WT) animals (n = 12; 22.6+/-7.4 mm3). To control for possible differences related to genetic background, the authors analyzed Balb/cJ, C57BL/6J, and 129SVJ WT in the MCA/CCA model of focal ischemia. No significant differences in stroke volume were detected between these WT strains. Impaired substrate phosphorylation as a consequence of gammaPKC-KO might be corrected by inhibition of protein dephosphorylation. To test this possibility, gammaPKC-KO mice were treated with the protein phosphatase 2B (calcineurin) inhibitor, FK-506, before ischemia. FK-506 reduced (P < 0.008) the infarct volume in gammaPKC-KO mice (n = 7; 24.6+/-4.6 mm3), but at this dose in this model, had no effect on the infarct volume in WT mice (n = 7; 20.5+/-10.7 mm3). These results indicate that gammaPKC plays some neuroprotective role in reversible focal ischemia.     

CD18-mediated neutrophil recruitment contributes to the pathogenesis of reperfused but not nonreperfused stroke

BACKGROUND AND PURPOSE: Neutrophil (PMN) recruitment mediated by increased expression of intercellular adhesion molecule-1 expression (ICAM-1, CD54) in the cerebral microvasculature contributes to the pathogenesis of tissue injury in stroke. However, studies using blocking antibodies against the common beta2-integrin subunit on the PMN, the counterligand for ICAM-1 (CD18), have demonstrated equivocal efficacy. The current study tested the hypothesis that mice deficient in CD18 would be protected in the setting of reperfused but not nonreperfused stroke. METHODS: Two groups of mice were studied, those whose PMNs could express CD18 (CD18 +/+) and those mice hypomorphic for the CD-18 gene (CD18 -/-). PMNs obtained from CD18 -/- or CD18 +/+ mice were fluorescently labeled and tested for binding to murine brain endothelial monolayers. Using a murine model of focal cerebral ischemia in which an occluding suture placed in the middle cerebral artery (MCA) is removed after 45 minutes (transient ischemia, reperfused stroke) or left in place (permanent ischemia, nonreperfused stroke), cerebral infarct volumes (% ipsilateral hemisphere by TTC staining), cerebral blood flow (CBF, % contralateral hemisphere by laser-Doppler flowmetry), and survival (%) were examined 24 hours after the initial ischemic event. Adoptive transfer studies used 111In-labeled PMNs (from either CD18 +/+ or CD18 -/- mice) to examine the relative accumulation of PMNs in the ischemic region. RESULTS: PMNs obtained from CD18 -/- mice exhibit reduced adhesivity (compared with CD18 +/+ PMNs) for both quiescent and cytokine-activated endothelial monolayers. CD18 -/- mice (n=14) subjected to transient focal cerebral ischemia demonstrated a 53% decrease in infarct volumes versus CD18 +/+ mice (n=26, P<0.05), improved penumbral CBF at 24 hours (1.8-fold, P=0.02), and a 3.7-fold decrease in mortality (P=0.02). However, when CD18 -/- mice (n=12) were subjected to permanent focal cerebral ischemia, no differences were noted in infarct volume, mortality, or CBF versus similarly treated CD18 +/+ mice (n=10). There was a greater accumulation of CD18 +/+ PMNs in the ischemic zone of CD18 +/+ animals than CD18 -/- animals subjected to reperfused stroke (82% increase, P=0.02), although there was no difference between groups when subjected to permanent MCA occlusion. CONCLUSIONS: Deficiency for the CD18 gene confers cerebral protection in a murine model of reperfused stroke, but this benefit does not extend to CD18-deficient animals subjected to permanent MCA occlusion. These data suggest that anti-PMN strategies should be targeted to reperfused stroke and may perhaps be used in conjunction with thrombolytic therapy that establishes reperfusion.     

Temporal thresholds for neocortical infarction in rats subjected to reversible focal cerebral ischemia.

We investigated the temporal threshold for focal cerebral infarction in the spontaneously hypertensive rat. The right middle cerebral artery and common carotid artery were occluded for 0, 1, 2, 3, 4, or 24 hours, and all the animals were sacrificed 24 hours after the onset of ischemia. Cortical infarct volumes and edema volumes were quantified in serial frozen sections of hematoxylin and eosin-stained tissue using image analysis. Upon occlusion, blood flow in the core of the ischemic zone, measured with laser-Doppler flowmetry, fell to a mean +/- standard deviation of 21 +/- 7% of the preocclusion baseline value (n = 26). During the first hour of ischemia, blood flow in the densely ischemic zone rose to 27 +/- 8% of baseline (n = 25). Release of the middle cerebral artery and common carotid artery occlusions rapidly restored cortical blood flow to 213 +/- 83% of baseline (n = 21). Focal ischemia of 1 hour's duration caused little or no infarction, while ischemic intervals of 2 and 3 hours produced successively larger volumes of infarcted cortex. Ischemic intervals of 3-4 hours' duration followed by approximately 20 hours of recirculation yielded infarct volumes that were not significantly different from those after 24 hours of permanent focal ischemia. The results indicate that 3-4 hours of focal cerebral ischemia in this rat model is sufficient to attain maximal infarction and suggest that recirculation or pharmacological interventions after this time will provide little benefit.     

Does poly-L-lysine coating of the middle cerebral artery occlusion suture improve infarct consistency in a murine model?

Background and Purpose: Rodent models of stroke that employ an intraluminal suture to cause focal cerebral ischemia are associated with some variability of resultant infarct volumes, thus requiring increased numbers of animals to determine significant differences between experimental groups. A recent modification of the occluding suture by coating with poly-L-lysine has been shown to create more uniform infarct volumes in rats. Methods: To evaluate the utility of this modification in murine models of both transient and permanent focal cerebral ischemia, male C57B16J mice were subjected to reversible middle cerebral artery occlusion (MCAo) for 45 minutes (n=42), or to permanent MCAo (n=25), with an intraluminal monofilament suture. Three types of sutures were used: untreated, partially coated, and completely coated with poly-L-lysine. Relative changes in regional cerebral blood flow, severity of neurological deficits, and infarct volumes were measured 24 hours after the ischemic injury. Results: Animals subjected to 45 minutes of temporary occlusion with completely coated poly-L-lysine sutures had infarct volumes of 13.8%+/-5% compared with infarct volumes of 7.2%+/-4% in those subjected to partially coated sutures and 22.4%+/-6% in the group occluded with untreated sutures (P=ns). Use of completely coated sutures resulted in significantly less reperfusion following suture removal. Control animals undergoing permanent occlusion with untreated sutures had infarct volumes of 17%+/-7% compared with 14.1%+/-5% using completely coated sutures and 6.5%+/-3% in animals with partially coated sutures (P=ns). There were no significant differences in cerebral blood flow between the experimental groups undergoing permanent MCAo. Conclusions: Poly-L-lysine coating of intraluminal sutures does not increase the consistency of infarct volumes in a murine model of temporary/permanent MCAo. These findings are in marked contrast to findings in rats.     

New insights on thrombolytic treatment of acute ischemic stroke

In 1995, a two-part randomized trial showed the efficacy of intravenous tissue plasminogen activator (tPA) when given within 3 hours of onset of symptoms of acute ischemic stroke. Two subsequent trials were unable to extend the therapeutic window of intravenous tPA beyond 3 hours. A phase IV study performed by experienced stroke centers showed an acceptably low symptomatic intracerebral hemorrhage rate for intravenous tPA of only 3%, whereas a review of the Cleveland area experience showed a disturbingly high rate of symptomatic intracerebral hemorrhage of 15.7%. The Prolyse in Acute Cerebral Thromboembolism (PROACT) II study showed efficacy of intra-arterial pro-urokinase and intravenous heparin over intravenous heparin alone when given within 6 hours of onset of symptoms to patients with thrombotic occlusion of the proximal middle cerebral artery. Additional controlled investigations of intra-arterial thrombolytic therapy are needed. Neuroprotectants in combination with intravenous tPA have yet to show improved efficacy over the use of tPA alone.     

Effect of ganaxolone in a rodent model of cerebral hematoma

BACKGROUND AND PURPOSE: Therapy with gamma-aminobutyric acid (GABA) agonists appears to improve outcome after experimental hematoma but with unacceptable side effects. We looked to synthetic GABA agonists, or positive GABA modulators, widely developed as anticonvulsants and anxiolytics, to find compounds that may be effective. Ganaxolone is a synthetic neuroactive steroid that positively modulates GABA. We sought to determine whether ganaxolone was beneficial using a model of intracerebral hematoma. METHODS: We stereotaxically injected varying doses of bacterial collagenase into the caudate nucleus of rats to induce blood-brain barrier failure and hematoma formation. Four hours later, we administered intravenously 15 or 30 mg/kg ganaxolone (n=23 each group), 20 mg/kg pregnanolone (n=21), or vehicle (n=30). Forty-eight hours after collagenase injection, we rated each animal using a standard rodent neurological examination. The ratings were compared with the amounts of injected collagenase using the quantal bioassay procedure. Other sets of animals were tested later for visuospatial learning. Brains were then prepared for histomorphometry, and brain volumes were estimated. RESULTS: We found that ganaxolone 30 mg/kg significantly increased the ED(50) in the bioassay, for a potency ratio of 1.8+/-0.41 compared with vehicle (P<0.05). Ganaxolone 15 mg/kg and pregnanolone did not affect neurological outcome. Ganaxolone 30 mg/kg did not clearly improve visuospatial learning several weeks after hemorrhage. Ganaxolone exhibited a weak effect on cerebral volumes 48 hours after stroke, but 3 months after hemorrhage no such effect could be detected. CONCLUSIONS: Ganaxolone improves neurological outcome 48 hours after intracerebral hematoma but not visuospatial learning several weeks after intracerebral hematoma. Histological evidence of damage was reduced at 48 hours but not at 3 months.     

Intravenous tPA in acute ischemic stroke related to internal carotid artery dissection

The authors describe the outcomes in 11 patients who had acute ischemic stroke related to internal carotid artery (ICA) dissection and were treated with IV tissue plasminogen activator (tPA). One symptomatic intracerebral hemorrhage occurred 36 hours after tPA was given. The mean day 90 modified Rankin Scale (m-RS) score was 2.4 (+/-1.6). No death was observed at 3 months. Four patients of 11 (36.4%) made an excellent recovery (day 90 m-RS score: 0 to 1). This study demonstrates the feasibility of IV thrombolysis with tPA (0.8 mg/kg) in ischemic stroke related to ICA dissection within the first 7 hours.     

Inhibition of VEGF signaling pathway attenuates hemorrhage after tPA treatment

An angiogenic factor, vascular endothelial growth factor (VEGF), might be associated with the blood–brain barrier (BBB) disruption after focal cerebral ischemia; however, it remains unknown whether hemorrhagic transformation (HT) after tissue plasminogen activator (tPA) treatment is related to the activation of VEGF signaling pathway in BBB. Here, we hypothesized that inhibition of VEGF signaling pathway can attenuate HT after tPA treatment. Rats subjected to thromboembolic focal cerebral ischemia were assigned to a permanent ischemia group and groups treated with tPA at 1 or 4 hours after ischemia. Anti-VEGF neutralizing antibody or control antibody was administered simultaneously with tPA. At 24 hours after ischemia, we evaluated the effects of the antibody on the VEGF expression, matrix metalloproteinase-9 (MMP-9) activation, degradation of BBB components, and HT. Delayed tPA treatment at 4 hours after ischemia promoted expression of VEGF in BBB, MMP-9 activation, degradation of BBB components, and HT. Compared with tPA and control antibody, combination treatment with tPA and the anti-VEGF neutralizing antibody significantly attenuated VEGF expression in BBB, MMP-9 activation, degradation of BBB components, and HT. It also improved motor outcome and mortality. Inhibition of VEGF signaling pathway may be a promising therapeutic strategy for attenuating HT after tPA treatment.