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Background: Ischemic stroke affects about 700 000 patients per year in the United States, and to date, there are no effective pharmacological agents that promote recovery. Here, we studied the pharmacokinetics, pharmacodynamics, and efficacy of NTS-105, a novel neuroactive steroid, and NTS-104, a prodrug of NTS-105, in 2 models of ischemic stroke., Methods: The pharmacodynamics and pharmacokinetics of NTS-104/105 were investigated in naive and stroke rats, and models of embolic and transient middle cerebral artery occlusion were used to investigate the dose-related effects of NTS-104. All rats were randomly assigned into the experimental groups, and all outcome measurements were performed blindly., Results: Blood plasma and brain pharmacokinetic analysis revealed that NTS-104 rapidly converted to NTS-105, which reached peak concentration at ≈1 hour after dosing and distributed similarly to normal and ischemic brains. NTS-104 administration 4 hours after embolic middle cerebral artery occlusion led to a dose-dependent improvement of neurological outcomes and a dose-dependent reduction of infarct volumes relative to vehicle-treated animals. A single dose level study confirmed that NTS-104 administered 4 hours after transient middle cerebral artery occlusion was also neuroprotective. Quantitative ELISA revealed that NTS-104 treatment resulted in time- and dose-dependent changes in AKT activation and cytokine levels within the ischemic brain, which included reductions of IL-6, VEGF, ICAM-1, IL-1β, MCP-1, RAGE, and GM-CSF. Time- and dose-dependent reductions in IL-6 and GM-CSF were also observed in the plasma along with an elevation of galectin-1., Conclusions: NTS-104 is a novel prodrug that converts to a novel neuroactive steroid, NTS-105, which improves functional outcomes in experimental ischemic stroke models.

Background and Purpose: Cerebral endothelial cells (CECs) and axons of neurons interact to maintain vascular and neuronal homeostasis and axonal remodeling in normal and ischemic brain, respectively. However, the role of exosomes in the interaction of CECs and axons in brain under normal conditions and after stroke is unknown., Methods: Exosomes were isolated from CECs of nonischemic rats and is chemic rats (nCEC-exos and isCEC-exos), respectively. A multicompartmental cell culture system was used to separate axons from neuronal cell bodies., Results: Axonal application of nCEC-exos promotes axonal growth of cortical neurons, whereas isCEC-exos further enhance axonal growth than nCEC-exos. Ultrastructural analysis revealed that CEC-exos applied into distal axons were internalized by axons and reached to their parent somata. Bioinformatic analysis revealed that both nCEC-exos and isCEC-exos contain abundant mature miRNAs; however, isCEC-exos exhibit more robust elevation of select miRNAs than nCEC-exos. Mechanistically, axonal application of nCEC-exos and isCEC-exos significantly elevated miRNAs and reduced proteins in distal axons and their parent somata that are involved in inhibiting axonal outgrowth. Blockage of axonal transport suppressed isCEC-exo-altered miRNAs and proteins in somata but not in distal axons., Conclusions: nCEC-exos and isCEC-exos facilitate axonal growth by altering miRNAs and their target protein profiles in recipient neurons.

Background and aims In an effort to characterize the effects of Cerebrolysin for treatment of stroke that are essential for successful clinical translation, we have demonstrated that Cerebrolysin dose dependently enhanced neurological functional recovery in experimental stroke. Here, we conduct a prospective, randomized, placebo-controlled, blinded study to examine the therapeutic window of Cerebrolysin treatment of rats subjected to embolic stroke. Methods Male Wistar rats age 3-4 months (n = 100) were subjected to embolic middle cerebral artery occlusion. Animals were randomized to receive saline or Cerebrolysin daily for 10 consecutive days starting 4, 24, 48, and 72 h after middle cerebral artery occlusion. Neurological outcome was measured weekly with a battery of behavioral tests (adhesive removal test, modified neurological severity score (mNSS), and foot-fault test). Global test was employed to assess Cerebrolysin effect on neurological recovery with estimation of mean difference between Cerebrolysin and control-treated groups and its 95% confidence interval in the intent-to-treat population, where a negative value of the mean difference and 95% confidence interval < 0 indicated a significant treatment effect. All rats were sacrificed 28 days after middle cerebral artery occlusion and infarct volume was measured. Results Cerebrolysin treatment initiated within 48 h after middle cerebral artery occlusion onset significantly improved functional outcome; mean differences and 95% confidence interval were -11.6 (-17.7, -5.4) at 4 h, -7.1 (-13.5, -0.8) at 24 h, -8.4 (-14.2, -8.6) at 48 h, and -4.9 (-11.4, 1.5) at 72 h. There were no differences on infarct volume and mortality rate among groups. Conclusions With a clinically relevant rigorous experimental design, our data demonstrate that Cerebrolysin treatment effectively improves stroke recovery when administered up to 48 h after middle cerebral artery occlusion.

Background and Purpose: Diabetes mellitus (DM) is a common metabolic disease among the middle-aged and older population, which leads to an increase of stroke incidence and poor stroke recovery. The present study was designed to investigate the impact of DM on brain damage and on ischemic brain repair after stroke in aging animals., Methods: DM was induced in middle-aged rats (13 months) by administration of nicotinamide and streptozotocin. Rats with confirmed hyperglycemia status 30 days after nicotinamide-streptozotocin injection and age-matched non-DM rats were subjected to embolic middle cerebral artery occlusion., Results: Middle-aged rats subjected to nicotinamide-streptozotocin injection became hyperglycemic and developed cognitive deficits 2 months after induction of DM. Histopathologic analysis revealed that there was sporadic vascular disruption, including cerebral microvascular thrombosis, blood-brain barrier leakage, and loss of paravascular aquaporin-4 in the hippocampi. Importantly, middle-aged DM rats subjected to stroke had exacerbated sensorimotor and cognitive deficits compared with age-matched non-DM ischemic rats during stroke recovery. Compared with age-matched non-DM ischemic rats, DM ischemic rats exhibited aggravated neurovascular disruption in the bilateral hippocampi and white matter, suppressed stroke-induced neurogenesis and oligodendrogenesis, and impaired dendritic/spine plasticity. However, DM did not enlarge infarct volume., Conclusions: Our data suggest that DM exacerbates neurovascular damage and hinders brain repair processes, which likely contribute to the impairment of stroke recovery., (© 2016 American Heart Association, Inc.)

Inflammatory markers have been associated with functional outcome and mortality of stroke. We investigated the changes in procalcitonin (PCT) and high-sensitivity C-reactive protein (Hs-CRP) levels during the acute period of ischemic stroke and evaluated the relationship between these levels and the long-term functional outcome and mortality. We prospectively studied 376 patients with acute ischemic stroke (AIS) who were admitted within 24 h after the onset of symptoms. PCT, Hs-CRP, and NIH Stroke Scale (NIHSS) were measured at the time of admission. Long-term functional outcome were measured by modified Rankin scale (mRS) at 1 year after admission. The correlations between the levels of PCT, Hs-CRP, and mortality at 1 year after stroke onset were analyzed. Patients with poor with functional outcome and non-survivors had significantly increased PCT and Hs-CRP levels on admission. Multivariate logistic regression analysis showed that PCT was an independent prognostic marker of 1-year functional outcome and death [odds ratio (OR) 2.33 (95% CI, 1.33-3.44) and 3.11 (2.02-4.43), respectively, P < 0.0001 for both, adjusted for age, NIHSS, other predictors, and vascular risk factors] in patients with AIS. The area under the receiver operating characteristic curve of PCT was 0.77 (95% CI, 0.72-0.83) for functional outcome and 0.88 (95% CI, 0.84-0.93) for mortality. PCT improved the area under the receiver operating characteristic curve of the NIHSS score for functional outcome from 0.74 (95% CI, 0.66-0.81) to 0.85 (95% CI, 0.76-0.92; P < 0.0001) and for mortality from 0.77 (95% CI, 0.70-0.83) to 0.94 (95% CI, 0.89-0.97; P < 0.0001). Serum level of PCT at admission was an independent predictor of long-term functional outcome and mortality after ischemic stroke in Chinese sample.

Animal models of focal cerebral ischemia are well accepted for investigating the pathogenesis and potential treatment strategies for human stroke. Occlusion of the middle cerebral artery (MCA) with an endovascular filament is a widely used model to induce focal cerebral ischemia. However, this model is not amenable to thrombolytic therapies. As thrombolysis with recombinant tissue plasminogen activator (rtPA) is a standard of care within 4.5 h of human stroke onset, suitable animal models that mimic cellular and molecular mechanisms of thrombosis and thrombolysis of stroke are required. By occluding the MCA with a fibrin-rich allogeneic clot, we previously developed an embolic model of MCA occlusion in the rat, which recapitulates the key components of thrombotic development and of thrombolytic therapy of rtPA observed from human ischemic stroke. Here we describe in detail the surgical procedures of our model, including preparing emboli from rat donors. These procedures can be typically completed within ∼30 min, and they are highly adaptable to other strains of rats, as well as mice, in both sexes. Thus, this model provides a powerful tool for translational stroke research.

Complement activation and inflammation have been suggested in the pathogenesis of stroke; mannose-binding lectin (MBL) was found to have roles during the process. We therefore evaluated the short-term prognostic value of serum MBL in Chinese patients with an acute ischemic stroke (AIS). Consecutive AIS patients admitted to the emergency department were identified. Clinical information was collected. Serum concentration of MBL and NIH Stroke Scale (NIHSS) was measured at the time of admission. Short-term functional outcome was measured by modified Rankin scale (mRS) 90 days after admission. Multivariate analyses were performed using logistic regression models. During the inclusion period, 231 patients were diagnosed with AIS, and 220 completed follow-up. The results indicated that the serum MBL levels were significantly (P = 0.000) higher in acutely ischemic stroke patients as compared with normal controls. MBL was an independent prognostic marker of short-term functional outcome and death (odds ratio (OR) 5.28 (2.88-10.67) and 6.99 (3.55-13.97), respectively, P = 0.000 for both, adjusted for NIHSS, other predictors, and vascular risk factors) in patients with AIS. MBL improved the area under the receiver operating characteristic curve of the NIHSS score for functional outcome from 0.826 (95 % CI 0.773-0.879) to 0.857 (95 % CI 0.808-0.905, P = 0.000) and for mortality from 0.768 (95 % CI 0.682-0.853) to 0.822 (95 % CI 0.747-0.896, P = 0.000). Serum MBL levels are a useful, complementary tool to predict functional outcome and mortality 90 days after stroke.

The unique cellular and vascular architecture of the adult ventricular-subventricular zone (V/SVZ) neurogenic niche plays an important role in regulating neural stem cell function. However, the in vivo identification of neural stem cells and their relationship to blood vessels within this niche in response to stroke remain largely unknown. Using whole-mount preparation of the lateral ventricle wall, we examined the architecture of neural stem cells and blood vessels in the V/SVZ of adult mouse over the course of 3 months after onset of focal cerebral ischemia. Stroke substantially increased the number of glial fibrillary acidic protein (GFAP) positive neural stem cells that are in contact with the cerebrospinal fluid (CSF) via their apical processes at the center of pinwheel structures formed by ependymal cells residing in the lateral ventricle. Long basal processes of these cells extended to blood vessels beneath the ependymal layer. Moreover, stroke increased V/SVZ endothelial cell proliferation from 2% in non-ischemic mice to 12 and 15% at 7 and 14 days after stroke, respectively. Vascular volume in the V/SVZ was augmented from 3% of the total volume prior to stroke to 6% at 90 days after stroke. Stroke-increased angiogenesis was closely associated with neuroblasts that expanded to nearly encompass the entire lateral ventricular wall in the V/SVZ. These data indicate that stroke induces long-term alterations of the neural stem cell and vascular architecture of the adult V/SVZ neurogenic niche. These post-stroke structural changes may provide insight into neural stem cell mediation of stroke-induced neurogenesis through the interaction of neural stem cells with proteins in the CSF and their sub-ependymal neurovascular interaction.

Cerebral ischemia induces neurogenesis, including proliferation and differentiation of neural progenitor cells and migration of newly generated neuroblasts. MicroRNAs (miRNAs) are small noncoding RNAs that decrease gene expression through mRNA destabilization and/or translational repression. Emerging data indicate that miRNAs have a role in mediating processes of proliferation and differentiation of adult neural progenitor cells. This article reviews recent findings on miRNA profile changes in neural progenitor cells after cerebral infarction and the contributions of miRNAs to their ischemia-induced proliferation and differentiation. We highlight interactions between the miR-124 and the miR17-92 cluster and the Notch and Sonic hedgehog signaling pathways in mediating stroke-induced neurogenesis.

Introduction: Stroke remains the leading cause of adult disability. Thus, it is imperative to develop restorative therapies for ischemic stroke designed specifically to treat the intact brain tissue to stimulate functional benefit. Therapies targeting amplification of brain repair processes with nitric oxide (NO) donors and phosphodiesterase type 5 (PDE5) inhibitors in preclinical studies are emerging and showing improvement of functional recovery after stroke., Areas Covered: This review will mainly cover the effect of NO donors, which produce NO, and PDE5 inhibitors, which elevate cyclic guanosine 3',5'-monophosphate (cGMP), on neural restorative events in ischemic brain and highlight mechanisms underlying their restorative therapeutic activity., Expert Opinion: During stroke recovery, interwoven restorative events occur in ischemic brain, which include angiogenesis, neurogenesis, oligodendrogenesis, astrogliosis and neurite outgrowth. Emerging preclinical data indicate that restorative therapies targeting multiple parenchymal cells including neural stem cells, cerebral endothelial cells, astrocytes, oligodendrocytes, neurons would be more effective than agents with a single cell target. Preclinical data suggest that elevated cGMP levels induced by NO donors and PDE5 inhibitors act on cerebral endothelial cells, neural stem cells and oligodendrocyte progenitor cells to enhance stroke-induced angiogenesis, neurogenesis and oligodendrogenesis, respectively. These interacting remodeling events collectively improve neurological function after stroke.

Neural and oligodendrocyte progenitor cells in the adult brain express Ascl1 (also known as Mash1), a basic helix-loop-helix transcription factor. We examined the progeny and fate of this progenitor population in adult male Ascl1-CreER(TM);R26R-stop-yellow fluorescent protein mice subjected to right middle cerebral occlusion over 60 days after stroke using inducible Cre recombination to label Ascl1-expressing cells at poststroke days 2 to 6 in vivo. Seven days after stroke, a substantial increase in Ascl1 lineage cells was detected in the ipsilateral subventricular zone (SVZ), striatum, and corpus callosum. These cells exhibited proliferating progenitor cell phenotypes (Sox2(+), BrdU(+), and Ki67(+)). Although Ascl1 lineage cells in the ipsilateral SVZ gradually decreased during 14 to 60 days after stroke, Ascl1 lineage cells in the ischemic striatum revealed a remarkable increase during this period. Thirty and sixty days after stroke, Ascl1 lineage cells in the ischemic striatum gave rise to GABAergic neurons and mature oligodendrocytes. In contrast, none of the Ascl1 lineage cells in the contralateral striatum exhibited neuronal and oligodendrocyte phenotypes. Moreover, Ascl1 lineage cells in the corpus callosum were only fated to become mature oligodendrocytes. Our data suggest that Ascl1 lineage cells contribute to stroke-induced neurogenesis and oligodendrogenesis in the adult ischemic brain.

Cerebrolysin is a peptide preparation mimicking the action of neurotrophic factors and has beneficial effects on neurodegenerative diseases and stroke. The present study investigated the effect of Cerebrolysin on neurogenesis in a rat model of embolic middle cerebral artery occlusion (MCAo). Treatment with Cerebrolysin at doses of 2.5 and 5 ml/kg significantly increased the number of bromodeoxyuridine-positive (BrdU(+)) subventricular zone (SVZ) neural progenitor cells and doublecortin (DCX) immunoreactivity (migrating neuroblasts) in the ipsilateral SVZ and striatal ischemic boundary 28 days after stroke when the treatment was initiated 24 hr after stroke. The treatment also reduced TUNEL(+) cells by ∼50% in the ischemic boundary. However, treatment with Cerebrolysin at a dose of 2.5 ml/kg initiated at 24 and 48 hr did not significantly reduce infarct volume but substantially improved neurological outcomes measured by an array of behavioral tests 21 and 28 days after stroke. Incubation of SVZ neural progenitor cells from ischemic rats with Cerebrolysin dose dependently augmented BrdU(+) cells and increased the number of Tuj1(+) cells (a marker of immature neurons). Blockage of the PI3K/Akt pathway abolished Cerebrolysin-increased BrdU(+) cells. Moreover, Cerebrolysin treatment promoted neural progenitor cell migration. Collectively, these data indicate that Cerebrolysin treatment when initiated 24 and 48 hr after stroke enhances neurogenesis in the ischemic brain and improves functional outcome and that Cerebrolysin-augmented proliferation, differentiation, and migration of adult SVZ neural progenitor cells contribute to Cerebrolysin-induced neurogenesis, which may be related to improvement of neurological outcome. The PI3K/Akt pathway mediates Cerebrolysin-induced progenitor cell proliferation.

Background and Purpose: Treatment with a selective proteasome inhibitor, VELCADE, in combination with tissue plasminogen activator (tPA) extended the therapeutic window to 6 hours in young rats after stroke. However, stroke is a major cause of death and disability in the elderly. The present study investigated the effect of VELCADE in combination with a low-dose tPA on aged rats after embolic stroke., Methods: Male Wistar rats at the age of 18 to 20 months were treated with VELCADE (0.2 mg/kg) alone, a low-dose tPA (5 mg/kg) alone, combination of VELCADE and tPA, or saline 2 hours after embolic middle cerebral artery occlusion. To test the contribution of endothelial nitric oxide synthase to VELCADE-mediated neuroprotection, endothelial nitric oxide synthase knockout and wild-type mice were treated with VELCADE (0.5 mg/kg) 2 hours after embolic stroke., Results: Treatment with VELCADE significantly reduced infarct volume, whereas tPA alone did not reduce infarct volume and aggravated blood-brain barrier disruption in aged rats compared with saline-treated rats. However, the combination treatment significantly enhanced the reduction of infarct volume, which was associated with an increase in endothelial nitric oxide synthase activity compared with saline-treated rats. Additionally, the combination treatment promoted thrombolysis and did not increase the incidence of hemorrhage transformation. VELCADE significantly reduced lesion volume in wild-type mice but failed to significantly reduce lesion volume in endothelial nitric oxide synthase knockout mice., Conclusions: Treatment with VELCADE exerts a neuroprotective effect in aged rats after stroke. The combination of VELCADE with the low-dose tPA further amplifies the neuroprotective effect. Endothelial nitric oxide synthase at least partly contributes to VELCADE-mediated neuroprotection after stroke.

We tested the hypotheses that administration routes affect the migration and distribution of grafted neural progenitor cells (NPCs) in the ischemic brain and that the ischemic lesion plays a role in mediating the grafting process. Male Wistar rats (n=41) were subjected to 2-h middle cerebral artery occlusion (MCAo), followed 1 day later by administration of magnetically labeled NPCs. Rats with MCAo were assigned to one of three treatment groups targeted for cell transplantation intra-arterially (IA), intracisternally (IC), or intravenously (IV). MRI measurements consisting of T2-weighted imaging and three-dimensional (3D) gradient echo imaging were performed 24 h after MCAo, 4 h after cell injection, and once a day for 4 days. Prussian blue staining was used to identify the labeled cells, 3D MRI to detect cell migration and distribution, and T2 map to assess lesion volumes. Intra-arterial (IA) administration showed significantly increased migration, a far more diffuse distribution pattern, and a larger number of transplanted NPCs in the target brain than IC or IV administration. However, high mortality with IA delivery (IA: 41%; IC: 17%; IV: 8%) poses a serious concern for using this route of administration. Animals with smaller lesions at the time of transplantation have fewer grafted cells in the parenchyma.

The migratory behavior of neuroblasts after a stroke is poorly understood. Using time-lapse microscopy, we imaged migration of neuroblasts and cerebral vessels in living brain slices of adult doublecortin (DCX, a marker of neuroblasts) enhanced green fluorescent protein (eGFP) transgenic mice that were subjected to 7 days of stroke. Our results show that neuroblasts originating in the subventricular zone (SVZ) of adult mouse brain laterally migrated in chains or individually to reach the ischemic striatum. The chains were initially formed at the border between the SVZ and the striatum by neuroblasts in the SVZ and then extended to the striatum. The average speed of DCX-eGFP-expressing cells within chains was 28.67+/-1.04 microm/h, which was significantly faster (P<0.01) than the speed of the cells in the SVZ (17.98+/-0.57 microm/h). Within the ischemic striatum, individual neuroblasts actively extended or retracted their processes, suggestive of probing the immediate microenvironment. The neuroblasts close to cerebral blood vessels exhibited multiple processes. Our data suggest that neuroblasts actively interact with the microenvironment to reach the ischemic striatum by multiple migratory routes.

MRI has been used to evaluate labeled cell migration and distribution. However, quantitative determination of labeled cell concentration using MRI has not been systematically investigated. In the current study, we investigated the relationships between labeled cell concentration and MRI parameters of transverse relaxation rate, R(2), and apparent diffusion coefficient (ADC), in vitro in phantoms and in vivo in rats after stroke. Significant correlations were detected between iron concentration or labeled cell concentration and MRI measurements of R(2), ADC, and ADC x R(2) in vitro. In contrast, in vivo labeled cell concentration did not significantly correlate with R(2), ADC, and ADC x R(2). A major factor for the absence of a significant correlation between labeled cell concentration and MRI measurements in vivo may be attributed to background effects of ischemic tissue. By correcting the background effects caused by ischemic damage, DeltaR(2) (difference in R(2) values in the ischemic tissue with and without labeled cells) exhibited a significant correlation to labeled cell concentration. Our study suggests that MRI parameters have the potential to quantitatively determine labeled cell concentration in vivo.

The subventricular zone (SVZ) of the lateral ventricle contains neural stem and progenitor cells that generate neuroblasts, which migrate to the olfactory bulb where they differentiate into interneurons. Ischemic stroke induces neurogenesis in the SVZ and these cells migrate to the boundary of the ischemic lesion. This article reviews current data on cytokinetics, signaling pathways and vascular niche that are involved in processes of proliferation, differentiation, and migration of neural progenitor cells after stroke.

Heparin and low-molecular-weight heparin have long been proposed for stroke treatment. This study was conducted to demonstrate the antagonistic effects of ultra-low-molecular-weight heparin (ULMWH) on cerebral ischemic injury in rats and the mechanisms underlying the effects. Male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 2h followed by reperfusion for 24h. ULMWH (0.5, 1 mg kg(-1), i.v.) was administered after the MCAO and reperfusion. Twenty-four hours after the reperfusion, neurological deficit scores, body weight and infarct volume were assessed. Spectrophotometric assay was used to determine the activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) of the brain. Furthermore, the intracellular Ca(2+) concentration ([Ca(2+)]i) was measured. The results showed that vein injection of ULMWH at doses of 0.5 and 1.0 mg kg(-1) exerted significant neuroprotective effects on rats with focal cerebral ischemic injury via significantly decreasing neurological deficit scores, increasing body weight, reducing the infarct volume. At the same time, ULMWH significantly decreased MDA content, and increased SOD activity in ischemic brain. Compared with model group, ULMWH decreased the intracellular calcium concentration remarkably. All these findings suggest that ultra-low-molecular-weight heparin might act as a neuroprotective agent useful in the treatment in focal cerebral ischemia.

Focal cerebral ischemia induces neurogenesis in the subventricular zone (SVZ) of the adult human brain. Neurogenesis is controlled by proliferation, differentiation, and migration of neural progenitor cells. This article reviews emerging data that changes of cell cycle kinetics of neural progenitor cells induced by stroke contribute to increased neural progenitor cell proliferation and that gene profiles control proliferation, differentiation, and migration of neural progenitor cells within the SVZ niche. A better understanding of gene profiles that control the biological function of adult SVZ neural progenitor cells could lead to more selective and effective treatments to enhance neurogenesis during stroke recovery.

Background and Purpose: Recombinant human erythropoietin (rhEPO; Epoetin-alpha; PROCRITtrade mark) has been shown to exert neuroprotective and restorative effects in a variety of CNS injury models. However, limited information is available regarding the dose levels required for these beneficial effects or the neuronal responses that may underlie them. Here we have investigated the dose-response to rhEPO and compared the effects of rhEPO with those of carbamylated rhEPO (CEPO) in a model of cerebral stroke in rats., Experimental Approach: Rats subjected to embolic middle cerebral artery occlusion (MCAo) were treated with rhEPO or CEPO, starting at 6 h and repeated at 24 and 48 h, after MCAo. Cerebral infarct volumes were assessed at 28 days and neurological impairment at 7, 14, 21 and 28 days, post-MCAo., Key Results: rhEPO at dose levels of 500, 1150 or 5000 IU kg(-1) or CEPO at a dose level of 50 microg kg(-1) significantly reduced cortical infarct volume and reduced neurologic impairment. All doses of rhEPO, but not CEPO, produced a transient increase in haematocrit, while rhEPO and CEPO substantially reduced the number of apoptotic cells and activated microglia in the ischemic boundary region., Conclusions and Implications: These data indicate that rhEPO and CEPO have anti-inflammatory and anti-apoptotic effects, even with administration at 6 h following embolic MCAo in rats. Taken together, these actions of rhEPO and CEPO are likely to contribute to their reduction of neurologic impairment following cerebral ischemia.

Ischemic stroke induces neurogenesis in the subventricular zone (SVZ), and newly generated neurons in the SVZ migrate toward the ischemic boundary. However, the characteristics of migrating SVZ cells have not been investigated after stroke. Using time-lapse imaging in both SVZ cells and organotypic brain slice cultures, we measured the dynamics of SVZ cell division and migration of adult rats subjected to stroke. In normal brain slices, SVZ cells primarily migrated dorsally and ventrally along the lateral ventricular surface. However, in stroke brain slices, SVZ cells migrated laterally toward the striatal ischemic boundary. Cultured stroke-derived SVZ cells exhibited a significant (p < 0.01) increase in the migration distance (212 +/- 21 microm) compared with the nonstroke-derived SVZ cells (97 +/- 12 microm). Migrating stroke-derived SVZ cells spent significantly (p = 0.01) less time in cytokinesis (0.63 +/- 0.04 h) compared with the time (1.09 +/- 0.09 h) for nonstroke-derived SVZ cells. Newborn cells with a single leading process exhibited fast migration (7.2 +/- 0.8 microm/h), and cells with multiple processes showed stationary migration (3.6 +/- 0.8 microm/h). Stroke SVZ daughter cells further divided during their migration. The morphology of doublecortin (DCX)-positive cells in fixed brain sections resembled those observed in cultured newborn cells, and the DCX-positive cells proliferated in the ischemic striatum. Collectively, the present study suggests that stroke promotes cytokinesis of migrating neuroblasts, and these cells migrate toward the ischemic striatum with distinct migratory behaviors and retain the capacity for cell division during migration.

To investigate the changes of the ischemic lesion in rat brain after subventricular zone (SVZ) cell transplantation and the influence of the grafted cells on the appearance of angiogenesis, SVZ cells, superparamagnetically labeled, were intracisternally transplanted into the rat brain 48 h after onset of embolic stroke. A complete set of magnetic resonance (MR) images was acquired for all animals with (n=8) and without (n=3) cell grafting at approximately 24 h, 72 h, and weekly for 6 weeks after stroke. Transplanted cells were tracked by high-resolution three-dimensional gradient-echo images and the interaction between the cells and ischemic lesion was detected by ISODATA (Iterative Self-Organizing Data Analysis Technique Algorithm) calculated from T(1), T(2) and T(1sat) maps. Tissue status from ISODATA was characterized by a specific signature, which represents the deviation from normal tissue in the feature space. Transplanted SVZ cells selectively migrated towards the ischemic side of the rat brain and approached the lesion boundary within 1-week after grafting. Cell treated rats exhibited a significant reduction of average lesion size compared with control rats (P<0.05). A significant reduction of tissue signature (P<0.001) induced by cell transplantation was localized to the position of grafted cells, and these sites exhibited stably restored cerebral blood flow (CBF) (approximately 85% of normal CBF). Angiogenesis was present in sites either immediately adjacent to or surrounded by the grafted cells. Our data indicate that map-ISODATA accurately and dynamically characterizes the ischemic lesion and its response to cell therapy.

In the adult rodent, stroke induces an increase in endogenous neural progenitor cell (NPC) proliferation in the subventricular zone (SVZ) and neuroblasts migrate towards the ischemic boundary. We investigated the role of stromal cell-derived factor 1alpha (SDF-1alpha) in mediating NPC migration after stroke. We found that cultured NPCs harvested from the normal adult SVZ, when they were overlaid onto stroke brain slices, exhibited significantly (P<0.01) increased migration (67.2+/-25.2 microm) compared with the migration on normal brain slices (29.5+/-29.5 microm). Immunohistochemistry showed that CXCR 4, a receptor of SDF-1alpha, is expressed in the NPCs and migrating neuroblasts in stroke brain. Blocking SDF-1alpha by a neutralizing antibody against CXCR 4 significantly attenuated stroke-enhanced NPC migration. ELISA analysis revealed that SDF-1alpha levels significantly increased (P<0.01) in the stroke hemisphere (43.6+/-6.5 pg/mg) when compared with the normal brain (25.2+/-1.9 pg/mg). Blind-well chamber assays showed that SDF-1alpha enhanced NPC migration in a dose-dependent manner with maximum migration at a dose of 500 ng/mL. In addition, SDF-1alpha induced directionally selective migration. These findings show that SDF-1alpha generated in the stroke hemisphere may guide NPC migration towards the ischemic boundary via binding to its receptor CXCR 4 in the NPC. Thus, our data indicate that SDF-1alpha/CXCR 4 is important for mediating specific migration of NPCs to the site of ischemic damaged neurons.

This article reviews current data on the induction of neurogenesis after stroke in the adult brain. The discussion of neurogenesis is divided into production, migration, and survival of these newly formed cells. For production, the subpopulations and the types of cell division are presented. Discussion of cell migration entails presenting data on both the pathways as well as the molecular targeting of newly formed neural progenitor cells to sites of injury. The role of the vascular and the astrocytic microenvironment in promoting the survival and integration of progenitor cells is also presented. Cell-based and pharmacological therapies designed to restore neurological function that promote neurogenesis are described. These therapies also induce angiogenesis and astrocytic changes that brain tissue, which prime the ischemic brain to foster the survival of the newly formed progenitor cells. Signaling pathways that regulate neurogenesis and angiogenesis are also addressed. This review summarizes recent data on neurogenesis and provides insight into the potential for restorative treatments of stroke.

Objectives: We analyze morphological alterations of cerebral neovascularization after stroke using a new 3D imaging software program., Methods: Male Wistar rats underwent unilateral embolic middle cerebral artery occlusion (MCAo) by a single fibrin rich clot. Subjects were sacrificed from 1 to 28 days post infarct. Vessel perimeters were measured on coronal sections stained with endothelial cell-specific antibody to von Willebrand's factor. Vessel segment lengths, diameters and number of vessels were analyzed on cerebral microvessels perfused with FITC-dextran 14 days after ischemia using LSCM and a 3-D vessel quantification program., Results: The mean number of microvessels with enlarged perimeters significantly increased in the ipsilateral cortex at day 7 when compared to the contralateral cortex (29.7+/-14.7 vs. 3.7+/-2.5, P<0.05). Subsequently, differences in the number of microvessels with enlarged perimeters decreased on days 14 and 28. Fourteen days post-MCA occlusion, microvessel segment length (15.0 vs. 26.0 microm, P<0.05) and diameter (3.14 vs. 3.75 microm, P<0.05) significantly decreased in the ipsilateral hemisphere when compared to the contralateral hemisphere, respectively. Furthermore, the mean total number of these smaller microvessels increased in the ipsilateral hemisphere (57.33+/-14.5 vs. 32.22+/-11.7, P<0.05)., Conclusions: Focal cerebral ischemia induces morphological changes (early dilated microvessels followed by decreased microvessel segment length and diameter) that are consistent with newly generated microvessels.

We tested the hypothesis that intravenous infusion of human bone marrow stromal cells (hMSCs) promotes vascular endothelial growth factor (VEGF) secretion, VEGF receptor 2 (VEGFR2) expression and angiogenesis in the ischemic boundary zone (IBZ) after stroke. hMSCs (1x10(6)) were intravenously injected into rats 24 hours after middle cerebral artery occlusion (MCAo). Laser scanning confocal microscopy (LSCM), immunohistochemistry and ELISA were performed to assay angiogenesis and levels of human and rat VEGF in the host brain, respectively. In addition, capillary-like tube formation was measured using mouse brain-derived endothelial cells (MBDECs). Morphological and three dimensional image analyses revealed significant (P<0.05) increases in numbers of enlarged and thin walled blood vessels and numbers of newly formed capillaries at the boundary of the ischemic lesion in rats (n=12) treated with hMSCs compared with numbers in rats (n=12) treated with PBS. ELISA measurements showed that treatment with hMSCs significantly (P<0.05) raised endogenous rat VEGF levels in the IBZ from 10.5+/-1.7 ng/mL in the control group to 17.5+/-1.6 ng/mL in the hMSC-treated group. In addition, treatment with hMSCs increased endogenous VEGFR2 immunoreactivity. In vitro, when MBDECs were incubated with the supernatant obtained from cultured hMSCs, capillary-like tube formation was significantly (P<0.01) induced. However, hMSC-induced capillary-like tube formation was significantly (P<0.01) inhibited when the endothelial cells were incubated with the supernatant from hMSCs in the presence of a neutralizing anti-VEGFR2. These data suggest that treatment of stroke with hMSCs enhances angiogenesis in the host brain and hMSC-enhanced angiogenesis is mediated by increases in levels of endogenous rat VEGF and VEGFR2.

The mouse is an excellent model for investigations of stroke and neural injury. However, there is a paucity of long term functional outcome measurements for the mouse. We, therefore, developed a sensorimotor functional test (corner test) and applied this test to a model of focal cerebral ischemia in the mouse. Male C57/6J mice (n=20) were subjected to embolic middle cerebral artery (MCA) occlusion. Reduction of cerebral blood flow (CBF) was measured by perfusion weighted MRI at 1 h after ischemia. The corner test, which is sensitive to chronic sensorimotor and postural symmetries, a general neurological test battery, and a foot fault test were performed between 2 and 90 days after ischemia. Infarct volume was measured at 90 days after ischemia. Multivariable analysis revealed that the corner test was highly predictive for infarct volume measured at 90 days after stroke, with R(2) values ranging from 0.73 to 0.93. The foot-fault test and neurological score did not detect chronic behavioral impairments. A significant (P<0.001) correlation between the infarct volume and the corner test was detected at 90 days after mild focal cerebral ischemia, whereas, there was no correlation between the infarct volume and neurological score or foot-fault. The data demonstrate that the corner test is a sensitive and objective test, which can be applied to evaluate long term functional outcome after stroke in the mouse.

In an effort to elucidate the molecular mechanisms underlying cerebral vascular alteration after stroke, the authors measured the spatial and temporal profiles of blood-brain barrier (BBB) leakage, angiogenesis, vascular endothelial growth factor (VEGF), associated receptors, and angiopoietins and receptors after embolic stroke in the rat. Two to four hours after onset of ischemia, VEGF mRNA increased, whereas angiopoietin 1 (Ang 1) mRNA decreased. Three-dimensional immunofluorescent analysis revealed spatial coincidence between increases of VEGF immunoreactivity and BBB leakage in the ischemic core. Two to 28 days after the onset of stroke, increased expression of VEGF/VEGF receptors and Ang/Tie2 was detected at the boundary of the ischemic lesion. Concurrently, enlarged and thin-walled vessels were detected at the boundary of the ischemic lesion, and these vessels developed into smaller vessels via sprouting and intussusception. Three-dimensional quantitative analysis of cerebral vessels at the boundary zone 14 days after ischemia revealed a significant (P < 0.05) increase in numbers of vessels (n = 365) compared with numbers (n = 66) in the homologous tissue of the contralateral hemisphere. Furthermore, capillaries in the penumbra had a significantly smaller diameter (4.8 +/- 2.0 microm) than capillaries (5.4 +/- 1.5 microm) in the homologous regions of the contralateral hemisphere. Together, these data suggest that acute alteration of VEGF and Ang 1 in the ischemic core may mediate BBB leakage, whereas upregulation of VEGF/VEGF receptors and Ang/Tie2 at the boundary zone may regulate neovascularization in ischemic brain.

We investigated whether circulating endothelial progenitor cells contribute to neovascularization after stroke. Donor bone marrow cells obtained from transgenic mice constitutively expressing beta-galactosidase transcriptionally regulated by an endothelial-specific promoter, Tie2, were injected into adult mice. Focal cerebral ischemia was induced by embolic middle cerebral artery (MCA) occlusion and changes of cerebral blood flow (CBF) were measured by perfusion-weighted magnetic resonance imaging (MRI). Laser scanning confocal microscopy (LSCM), immunohistochemistry and X-gal staining were performed. Perfusion-weighted MRI demonstrated increases in CBF around the boundary of an infarct area 1 month after ischemia. Morphological and 3-dimensional image analyses revealed enlarged and thin-walled blood vessels with sprouting or intussusception at the boundary of the ischemic lesion, which closely corresponded to elevated CBF areas detected on perfusion-weighted MRI, indicating the presence of neovascularization. X-gal and double immunostaining demonstrated that Tie2-lacZ-positive cells incorporated into sites of neovascularization at the border of the infarct, and these cells exhibited an endothelial antigenic marker (von Willebrand factor). In addition, bone marrow recipient mice without ischemia showed incorporation of Tie2-lacZ-expressing cells into vessels of the choroid plexus. These data suggest that formation of new blood vessels in the adult brain after stroke is not restricted to angiogenesis but also involves vasculogenesis and that circulating endothelial progenitor cells from bone marrow contribute to the vascular substructure of the choroid plexus.

Angiopoietin-1 (Ang1) is a ligand for the endothelial specific receptor tyrosine kinase, Tie2, that protects the adult peripheral vasculature from vascular leakage. We tested the hypothesis that increases in levels of Ang1 reduce blood-brain barrier (BBB) leakage in ischemic brain. Mice were subjected to embolic middle cerebral artery (MCA) occlusion. Recombinant adenoviruses expressing Ang1 (Ad-Ang1) or a control gene encoding green fluorescent protein (Ad-GFP), or recombinant Ang1 protein, BowAng1, was administered to mice before MCA occlusion. Regional cerebral blood flow (rCBF), the brain tissue content of Evans Blue, and ischemic lesion volume were measured. Serum levels of Ang1 (183+/-31.9 microg/ml, n=4) were detected in mice receiving Ad-Ang1 or in mice treated with BowAng1 (262+/-35.4 microg/ml, n=7) but not in the control mice (n=11). Six hours after MCA occlusion, mice receiving Ad-GFP (n=8) or control protein (n=7) showed large Evans Blue leakage in the ipsilateral hemisphere (0.46+/-0.05 or 0.55+/-0.16 ng/mg tissue) whereas mice receiving Ad-Ang1 (n=6) or BowAng1 (n=7) had significantly (P<0.05) less Evans Blue leakage (0.26+/-0.07 or 0.14+/-0.03 ng/mg tissue). Infusion of recombinant human vascular endothelial growth factor (rhVEGF(165)) to ischemic mice resulted in significant (P<0.05) increases in Evans Blue leakage (1.24+/-0.34 ng/mg tissue, n=7) compared with the control mice. In contrast, infusion of rhVEGF(165) in ischemic mice receiving Ad-Ang1 did not significantly increase Evans Blue dye in the ipsilateral hemisphere (0.22+/-0.06 ng/mg tissue, n=6). Moreover, 24 h after ischemia mice receiving Ad-Ang1 had a significantly smaller ischemic lesion volume (22.6+/-2.7%, n=8) than the lesion volume in mice receiving Ad-GFP (44.7+/-3.7%, n=8), although rCBF reduced to approximately 20% of the contralateral levels in both groups of mice 10 min after ischemia. Our data demonstrate that Ang1 reduces BBB leakage in ischemic brain and consequently decreases ischemic lesion volume.

Background and Purpose: The proteasome inhibitor PS-519 blocks activation of nuclear factor-kappaB, a major mediator of inflammation. We tested the hypothesis that combination treatment of recombinant human tissue plasminogen activator (rhtPA) and PS-519 extends the therapeutic window for treatment of stroke with rhtPA without increasing incidence of hemorrhagic transformation., Methods: The middle cerebral artery (MCA) of male Wistar rats (n=56) was occluded by an embolus. After embolization, animals were randomly divided into the following groups: PS-519 treatment groups: PS-519 was given at 2, 4, or 6 hours after MCA occlusion; rhtPA treatment groups: rhtPA was given at 2 or 4 hours after MCA occlusion; combination treatment groups: PS-519 and rhtPA were given at 2, 4, or 6 hours after MCA occlusion; control group: the same volume of saline was given at 2 hours after MCA occlusion., Results: Administration of PS-519 alone at 2 or 4 hours, but not 6 hours, significantly (P<0.05) reduced infarct volume and improved neurological recovery compared with the control group. Administration of rhtPA alone at 2 hours, but not 4 hours, significantly (P<0.05) reduced infarct volume and improved neurological recovery compared with the control group. Furthermore, combination treatment with rhtPA and PS-519 even at 6 hours significantly (P<0.05) reduced infarct volume, improved neurological recovery, and did not increase the incidence of hemorrhagic transformation compared with the control group or the group treated with PS-519 alone., Conclusions: Our data suggest that combination treatment with PS-519 and rhtPA extends the neuroprotective effect to at least 6 hours after embolization.

Information is lacking regarding dynamic platelet accumulation at the site of the occluded middle cerebral artery (MCA) and the relationship between platelet aggregation in downstream cerebral microvessels and loss of perfusion and vascular integrity of these microvessels. In the present study, we employed a model of embolic MCA occlusion in the rat to simultaneously measure temporal and spatial profiles of platelet accumulation at the site of the embolus occluding the MCA and within downstream cerebral microvessels. We also measured the integrity of microvessels and matrix metalloproteinase (MMP) activity in ischemic brain. Rats (n=36) were subjected to embolic MCA occlusion. Immunohistochemistry was used to detect microvascular integrity, plasminogen activator inhibitor 1 (PAI-1) and the deposition of fibrin. SDS-PAGE zymography was used to measure MMP2 and MMP9 activities. Accumulation of platelets and increases in PAI-1 immunoreactivity at the site of the embolus occluding the MCA were detected 1 h (n=7) and 4 h (n=7) after ischemia, respectively, and numbers of GPIIb/IIIa immunoreactive downstream cerebral microvessels increased significantly (209+/-59; n=7; P<0.05) 4 h after ischemia, suggesting dynamic platelet aggregation. A significant (n=7; P<0.01) diffuse loss of type IV collagen immunoreactivity in microvessels was temporally associated with platelet GPIIb/IIIa immunoreactivity within the vessels. Triple immunostaining revealed that microvessels containing platelet aggregates exhibited loss of type IV collagen immunoreactivity and both intra- and extra-vascular fibrin deposition, suggesting that intravascular platelet aggregation is associated with decreases in the integrity of the microvascular basal lamina and blood-brain barrier leakage. A significant increase (P<0.05) in MMP9 was detected at 4 h (n=3) and 24 h (n=3) after ischemia but levels of MMP2 were not significantly changed in ischemic brain. Our data suggest that dynamic platelet aggregation in ischemic brain may contribute to time-dependent resistance to fibrinolysis. In addition, platelet deposition and increased MMP9 coincided with degradation of type IV collagen and loss of vascular integrity. These data suggest an important role for post-occlusive distal platelet deposition in the pathophysiology of stroke.

During development, neuropilin-1 is a receptor for semaphorin 3a-mediated axonal guidance and for vascular endothelial growth factor (VEGF) promotion of angiogenesis. The authors measured neuropilin-1 expression in the adult ischemic brain using Northern blot, in situ hybridization, and immunohistochemistry. Neuropilin-1 mRNA was significantly up-regulated as early as 2 hours and persisted at least 28 days after focal cerebral ischemia. Acute up-regulation of neuropilin-1 mRNA primarily localized to the ischemic neurons. A marked increase in both mRNA and protein of neuropilin-1 was detected in endothelial cells of cerebral blood vessels at the border and in the core of the ischemic lesion 7 days after ischemia, and neuropilin-1 gene expression persisted on these vessels for at least 28 days after ischemia. In these areas, neovascularization was detected using three-dimensional reconstructed images obtained from laser scanning confocal microscopy. Activated astrocytes also exhibited neuropilin-1 immunoreactivity during 7 to 28 days of ischemia. Double immunofluorescent staining showed colocalization of neuropilin-1 and VEGF to cerebral blood vessels and activated astrocytes. These data suggest that in addition to its role in axonal growth, up-regulation of neuropilin-1, in concert with VEGF and its receptors, may contribute to neovascular formation in the adult ischemic brain.

Background and Purpose: After stroke, brain tissue undergoes time-dependent heterogeneous histopathological change. These tissue alterations have MRI characteristics that allow segmentation of ischemic from nonischemic tissue. Moreover, MRI segmentation generates different zones within the lesion that may reflect heterogeneity of tissue damage., Methods: A vector tissue signature model is presented that uses multiparametric MRI for segmentation and characterization of tissue. An objective (unsupervised) computer segmentation algorithm was incorporated into this model with the use of a modified version of the Iterative Self-Organizing Data Analysis Technique (ISODATA). The ability of the model to characterize ischemic tissue after permanent middle cerebral ischemia occlusion in the rat was tested. Multiparametric ISODATA measurements of the ischemic tissue were compared with quantitative histological characterization of the tissue from 4 hours to 1 week after stroke., Results: The ISODATA segmentation of tissue identified a gradation of cerebral tissue damage at all time points after stroke. The histological scoring of ischemic tissue from 4 hours to 1 week after stroke on all the animals was significantly correlated with ISODATA segmentation (r=0.78, P<0.001; n=20) when a multiparametric (T2-, T1-, diffusion-weighted imaging) data set was used, less correlated (r=0.70, P<0.01; n=20) when a T2- and T1-weighted data set was used, and not correlated (r=-0.12, P>0.47; n=20) when only a diffusion-weighted imaging data set was used., Conclusions: Our data indicate that an integrated set of MRI parameters can distinguish and stage ischemic tissue damage in an objective manner.

Progenitor cells in the subventricular zone of the lateral ventricle and in the dentate gyrus of the hippocampus can proliferate throughout the life of the animal. To examine the proliferation and fate of progenitor cells in the subventricular zone and dentate gyrus after focal cerebral ischemia, we measured the temporal and spatial profiles of proliferation of cells and the phenotypic fate of proliferating cells in ischemic brain in a model of embolic middle cerebral artery occlusion in the adult rat. Proliferating cells were labeled by injection of bromodeoxyuridine (BrdU) in a pulse or a cumulative protocol. To determine the temporal profile of proliferating cells, ischemic rats were injected with BrdU every 4 h for 12 h on the day preceding death. Rats were killed 2-14 days after ischemia. We observed significant increases in numbers of proliferating cells in the ipsilateral cortex and subventricular zone 2-14 days with a peak at 7 days after ischemia compared with the control group. To maximize labeling of proliferating cells, a single daily injection of BrdU was administered over a 14-day period starting the day after ischemia. Rats were killed either 2 h or 28 days after the last injection of BrdU. A significant increase in numbers of BrdU immunoreactive cells in the subventricular zone was coincident with a significant increase in numbers of BrdU immunoreactive cells in the olfactory bulb 14 days after ischemia and numbers of BrdU immunoreactive cells did not significantly increase in the dentate gyrus. However, 28 days after the last labeling, the number of BrdU labeled cells decreased by 90% compared with number at 14 days. Clusters of BrdU labeled cells were present in the cortex distal to the infarction. Numerous cells immunostained for the polysialylated form of the neuronal cell adhesion molecule were detected in the ipsilateral subventricular zone. Only 6% of BrdU labeled cells exhibited glial fibrillary acidic protein immunoreactivity in the cortex and subcortex and no BrdU labeled cells expressed neuronal protein markers (neural nuclear protein and microtubule associated protein-2). From these data we suggest that focal cerebral ischemia induces transient and regional specific increases in cell proliferation in the ipsilateral hemisphere and that proliferating progenitor cells may exist in the adult cortex.

VEGF is a secreted mitogen associated with angiogenesis and is also a potent vascular permeability factor. The biological role of VEGF in the ischemic brain remains unknown. This study was undertaken to investigate whether VEGF enhances cerebral microvascular perfusion and increases blood-brain barrier (BBB) leakage in the ischemic brain. Using magnetic resonance imaging (MRI), three-dimensional laser-scanning confocal microscope, and functional neurological tests, we measured the effects of administrating recombinant human VEGF(165) (rhVEGF(165)) on angiogenesis, functional neurological outcome, and BBB leakage in a rat model of focal cerebral embolic ischemia. Late (48 hours) administration of rhVEGF(165) to the ischemic rats enhanced angiogenesis in the ischemic penumbra and significantly improved neurological recovery. However, early postischemic (1 hour) administration of rhVEGF(165) to ischemic rats significantly increased BBB leakage, hemorrhagic transformation, and ischemic lesions. Administration of rhVEGF(165) to ischemic rats did not change BBB leakage and cerebral plasma perfusion in the contralateral hemisphere. Our results indicate that VEGF can markedly enhance angiogenesis in the ischemic brain and reduce neurological deficits during stroke recovery and that inhibition of VEGF at the acute stage of stroke may reduce the BBB permeability and the risk of hemorrhagic transformation after focal cerebral ischemia.

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.

We tested the hypothesis that mild and severe ischemic cell damage are reflected in neurological and functional recovery after stroke. Rats were subjected to either 30 min or 120 min of middle cerebral artery occlusion or sham operation. Neurological and functional tests including, gross neurological score, and rotarod and adhesive removal tests were performed at various time points up to 21 days after stroke. Significant differences between groups of animals were detected using the rotarod and adhesive removal test. A significant correlation between lesion volume and adhesive removal test was detected in rats subjected to 30 min of ischemia. Our data indicate that quantitative rotarod and adhesive removal tests measure different aspects of functional recovery after stroke, and both are useful in characterizing functional recovery from an ischemic insult.

The mechanisms underlying cerebral microvascular perfusion deficit resulting from occlusion of the middle cerebral artery (MCA) require elucidation. We, therefore, tested the hypothesis that intravascular fibrin deposition in situ directly obstructs cerebral microcirculation and that local changes in type 1 plasminogen activator inhibitor (PAI-1) gene expression contribute to intravascular fibrin deposition after embolic MCA occlusion. Using laser-scanning confocal microscopy (LSCM) in combination with immunofluorescent staining, we simultaneously measured in three dimensions the distribution of microvascular plasma perfusion deficit and fibrin(ogen) immunoreactivity in a rat model of focal cerebral embolic ischemia (n = 12). In addition, using in situ hybridization and immunostaining, we analyzed expression of PAI-1 in ischemic brain (n = 13). A significant (p < 0.05) reduction of cerebral microvascular plasma perfusion accompanied a significant (p < 0.05) increase of intravascular and extravascular fibrin deposition in the ischemic lesion. Microvascular plasma perfusion deficit and fibrin deposition expanded concomitantly from the subcortex to the cortex during 1 and 4 hr of embolic MCA occlusion. Three-dimensional analysis revealed that intravascular fibrin deposition directly blocks microvascular plasma perfusion. Vascular plugs contained erythrocytes, polymorphonuclear leukocytes, and platelets enmeshed in fibrin. In situ hybridization demonstrated induction of PAI-1 mRNA in vascular endothelial cells in the ischemic region at 1 hr of ischemia. PAI-1 mRNA significantly increased at 4 hr of ischemia. Immunohistochemical staining showed the same pattern of increased PAI-1 antigen in the endothelial cells. These data demonstrate, for the first time, that progressive intravascular fibrin deposition directly blocks cerebral microvascular plasma perfusion in the ischemic region during acute focal cerebral embolic ischemia, and upregulation of the PAI-1 gene in the ischemic lesion may foster fibrin deposition through suppression of fibrinolysis.

An accurate noninvasive time-independent identification of an ischemic cerebral lesion is an important objective of magnetic resonance imaging (MRI). This study describes a novel application of a multiparameter MRI analysis algorithm, the Eigenimage (EI) filter, to experimental stroke. The EI is a linear filter that maximizes the projection of a desired tissue (ischemic tissue) while it minimizes the projection of undesired tissues (nonischemic tissue) onto a composite image called an eigenimage. Rats (n=26) were subjected to permanent middle cerebral artery occlusion. T2- and T1-weighted coronal MRI were acquired on separate groups of animals. The animals were immediately sacrificed after each imaging session for histopathological analysis of tissue at 4-8 h, 16-24 h, and 48-168 h after stroke onset. Lesion areas from MRI were defined using EI. The EI defined lesion areas were coregistered and warped to the corresponding histopathological sections. The ischemic lesion as defined by EI exhibited ischemic cell damage ranging from scattered acute cell damage to pan necrosis. Ischemic cellular damage was not detected in homologous contralateral hemisphere regions. EI lesion areas overlaid on histopathological sections were significantly correlated (r=0.92, p<0.05) acutely, (r=0.98, p<0.05) subacutely, and (r=0.99, p<0.05) chronically. These data indicate that EI methodology can accurately segment ischemic damage after MCA occlusion from 4-168 h after stroke., (Copyright 1999 Elsevier Science B.V.)

We measured urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) activity in the brain of 2-3 month old and 6-8 month old mice subjected to 4 h of middle cerebral artery (MCA) occlusion. t-PA activity was present in all non-ischemic and ischemic young mouse brain. In contrast, t-PA activity was present in 46.7% of non-ischemic middle aged mouse brain and in 44.4% of ischemic middle aged mouse brain. u-PA activity was present in all young and middle aged non-ischemic brains., (Copyright 1999 Elsevier Science B.V.)

Background and Purpose: We tested the hypothesis that treatment of embolic stroke with recombinant human tissue plasminogen activator (rhtPA) alters cerebral expression of adhesion molecules., Methods: Male Wistar rats were subjected to middle cerebral artery occlusion by a single fibrin-rich clot. P-selectin, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) immunoreactivity was measured at 6 or 24 hours after embolic stroke in control rats and in rats treated with rhtPA at 1 or 4 hours after stroke. To examine the therapeutic efficacy of combined rhtPA and anti-ICAM-1 antibody treatment at 4 hours after embolization, ischemic lesion volumes were measured in rats treated with rhtPA alone, rats treated with rhtPA and anti-ICAM-1 antibody, and nontreated rats., Results: Administration of rhtPA at 1 hour after embolization resulted in a significant reduction of adhesion molecule vascular immunoreactivity after embolization in the ipsilateral hemisphere compared with corresponding control rats. However, when rhtPA was administered to rats at 4 hours after embolization, significant increases of adhesion molecule immunoreactivity in the ipsilateral hemisphere were detected. A significant increase of ICAM-1 immunoreactivity was also detected in the contralateral hemisphere at 24 hours after ischemia. A significant reduction in lesion volume was found in rats treated with the combination of rhtPA and anti-ICAM-1 antibody compared with rats treated only with rhtPA., Conclusions: The present study suggests that the time of initiation of thrombolytic therapy alters vascular immunoreactivity of inflammatory adhesion molecules in the ischemic brain and that therapeutic benefit can be obtained by combining rhtPA and anti-ICAM-1 antibody treatment 4 hours after stroke.

We developed a new model of embolic cerebral ischemia in the rat which provides a reproducible and predictable infarct volume within the territory supplied by the middle cerebral artery (MCA). The MCA was occluded by an embolus in Wistar rats (n = 71). An additional three non-embolized rats were used as a control. Cerebral blood flow (CBF) was measured by means of laser Doppler flowmetry (LDF) and perfusion weighted imaging (PWI) before and after embolization. The evolution of the lesion was monitored by diffusion weighted imaging (DWI). Cerebral vascular perfusion patterns were examined using laser scanning confocal microscopy. Infarct volumes were measured on hematoxylin and eosin (H&E) stained coronal sections. The lodgment of the clot at the origin of the MCA and the ischemic cell damage were examined using light microscopy. Regional CBF in the ipsilateral parietal cortex decreased to 43 +/- 4.1% (P < 0.05) of preischemic levels (n = 10). Confocal microscopic examination revealed a reduction of cerebral plasma perfusion in the ipsilateral MCA territory (n = 6). MRI measurements showed a reduction in CBF and a hyperintensity DWI encompassing the territory supplied by the MCA (n = 4). An embolus was found in all rats at 24 h after embolization. The infarct volume as a percentage of the contralateral hemisphere was 32.5 +/- 3.31% at 24 h (n = 20), 33.0 +/- 3.6% at 48 h (n = 13), and 34.5 +/- 4.74% at 168 h (n = 12) after embolization. This model of embolic focal cerebral ischemia results in ischemic cell damage and provides a reproducible and predictable infarct volume. This model is relevant to thromboembolic stroke in humans and may be useful in documenting the safety and efficacy of fibrinolytic intervention and in investigating therapies complementary to antithrombotic therapy.

We have developed a multiparameter magnetic resonance imaging (MRI) cluster analysis model of acute ischemic stroke using T2 relaxation times and the diffusion coefficient of water (ADCw). To test the ability of this model to predict cerebral infarction, male Wistar rats (n = 7) were subjected to 2 h of transient middle cerebral artery (MCA) occlusion, and diffusion and T2 weighted MRI were performed on these rats before, during and up to 7 days after MCA occlusion. MRI tissue signatures, specified by values of ADCw and T2 were assigned to tissue histopathology. Significant correlations were obtained between MRI signatures at different time points and histopathologic measurements of lesion area obtained at 1 week. In addition, we compared the temporal evolution of MRI tissue signatures to a separate population of animals at which histological data were obtained at select times of reperfusion. A significant shift (p < or = 0.05) within signatures reflecting tissue histopathology was demonstrated as the ischemic lesion evolved over time. Our data suggest, that the MRI signatures are associated with the degree of ischemic cell damage. Thus, the tissue signature model may provide a noninvasive means to monitor the evolution of ischemic cell damage and to predict final outcome of ischemic cell damage.

The selectin family of glycoproteins facilitates the early phase of polymorphonuclear leukocyte adhesion to the endothelial cell and, thus, may promote ischemic cell damage. To evaluate E-selectin in the pathogenesis of focal cerebral ischemia and reperfusion injury, we cloned rat E-selectin cDNA and measured the temporal profiles E-selectin mRNA (Northern blot) and protein (immunohistochemistry) during (1 h of ischemia) and after (up to 1 week) transient (2 h) middle cerebral artery (MCA) occlusion in the male Wistar rat. We also tested the effect on these rats of administration of CY-1503, an analog of sialyl Lewis(x) (SLe(x)), on ischemia cell damage. mRNA for E-selectin was first detected in the ischemic hemisphere at 2 h of reperfusion and persisted to 46 h of reperfusion. E-selectin (protein) was localized to microvessels within the ischemic lesion at 0 h of reperfusion and persisted to 70 h of reperfusion. Treatment of the ischemic animals with CY-1503 (50 mg/kg) (n = 8) significantly reduced infarct volume by 42% (p < 0.05) and significantly reduced myeloperoxidase immunoreactive cells in the ischemic lesion by 60% (p < 0.05). These findings provide the first direct evidence for the involvement of E-selectin in transient MCA occlusion in rats and suggest that the E-selectin may facilitate neutrophil adhesion and subsequent cerebral ischemic cell damage.

In this review we focus on recent efforts designed to elucidate the role of inflammatory cells and nitric oxide in promoting cerebral ischemic cell damage, the mechanisms by which these events exacerbate ischemic cell damage, and the associated therapeutic interventions to protect tissue from ischemic injury.

Using a porphyritic microsensor, we measured the cortical NO concentration within ischemic tissue during 2 h of middle cerebral artery (MCA) occlusion and 1 h of reperfusion in the rat (n = 36). Local cerebral blood flow was simultaneously measured by laser Doppler flowmetry to verify MCA occlusion and reperfusion. Baseline concentration of NO was < 10(-8) M. The maximum concentrations of NO during MCA occlusion and reperfusion were, respectively, 1.47 +/- 0.45 microM and 0.54 +/- 0.24 microM. Administration of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, prior to ischemia, significantly (p < 0.05) reduced NO release to 0.04 +/- 0.02 microM during MCA occlusion and completely inhibited NO release during 1 h of reperfusion. Administration of L-arginine 30 min after administration of L-NAME restored NO release (3.45 +/- 1.14 microM) during MCA occlusion; however, administration of L-arginine did not overcome the effect of L-NAME on mean arterial blood pressure. Our data indicate that NO is released in the brain after the onset of ischemia and NO levels can be modulated by administration of NO substrate and NO antagonists.

Nitric oxide synthase-containing neurons are presumed to be resistant to neurodegeneration and neurotoxicity, however this resistance has not been demonstrated after focal cerebral ischemia. We therefore measured the temporal profile of neuronal nitric oxide synthase (NOS-I) mRNA and immunoreactivity and NADPH-diaphorase reactivity over a one week period after permanent middle cerebral artery (MCA) occlusion in 48 male Wistar rats and compared these data to ischemic cell damage as evaluated on hematoxylin and eosin (H & E) stained sections by light microscopy. NOS-I mRNA increased as early as 15 min after MCA occlusion in the ipsilateral striatum and maximal expression of NOS-I was found in the ipsilateral cortex and striatum 1 h after MCA occlusion. The numbers of NOS-I-containing neurons in the ipsilateral cortex and striatum were significantly greater (P < 0.05) than NOS-I-containing neurons in the contralateral hemisphere at 2-48 h after the onset of ischemia. The number of NOS-I-containing neurons peaked at 4 h after MCA occlusion. Neurons exhibited shrinkage or were swollen at 1 to 4 h after MCA occlusion. At 24-48 h after ischemia, neurons in the ischemic lesion appeared to be eosinophilic or ghost like on H & E stained sections. However, some of these neurons retained morphological integrity on the NOS-I immunohistochemical sections. At 168 h after ischemia, all neurons within the lesion appeared necrotic on H & E stained sections; however, scatterred neurons expressed NOS-I and NADPH-diaphorase. The rapid upregulation of NOS-I and mRNA in the ischemic lesion suggests that NOS-I is involved in focal cerebral ischemic injury; the expression of NOS-I by neurons that retain their morphological structure in the area of the infarct suggests that NOS-I-containing neurons are more resistant to the ischemic insult. Our data also indicate a close association of NOS-I immunoreactivity and NADPH-diaphorase reactivity in ischemic brain.

Background and Purpose: We investigated the temporal distribution of the p53-immunoreactive protein in conjunction with cellular damage and the expression of the p53 mRNA after focal cerebral ischemia in rats., Methods: Male Wistar rats (n = 66; controls, n = 7) were subjected to 2 hours of middle cerebral artery occlusion and were killed at various times of reperfusion (0.5 to 168 hours) for p53 immunohistochemistry and Northern blot analysis., Results: A cellular expression of mutant p53-immunoreactive protein was found localized to anatomic sites exhibiting severe neuronal damage. A maximal induction of mutant p53-immunoreactive protein was found at 12 hours after reperfusion and subsequently declined. No wild-type p53 protein expression was detected after ischemia. A time-dependent expression of p53 mRNA was observed in both hemispheres. The peak level of p53 mRNA occurred at 24 hours after reperfusion., Conclusions: Our data indicate that the expressions of p53-immunoreactive protein and p53 mRNA are upregulated after transient focal cerebral ischemic insult in rats. The concomitant appearance of p53 and cell damage in ischemic brain suggests that p53 expression may impact cell biological response to an ischemic insult.

Induction of the 72-kDa heat shock protein expression is thought to protect neurons against the subsequent effects of ischemia. However, it is not clear whether the induction of 72-kDa heat shock protein expression by an ischemic event improves neuronal survival. To address this question, we outlined the temporal profile of neuronal induction and expression of the 72-kDa heat shock protein in a model of transient focal ischemia in the rat. Fifty two adult Wistar rats were subjected to middle cerebral artery occlusion of 2 h duration. At 0.5, 3, 6, 9, 12, 24, 48, 96 and 168 h after reopening the artery, coronal brain sections were analyzed using both immunohistochemical methods and hematoxylin and eosin staining to determine the topographic and cellular distribution of the 72-kDa heat shock protein, as well as the extent of neuronal damage. Immunoreactivity to the 72-kDa heat shock protein was not detected in neurons that were destined to become necrotic, and were located in the ischemic core of the brain lesions. However, 72-kDa heat shock protein expression was evident in morphologically intact neurons located in the peripheral zone. The earliest neuronal expression of 72-kDa heat shock protein was detected in animals in which the 2 h occlusion of the middle cerebral artery was followed by 6 h recirculation; the intensity of the 72-kDa heat shock protein immunoreactivity peaked at 48 h, and progressively disappeared 7 days after the ischemic reperfusion event.(ABSTRACT TRUNCATED AT 250 WORDS)