α7 Nicotinic Acetylcholine Receptor Stimulation Attenuates Neuroinflammation through JAK2-STAT3 Activation in Murine Models of Intracerebral Hemorrhage.

Accounting for high mortality and morbidity rates, intracerebral hemorrhage (ICH) remains one of the most detrimental stroke subtypes lacking a specific therapy. Neuroinflammation contributes to ICH-induced brain injury and is associated with unfavorable outcomes. This study aimed to evaluate whether α7 nicotinic acetylcholine receptor (α7nAChR) stimulation ameliorates neuroinflammation after ICH. Male CD-1 mice and Sprague-Dawley were subjected to intracerebral injection of autologous blood or bacterial collagenase. ICH animals received either α7nAChR agonist PHA-543613 alone or combined with α7nAChR antagonist methyllycaconitine (MLA) or Janus kinase 2 (JAK2) antagonist AG490. Neurobehavioral deficits were evaluated at 24 hours, 72 hours, and 10 weeks after ICH induction. Perihematomal expressions of JAK2, signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor-α (TNF-α), and myeloperoxidase (MPO) were quantified via Western blot. Histologic volumetric analysis of brain tissues was conducted after 10 weeks following ICH induction. PHA-543613 improved short-term neurobehavioral (sensorimotor) deficits and increased activated perihematomal JAK2 and STAT3 expressions while decreasing TNF-α and MPO expressions after ICH. MLA reversed these treatment effects. PHA-543613 also improved long-term neurobehavioral (sensorimotor, learning, and memory) deficits and ameliorated brain atrophy after ICH. These treatment effects were reduced by AG490. α7nAChR stimulation reduced neuroinflammation via activation of the JAK2-STAT3 pathway, thereby ameliorating the short- and long-term sequelae after ICH.

Dabigatran ameliorates post-haemorrhagic hydrocephalus development after germinal matrix haemorrhage in neonatal rat pups.

We aim to determine if direct thrombin inhibition by dabigatran will improve long-term brain morphological and neurofunctional outcomes and if potential therapeutic effects are dependent upon reduced PAR-1 stimulation and consequent mTOR activation. Germinal matrix haemorrhage was induced by stereotaxically injecting 0.3 U type VII-S collagenase into the germinal matrix of P7 rat pups. Animals were divided into five groups: sham, vehicle (5% DMSO), dabigatran intraperitoneal, dabigatran intraperitoneal + TFLLR-NH2 (PAR-1 agonist) intranasal, SCH79797 (PAR-1 antagonist) intraperitoneal, and dabigatran intranasal. Neurofunctional outcomes were determined by Morris water maze, rotarod, and foot fault evaluations at three weeks. Brain morphological outcomes were determined by histological Nissl staining at four weeks. Expression levels of p-mTOR/p-p70s6k at three days and vitronectin/fibronectin at 28 days were quantified. Intranasal and intraperitoneal dabigatran promoted long-term neurofunctional recovery, improved brain morphological outcomes, and reduced intracranial pressure at four weeks after GMH. PAR-1 stimulation tended to reverse dabigatran's effects on post-haemorrhagic hydrocephalus development. Dabigatran also reduced expression of short-term p-mTOR and long-term extracellular matrix proteins, which tended to be reversed by PAR-1 agonist co-administration. PAR-1 inhibition alone, however, did not achieve the same therapeutic effects as dabigatran administration.

Fingolimod confers neuroprotection through activation of Rac1 after experimental germinal matrix hemorrhage in rat pups.

Fingolimod, a sphingosine-1-phosphate receptor (S1PR) agonist, is clinically available to treat multiple sclerosis and is showing promise in treating stroke. We investigated if fingolimod provides long-term protection from experimental neonatal germinal matrix hemorrhage (GMH), aiming to support a potential mechanism of acute fingolimod-induced protection. GMH was induced in P7 rats by infusion of collagenase (0.3 U) into the right ganglionic eminence. Animals killed at 4 weeks post-GMH received low- or high-dose fingolimod (0.25 or 1.0 mg/kg) or vehicle, and underwent neurocognitive testing before histopathological evaluation. Subsequently, a cohort of animals killed at 72 h post-GMH received 1.0 mg/kg fingolimod; the specific S1PR1 agonist, SEW2871; or fingolimod co-administered with the S1PR1/3/4 inhibitor, VPC23019, or the Rac1 inhibitor, EHT1864. All drugs were injected intraperitoneally 1, 24, and 48 h post-surgery. At 72 h post-GMH, brain water content, extravasated Evans blue dye, and hemoglobin were measured as well as the expression levels of phospho-Akt, Akt, GTP-Rac1, Total-Rac1, ZO1, occludin, and claudin-3 determined. Fingolimod significantly improved long-term neurocognitive performance and ameliorated brain tissue loss. At 72 h post-GMH, fingolimod reduced brain water content and Evans blue dye extravasation as well as reversed GMH-induced loss of tight junctional proteins. S1PR1 agonism showed similar protection, whereas S1PR or Rac1 inhibition abolished the protective effect of fingolimod. Fingolimod treatment improved functional and morphological outcomes after GMH, in part, by tempering acute post-hemorrhagic blood-brain barrier disruption via the activation of the S1PR1/Akt/Rac1 pathway.

PPARγ-induced upregulation of CD36 enhances hematoma resolution and attenuates long-term neurological deficits after germinal matrix hemorrhage in neonatal rats.

Germinal matrix hemorrhage remains the leading cause of morbidity and mortality in preterm infants in the United States with little progress made in its clinical management. Survivors are often afflicted with long-term neurological sequelae, including cerebral palsy, mental retardation, hydrocephalus, and psychiatric disorders. Blood clots disrupting normal cerebrospinal fluid circulation and absorption after germinal matrix hemorrhage are thought to be important contributors towards post-hemorrhagic hydrocephalus development. We evaluated if upregulating CD36 scavenger receptor expression in microglia and macrophages through PPARγ stimulation, which was effective in experimental adult cerebral hemorrhage models and is being evaluated clinically, will enhance hematoma resolution and ameliorate long-term brain sequelae using a neonatal rat germinal matrix hemorrhage model. PPARγ stimulation (15d-PGJ2) increased short-term PPARγ and CD36 expression levels as well as enhanced hematoma resolution, which was reversed by a PPARγ antagonist (GW9662) and CD36 siRNA. PPARγ stimulation (15d-PGJ2) also reduced long-term white matter loss and post-hemorrhagic ventricular dilation as well as improved neurofunctional outcomes, which were reversed by a PPARγ antagonist (GW9662). PPARγ-induced upregulation of CD36 in macrophages and microglia is, therefore, critical for enhancing hematoma resolution and ameliorating long-term brain sequelae.

Remote Ischemic Postconditioning (RIPC) After GMH in Rodents.

Germinal matrix hemorrhage (GMH) is the most common and devastating neurological injury of premature infants, and current treatment approaches are ineffective. Remote ischemic postconditioning (RIPC) is a method by which brief limb ischemic stimuli protect the injured brain. We hypothesized that RIPC can improve outcomes following GMH in rats. Neonatal rats (P7) were subjected to either stereotactic ganglionic eminence collagenase infusion or sham surgery. Groups were as follows: sham (n = 0), GMH non-RIPC (n = 10), GMH + 1 week RIPC (n = 10), GMH + 2 weeks RIPC (n = 10). Neurobehavior analysis at the fourth week consisted of Morris water maze (MWM) and rotarod (RR). This was followed by euthanasia for histopathology on day 28. Both 1- and 2-week RIPC showed significant improvement in FF and RR motor testing compared with untreated animals (i.e., GMH without RIPC). RIPC treatment also improved cognition (MWM) and attenuated neuropathological ventricular enlargement (hydrocephalus) in juvenile animals following GMH. RIPC is a safe and noninvasive approach that improved sensorimotor and neuropathological outcomes following GMH in rats. Further studies are needed to evaluate for mechanisms of neuroprotection.

Intranasal IGF-1 Reduced Rat Pup Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is the most devastating neurological problem of premature infants. Current treatment strategies are ineffective and brain injury is unpreventable. Insulin-like growth factor 1 (IGF-1) is an endogenous protein shown to have multiple neuroprotective properties. We therefore hypothesized that IGF-1 would reduce brain injury after GMH. Neonatal rats (P7 age) received stereotactic collagenase into the right ganglionic eminence. The following groups were studied: (1) sham, (2) GMH + vehicle, (3) GMH + intranasal IGF-1. Three days later, the animals were evaluated using the righting-reflex (early neurobehavior), Evans blue dye leakage (blood-brain barrier (BBB) permeability), brain water content (edema), and hemoglobin assay (extent of bleeding). Three weeks later, juvenile rats were tested using a water maze (delayed neurobehavior), and then were sacrificed on day 28 for assessment of hydrocephalus (ventricular size). Intranasal IGF-1 treated animals had improved neurological function, and amelioration of BBB permeability, edema, and re-bleeding. IGF-1 may play a part in protective brain signaling following GMH, and our observed protective effect may offer new promise for treatment targeting this vulnerable patient population.

Cyclooxygenase-2 Inhibition Provides Lasting Protection Following Germinal Matrix Hemorrhage in Premature Infant Rats.

Germinal matrix hemorrhage (GMH) is a major cause of brain damage in prematurity and has long-lasting neurological implications. The development of brain inflammation contributes to brain injury, leading to a lifetime of neurologic deficits. PAR-1 and 4 receptors are involved with inflammatory pathways after brain hemorrhage in adult models of stroke, of which cyclooxygenase-2 (COX-2) is a potential mediator. We therefore hypothesized a role for PAR-1, 4/ COX-2 signaling following GMH. Postnatal day 7 Sprague-Dawley rats were subjected to GMH induction, which entailed stereotactic collagenase infusion into the ganglionic eminence. Animals were euthanized at two time points: 72 h (short-term) or 4 weeks (long-term). Short-term COX-2 expression was evaluated in the context of PAR-1 (SCH-79797) and PAR-4 (P4pal10) inhibition. Pups in the long-term group were administered the selective COX-2 inhibitor (NS-398); and the neurobehavioral and pathological examinations were performed 4 weeks later. Pharmacological PAR-1, 4 antagonism normalized COX-2 expression following GMH and reduced hydrocephalus. Early inhibition of COX-2 by NS-398 improved long-term neurobehavioral outcomes. COX-2 signaling plays an important role in brain injury following neonatal GMH, possibly through upstream PAR-1, 4 receptor mechanisms.

PAR-1, -4, and the mTOR Pathway Following Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is the most common cause of neurological complications of prematurity and has lasting implications. PAR-1 and PAR-4 receptors are involved with upstream signaling pathways following brain hemorrhage in adult models of stroke, of which the mammalian target of rapamycin (mTOR) is a potential downstream mediator. Therefore, we hypothesized a role for PAR-1, -4/ mTOR signaling following GMH brain injury. Postnatal day 7 Sprague-Dawley rats were subjected to GMH through stereotactic infusion of collagenase into the right ganglionic eminence. Rodents were euthanized at 72 h (short term), or 4 weeks (long term). Short-term mTOR expression was evaluated by Western blot in the context of PAR-1 (SCH-79797) and PAR-4 (P4pal10) inhibition. Pups in the long-term group were administered the selective mTOR inhibitor (rapamycin) with neurobehavioral and brain pathological examinations performed at 4 weeks. Pharmacological PAR-1, -4 antagonism normalized the increased mTOR expression following GMH. Early inhibition of mTOR by rapamycin improved long-term outcomes in rats. Mammalian-TOR signaling plays an important role in brain injury following neonatal GMH, possibly involving upstream PAR-1, -4 mechanisms.

Protease-activated receptor 1 and 4 signal inhibition reduces preterm neonatal hemorrhagic brain injury.

BACKGROUND AND PURPOSE: This study examines the role of thrombin's protease-activated receptor (PAR)-1, PAR-4 in mediating cyclooxygenase-2 and mammalian target of rapamycin after germinal matrix hemorrhage. METHODS: Germinal matrix hemorrhage was induced by intraparenchymal infusion of bacterial collagenase into the right ganglionic eminence of P7 rat pups. Animals were treated with PAR-1, PAR-4, cyclooxygenase-2, or mammalian target of rapamycin inhibitors by 1 hour, and ≤5 days. RESULTS: We found increased thrombin activity 6 to 24 hours after germinal matrix hemorrhage, and PAR-1, PAR-4, inhibition normalized cyclooxygenase-2, and mammalian target of rapamycin by 72 hours. Early treatment with NS398 or rapamycin substantially improved long-term outcomes in juvenile animals. CONCLUSIONS: Suppressing early PAR signal transduction, and postnatal NS398 or rapamycin treatment, may help reduce germinal matrix hemorrhage severity in susceptible preterm infants.

Neonatal brain hemorrhage (NBH) of prematurity: translational mechanisms of the vascular-neural network.

Neonatal brain hemorrhage (NBH) of prematurity is an unfortunate consequence of preterm birth. Complications result in shunt dependence and long-term structural changes such as posthemorrhagic hydrocephalus, periventricular leukomalacia, gliosis, and neurological dysfunction. Several animal models are available to study this condition, and many basic mechanisms, etiological factors, and outcome consequences, are becoming understood. NBH is an important clinical condition, of which treatment may potentially circumvent shunt complication, and improve functional recovery (cerebral palsy, and cognitive impairments). This review highlights key pathophysiological findings of the neonatal vascular-neural network in the context of molecular mechanisms targeting the posthemorrhagic hydrocephalus affecting this vulnerable infant population.

Cannabinoid type 2 receptor stimulation attenuates brain edema by reducing cerebral leukocyte infiltration following subarachnoid hemorrhage in rats.

Early brain injury (EBI), following subarachnoid hemorrhage (SAH), comprises blood-brain barrier (BBB) disruption and consequent edema formation. Peripheral leukocytes can infiltrate the injured brain, thereby aggravating BBB leakage and neuroinflammation. Thus, anti-inflammatory pharmacotherapies may ameliorate EBI and provide neuroprotection after SAH. Cannabinoid type 2 receptor (CB2R) agonism has been shown to reduce neuroinflammation; however, the precise protective mechanisms remain to be elucidated. This study aimed to evaluate whether the selective CB2R agonist, JWH133 can ameliorate EBI by reducing brain-infiltrated leukocytes after SAH. Adult male Sprague-Dawley rats were randomly assigned to the following groups: sham-operated, SAH with vehicle, SAH with JWH133 (1.0mg/kg), or SAH with a co-administration of JWH133 and selective CB2R antagonist SR144528 (3.0mg/kg). SAH was induced by endovascular perforation, and JWH133 was administered 1h after surgery. Neurological deficits, brain water content, Evans blue dye extravasation, and Western blot assays were evaluated at 24h after surgery. JWH133 improved neurological scores and reduced brain water content; however, SR144528 reversed these treatment effects. JWH133 reduced Evans blue dye extravasation after SAH. Furthermore, JWH133 treatment significantly increased TGF-ß1 expression and prevented an SAH-induced increase in E-selectin and myeloperoxidase. Lastly, SAH resulted in a decreased expression of the tight junction protein zonula occludens-1 (ZO-1); however, JWH133 treatment increased the ZO-1 expression. We suggest that CB2R stimulation attenuates neurological outcome and brain edema, by suppressing leukocyte infiltration into the brain through TGF-ß1 up-regulation and E-selectin reduction, resulting in protection of the BBB after SAH.

Correlation between subacute sensorimotor deficits and brain edema in two mouse models of intracerebral hemorrhage.

Formation of brain edema after intracerebral hemorrhage (ICH) is highly associated with its poor outcome. However, the relationship between cerebral edema and behavioral deficits has not been thoroughly examined in the preclinical setting. Hence, this study aimed to evaluate the ability of common sensorimotor tests to predict the extent of brain edema in two mouse models of ICH. One hundred male CD-1 mice were subjected to sham surgery or ICH induction via intrastriatal injection of either autologous blood (30 µL) or bacterial collagenase (0.0375U or 0.075U). At 24 and 72 h after surgery, animals underwent a battery of behavioral tests, including the modified Garcia neuroscore (Neuroscore), corner turn test (CTT), forelimb placing test (FPT), wire hang task (WHT) and beam walking (BW). Brain edema was evaluated via the wet weight/dry weight method. Intrastriatal injection of autologous blood or bacterial collagenase resulted in a significant increase in brain water content and associated sensorimotor deficits (p<0.05). A significant correlation between brain edema and sensorimotor deficits was observed for all behavioral tests except for WHT and BW. Based on these findings, we recommend implementing the Neuroscore, CTT and/or FPT in preclinical studies of unilateral ICH in mice.

Isoflurane post-treatment ameliorates GMH-induced brain injury in neonatal rats.

BACKGROUND AND PURPOSE: This study investigated whether isoflurane ameliorates neurological sequelae after germinal matrix hemorrhage (GMH) through activation of the cytoprotective sphingosine kinase/sphingosine-1-phosphate receptor/Akt pathway. METHODS: GMH was induced in P7 rat pups by intraparenchymal infusion of bacterial collagenase (0.3 U) into the right hemispheric germinal matrix. GMH animals received 2% isoflurane either once 1 hour after surgery or every 12 hours for 3 days. Isoflurane treatment was then combined with sphingosine-1-phosphate receptor-1/2 antagonist VPC23019 or sphingosine kinase 1/2 antagonist N,N-dimethylsphingosine. RESULTS: Brain protein expression of sphingosine kinase-1 and phosphorylated Akt were significantly increased after isoflurane post-treatment, and cleaved caspase-3 was decreased at 24 hours after surgery, which was reversed by the antagonists. Isoflurane significantly reduced posthemorrhagic ventricular dilation and improved motor, but not cognitive, functions in GMH animals 3 weeks after surgery; no improvements were observed after VPC23019 administration. CONCLUSIONS: Isoflurane post-treatment improved the neurological sequelae after GMH possibly by activation of the sphingosine kinase/Akt pathway.

Early brain injury, an evolving frontier in subarachnoid hemorrhage research.

Subarachnoid hemorrhage (SAH), predominantly caused by a ruptured aneurysm, is a devastating neurological disease that has a morbidity and mortality rate higher than 50%. Most of the traditional in vivo research has focused on the pathophysiological or morphological changes of large-arteries after intracisternal blood injection. This was due to a widely held assumption that delayed vasospasm following SAH was the major cause of delayed cerebral ischemia and poor outcome. However, the results of the CONSCIOUS-1 trial implicated some other pathophysiological factors, independent of angiographic vasospasm, in contributing to the poor clinical outcome. The term early brain injury (EBI) has been coined and describes the immediate injury to the brain after SAH, before onset of delayed vasospasm. During the EBI period, a ruptured aneurysm brings on many physiological derangements such as increasing intracranial pressure (ICP), decreased cerebral blood flow (CBF), and global cerebral ischemia. These events initiate secondary injuries such as blood-brain barrier disruption, inflammation, and oxidative cascades that all ultimately lead to cell death. Given the fact that the reversal of vasospasm does not appear to improve patient outcome, it could be argued that the treatment of EBI may successfully attenuate some of the devastating secondary injuries and improve the outcome of patients with SAH. In this review, we provide an overview of the major advances in EBI after SAH research.

PHA-543613 preserves blood-brain barrier integrity after intracerebral hemorrhage in mice.

BACKGROUND AND PURPOSE: Blood-brain barrier disruption and consequent vasogenic edema formation codetermine the clinical course of intracerebral hemorrhage (ICH). This study examined the effect of PHA-543613, a novel α7 nicotinic acetylcholine receptor agonist, on blood-brain barrier preservation after ICH. METHODS: Male CD-1 mice, subjected to intrastriatal blood infusion, received PHA-543613 alone or in combination with α7 nicotinic acetylcholine receptor antagonist methyllycaconitine or phosphatidylinositol 3-kinase inhibitor wortmannin. RESULTS: PHA-543613 alone, but not in combination with methyllycaconitine or wortmannin, inhibited glycogen synthase kinase-3ß, thus, stabilizing ß-catenin and tight junction proteins, which was paralleled by improved blood-brain barrier stability and ameliorated neurofunctional deficits in ICH animals. CONCLUSIONS: PHA-543613 preserved blood-brain barrier integrity after ICH, possibly through phosphatidylinositol 3-kinase-Akt-induced inhibition of glycogen synthase kinase-3ß and ß-catenin stabilization.

Fingolimod reduces cerebral lymphocyte infiltration in experimental models of rodent intracerebral hemorrhage.

T-lymphocytes promote cerebral inflammation, thus aggravating neuronal injury after stroke. Fingolimod, a sphingosine 1-phosphate receptor analog, prevents the egress of lymphocytes from primary and secondary lymphoid organs. Based on these findings, we hypothesized fingolimod treatment would reduce the number of T-lymphocytes migrating into the brain, thereby ameliorating cerebral inflammation following experimental intracerebral hemorrhage (ICH). We investigated the effects of fingolimod in two well-established murine models of ICH, implementing intrastriatal infusions of either bacterial collagenase (cICH) or autologous blood (bICH). Furthermore, we tested the long term neurological improvements by Fingolimod in a collagenase-induced rat model of ICH. Fingolimod, in contrast to vehicle administration alone, improved neurological functions and reduced brain edema at 24 and 72 h following experimental ICH in CD-1 mice (n=103; p<0.05). Significantly fewer lymphocytes were found in blood and brain samples of treated animals when compared to the vehicle group (p<0.05). Moreover, fingolimod treatment significantly reduced the expression of intercellular adhesion molecule-1 (ICAM-1), interferon-γ (INF-γ), and interleukin-17 (IL-17) in the mouse brain at 72 h post-cICH (p<0.05 compared to vehicle). Long-term neurocognitive performance and histopathological analysis were evaluated in Sprague-Dawley rats between 8 and 10 weeks post-cICH (n=28). Treated rats showed reduced spatial and motor learning deficits, along with significantly reduced brain atrophy and neuronal cell loss within the basal ganglia (p<0.05 compared to vehicle). We conclude that fingolimod treatment ameliorated cerebral inflammation, at least to some extent, by reducing the availability and subsequent brain infiltration of T-lymphocytes, which improved the short and long-term sequelae after experimental ICH in rodents.

Delayed hyperbaric oxygen therapy induces cell proliferation through stabilization of cAMP responsive element binding protein in the rat model of MCAo-induced ischemic brain injury.

Treatments that could extend the therapeutic window of opportunity for stroke patients are urgently needed. Early administration of hyperbaric oxygen therapy (HBOT) has been proven neuroprotective in the middle cerebral artery occlusion (MCAo) in rodents. Our aim was to determine: 1) whether delayed HBOT after permanent MCAo (pMCAo) can still convey neuroprotection and restorative cell proliferation, and 2) whether these beneficial effects rely on HBO-induced activation of protein phosphatase-1γ (PP1-γ) leading to a decreased phosphorylation and ubiquitination of CREB and hence its stabilization. The experiments were performed in one hundred thirty-two male Sprague-Dawley rats with the body weight ranging from 240 to 270 g. Permanent MCAo was induced with the intraluminal filament occluding the right middle cerebral artery (MCA). In the first experiment, HBOT (2.5 ATA, 1h daily for 10 days) was started 48 h after pMCAo. Neurobehavioral deficits and infarct size as well as cyclic AMP response element-binding protein (CREB) expression and BrdU-DAB staining in the hippocampus and the peri-infarct region were evaluated on day 14 and day 28 post-MCAo. In the second experiment, HBOT (2.5 ATA, 1h) was started 3h after pMCAo. The effects of CREB siRNA or PP1-γ siRNA on HBO-induced infarct size alterations and target protein expression were studied. HBOT started with 48 h delay reduced infarct size, ameliorated neurobehavioral deficits and increased protein expression of CREB, resulting in increased cell proliferations in the hippocampus and peri-infarct region, on day 14 and day 28 post-MCAo. In the acute experiment pMCAo resulted in cerebral infarction and functional deterioration and reduced brain expression of PP1-γ, which led to increased phosphorylation and ubiquitination of CREB 24h after MCAo. However HBOT administered 3h after ischemia reversed these molecular events and resulted in CREB stabilization, infarct size reduction and neurobehavioral improvement. Gene silencing with CREB siRNA or PP1-γ siRNA reduced acute beneficial effects of HBO. In conclusion, delayed daily HBOT presented as potent neuroprotectant in pMCAo rats, increased CREB expression and signaling activity, and bolstered regenerative type cell proliferation in the injured brain. As shown in the acute experiment these effects of HBO were likely to be mediated by reducing ubiquitin-dependent CREB degradation owing to HBO-induced activation of PP1γ.

Modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase.

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date(1,2). Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies(3). Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase(4). The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours(5). Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood(6) as well as an ICH injection model of bacterial collagenase(7), both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 µl) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p<0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Etiology of stroke and choice of models.

Animal models of stroke contribute to the development of better stroke prevention and treatment through studies investigating the pathophysiology of different stroke subtypes and by testing promising treatments before trials in humans. There are two broad types of animal models: those in which stroke is induced through artificial means, modeling the consequences of a vascular insult but not the vascular pathology itself; and those in which strokes occur spontaneously. Most animal models of stroke are in rodents due to cost, ethical considerations, availability of standardized neurobehavioral assessments, and ease of physiological monitoring. While there are similarities in cerebrovascular anatomy and pathophysiology between rodents and humans, there are also important differences, including brain size, length and structure of perforating arteries, and gray to white matter ratio, which is substantially lower in humans. The wide range of rodent models of stroke includes models of global and focal ischemia, and of intracerebral and sub-arachnoid hemorrhage. The most widely studied model of spontaneous stroke is the spontaneously hypertensive stroke-prone rat, in which the predominant lesions are small subcortical infarcts resulting from a vascular pathology similar to human cerebral small vessel disease. Important limitations of animal models of stroke - they generally model only certain aspects of the disease and do not reflect the heterogeneity in severity, pathology and comorbidities of human stroke - and key methodological issues (especially the need for adequate sample size, randomization, and blinding in treatment trials) must be carefully considered for the successful translation of pathophysiological concepts and therapeutics from bench to bedside.

Fibroblast growth factors preserve blood-brain barrier integrity through RhoA inhibition after intracerebral hemorrhage in mice.

Fibroblast growth factors (FGFs) maintain and promote vascular integrity; however whether FGFs protect the blood-brain barrier (BBB) after intracerebral hemorrhage (ICH) remains unexplored. In this present study, we hypothesized that exogenous FGF administration attenuates brain injury after ICH, specifically by preserving endothelial adherens junctions, therefore reducing vasogenic brain edema and attenuating neurofunctional deficits in mice subjected to experimental ICH. Acid fibroblast growth factor (FGF1) or basic fibroblast growth factor (FGF2) was administered intracerebroventricularly (ICV) at 0.5 h after intrastriatal injection of bacterial collagenase (cICH) or autologous whole blood (bICH). Fibroblast growth factor receptor (FGFR) inhibitor PD173074 and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were additionally administered with FGF2. The selective Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) inhibitor Y27632 was independently administered at 0.5 h after cICH. Brain water content and neurofunctional deficits were evaluated at 24 and 72h after ICH induction. Evans blue extravasation as well as Western blot analysis for the quantification of activated FGFR, Akt, Ras-related C3 botulinum toxin substrate 1 (Rac1), Ras homolog gene family member A (RhoA) and adherens junction proteins (p120-catenin, ß-catenin and VE-cadherin) were conducted at 72 h post-cICH. FGF treatment reduced perihematomal brain edema and improved neurofunctional deficits at 72 h after experimental ICH (p<0.05, compared to vehicle); however, FGFR and PI3K inhibition reversed these neuroprotective effects. Exogenous FGF2 increased activated FGFR, Akt, and Rac1 but reduced activated RhoA protein expression at 72 h after cICH (p<0.05, compared to vehicle), which was reversed by FGFR and PI3K inhibition. Y27632 treatment reduced brain injury at 72 h after cICH (p<0.05, compared to vehicle) and increased the expression of catenins (p120-catenin, ß-catenin). In conclusion, our findings suggest that exogenous FGF treatment reduced RhoA activity via FGFR-induced activation of the PI3K-Akt-Rac1 signaling pathway, thus preserving BBB integrity, and therefore attenuating secondary brain injury after experimental ICH in mice.