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.

Epsilon Aminocaproic Acid Pretreatment Provides Neuroprotection Following Surgically Induced Brain Injury in a Rat Model.

Neurosurgical procedures can damage viable brain tissue unintentionally by a wide range of mechanisms. This surgically induced brain injury (SBI) can be a result of direct incision, electrocauterization, or tissue retraction. Plasmin, a serine protease that dissolves fibrin blood clots, has been shown to enhance cerebral edema and hemorrhage accumulation in the brain through disruption of the blood brain barrier. Epsilon aminocaproic acid (EAA), a recognized antifibrinolytic lysine analogue, can reduce the levels of active plasmin and, in doing so, potentially can preserve the neurovascular unit of the brain. We investigated the role of EAA as a pretreatment neuroprotective modality in a SBI rat model, hypothesizing that EAA therapy would protect brain tissue integrity, translating into preserved neurobehavioral function. Male Sprague-Dawley rats were randomly assigned to one of four groups: sham (n = 7), SBI (n = 7), SBI with low-dose EAA, 150 mg/kg (n = 7), and SBI with high-dose EAA, 450 mg/kg (n = 7). SBI was induced by partial right frontal lobe resection through a frontal craniotomy. Postoperative assessment at 24 h included neurobehavioral testing and measurement of brain water content. Results at 24 h showed both low- and high-dose EAA reduced brain water content and improved neurobehavioral function compared with the SBI groups. This suggests that EAA may be a useful pretherapeutic modality for SBI. Further studies are needed to clarify optimal therapeutic dosing and to identify mechanisms of neuroprotection in rat SBI models.

Valproic Acid Pretreatment Reduces Brain Edema in a Rat Model of Surgical Brain Injury.

Surgically induced brain injury (SBI) results in brain edema and neurological decline. Valproic acid (VA) has been shown to be neuroprotective in several experimental brain diseases. In this study, we investigated the pretreatment effect of VA in a rat model of SBI. A total of 57 male Sprague-Dawley rats were use in four groups: sham, SBI + vehicle, SBI + low dose (100 mg/kg) VA, and SBI + high dose (300 mg/kg) VA. SBI was induced by partially resecting right frontal lobes. Shams underwent identical surgical procedures without brain resection. VA or vehicle was administered subcutaneously 30 min prior to SBI. At 24 and 72 h post SBI, neurobehavior and brain water content were assessed as well as matrix metalloproteinases (MMPs) activities. There was significantly higher brain water content within the right frontal lobe in SBI rats than in shams. Without neurobehavioral improvements, the low-dose but not high-dose VA significantly reduced brain edema at 24 h post SBI. The protection tends to persist to 72 h post SBI. At 24 h post SBI, low-dose VA did not significantly reduce the elevated MMP-9 activity associated with SBI. In conclusion, VA pretreatment attenuated brain edema at 24 h after SBI but lacked MMP inhibition. The single dose VA was not associated with neurobehavioral benefits.

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.

Acute and delayed deferoxamine treatment attenuates long-term sequelae after germinal matrix hemorrhage in neonatal rats.

BACKGROUND AND PURPOSE: This study investigated if acute and delayed deferoxamine treatment attenuates long-term sequelae after germinal matrix hemorrhage (GMH). METHODS: Bacterial collagenase (0.3 U) was infused intraparenchymally into the right hemispheric ganglionic eminence in P7 rat pups to induce GMH. GMH animals received either deferoxamine or vehicle twice a day for 7 consecutive days. Deferoxamine administration was initiated at either 1 hour or 72 hours post-GMH. Long-term neurocognitive deficits and motor coordination were assessed using Morris water maze, rotarod, and foot fault tests between day 21 to 28 post-GMH. At 28 days post-GMH, brain morphology was assessed and extracellular matrix protein (fibronectin and vitronectin) expression was determined. RESULTS: Acute and delayed deferoxamine treatment improved long-term motor and cognitive function at 21 to 28 days post-GMH. Attenuated neurofunction was paralleled with improved overall brain morphology at 28 days post-GMH, reducing white matter loss, basal ganglia loss, posthemorrhagic ventricular dilation, and cortical loss. GMH resulted in significantly increased expression of fibronectin and vitronectin, which was reversed by acute and delayed deferoxamine treatment. CONCLUSIONS: Acute and delayed deferoxamine administration ameliorated long-term sequelae after GMH.

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.

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.

P2X7R/cryopyrin inflammasome axis inhibition reduces neuroinflammation after SAH.

Neuroinflammation contributes to the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Cytotoxic events following SAH, such as extracellular accumulation of adenosine triphosphate (ATP), may activate the P2X purinoceptor 7 (P2X7R)/cryopyrin inflammasome axis, thus inducing the proinflammatory cytokine IL-1ß/IL-18 secretion. We therefore hypothesized that inhibition of P2X7R/cryopyrin inflammasome axis would ameliorate neuroinflammation after SAH. In the present study, SAH was induced by the endovascular perforation in rats. Small interfering RNAs (siRNAs) of P2X7R or cryopyrin were administered intracerebroventricularly 24h before SAH. Brilliant blue G (BBG), a non-competitive antagonist of P2X7R, was administered intraperitoneally 30min following SAH. Post-assessments including SAH severity score, neurobehavioral test, brain water content, Western blot and immunofluorescence, were performed. Administration of P2X7R and cryopyrin siRNA as well as pharmacologic blockade of P2X7R by BBG ameliorated neurological deficits and brain edema at 24h following SAH. Inhibition of P2X7R/cryopyrin inflammasome axis suppressed caspase-1 activation, which subsequently decreased maturation of IL-1ß/IL-18. To investigate the link between P2X7R and cryopyrin inflammasome in vivo, Benzoylbenzoyl-ATP (BzATP), a P2X7R agonist, was given to lipopolysaccharide (LPS) primed naive rats with scramble or cryopyrin siRNAs. In LPS-primed naive rats, BzATP induced caspase-1 activation and mature IL-1ß release were neutralized by cryopyrin siRNA. Thus, the P2X7R/cryopyrin inflammasome axis may contribute to neuroinflammation via activation of caspase-1 and thereafter mature IL-1ß/IL-18 production following SAH. Therapeutic interventions targeting P2X7R/cryopyrin pathway may be a novel approach to ameliorate EBI following SAH.

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.

α7 nicotinic acetylcholine receptor agonism confers neuroprotection through GSK-3ß inhibition in a mouse model of intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Perihematomal edema formation and consequent cell death contribute to the delayed brain injury evoked by intracerebral hemorrhage (ICH). We aimed to evaluate the effect of α7 nicotinic acetylcholine receptor (α7nAChR) stimulation on behavior, brain edema, and neuronal apoptosis. Furthermore, we aimed to determine the role of the proapoptotic glycogen synthase kinase-3ß (GSK-3ß) after experimental ICH. METHODS: Male CD-1 mice (n=109) were subjected to intracerebral infusion of autologous blood (n=88) or sham surgery (n=21). ICH animals received vehicle administration, 4 or 12 mg/kg of α7nAChR agonist PHA-543613, 12 mg/kg of α7nAChR agonist PNU-282987, 6 mg/kg of α7nAChR antagonist methyllycaconitine (MLA), 15 µg/kg of phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, or PHA-543613 combined with MLA or wortmannin. Behavioral deficits and brain water content were evaluated at 24 and 72 hours after surgery. Western blotting and immunofluorescence staining were used for the quantification and localization of activated Akt (p-Akt), GSK-3ß (p-GSK-3ß), and cleaved caspase-3 (CC3). Neuronal cell death was quantified through terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). RESULTS: α7nAChR stimulation improved neurological outcome and reduced brain edema at 24 and 72 hours after surgery (P<0.05 compared with vehicle). Furthermore, PHA-543613 treatment increased p-Akt and decreased p-GSK-3ß and CC3 expressions in the ipsilateral hemisphere (P<0.05, respectively), which was reversed by MLA and wortmannin. P-Akt, p-GSK-3ß, and CC3 were generally localized in neurons. PHA-543613 reduced neuronal cell death in the perihematomal area (P<0.05). CONCLUSIONS: α7nAChR stimulation improved functional and morphological outcomes after experimental ICH in mice. PHA-543613 reduced the expression of proapoptotic GSK-3ß through the PI3K-Akt signaling pathway.

Nanoerythropoietin is 10-times more effective than regular erythropoietin in neuroprotection in a neonatal rat model of hypoxia and ischemia.

BACKGROUND AND PURPOSE: Erythropoietin (EPO) has been demonstrated to possess significant neuroprotective effects in stroke. We determined if the nano-drug form of human recombinant EPO (PLGA-EPO nanoparticles [PLGA-EPO-NP]) can enhance neuroprotection at lower dosages versus human recombinant EPO (r-EPO). METHODS: Established neonatal rat model of unilateral ischemic stroke was used to compare r-EPO, PLGA-EPO-NP and phosphate-buffered saline, given by daily intraperitoneal injections, followed by infarction volume and Rotarod Performance Test assessment. RESULTS: PLGA-EPO-NP significantly reduced infarction volumes 72 hours after injury compared with the same concentrations of r-EPO. Functional deficits were significantly reduced by 300 U/kg PLGA-EPO-NP versus controls, with deficit attenuation apparent at significantly lower dosages of PLGA-EPO-NP versus r-EPO. CONCLUSIONS: PLGA-EPO-NP is neuroprotective and beneficial against deficits after brain ischemia, at significantly reduced dosages versus r-EPO.

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.

PDGFR-α inhibition preserves blood-brain barrier after intracerebral hemorrhage.

OBJECTIVE: Perihematomal edema results from disruption of the blood-brain barrier (BBB) by key mediators, such as thrombin, following intracerebral hemorrhage (ICH). Platelet-derived growth factor receptor alpha (PDGFR-α), a tyrosine kinase receptor, was found in previous studies to play a role in orchestrating BBB impairment. In the present study, we investigated the role of PDGFR-α following ICH-induced brain injury in mice, specifically investigating its effect on BBB disruption. METHODS: Brain injury was induced by autologous arterial blood (30 µl) or thrombin (5 U) injection into mice brains. A PDGFR antagonist (Gleevec) or agonist (PDGF-AA) was administered following ICH. PDGF-AA was injected with a thrombin inhibitor, hirudin, in ICH mice. Thrombin-injected mice were given Gleevec or PDGF-AA neutralizing antibody. A p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, was delivered with PDGF-AA in naïve animals. Postassessment included neurological function tests, brain edema measurement, Evans blue extravasation, immunoprecipitation, western blot, and immunohistology assay. RESULTS: PDGFR-α suppression prevented neurological deficits, brain edema, and Evans blue extravasation at 24 to 72 hours following ICH. PDGFR-α activation led to BBB impairment and this was reversed by SB203580 in naïve mice. Thrombin inhibition suppressed PDGFR-α activation and exogenous PDGF-AA increased PDGFR-α activation, regardless of thrombin inhibition. Animals receiving a PDGF-AA-neutralizing antibody or Gleevec showed minimized thrombin injection-induced BBB impairment. INTERPRETATION: PDGFR-α signaling may contribute to BBB impairment via p38 MAPK-mediated matrix metalloproteinase (MMP) activation/expression following ICH, and thrombin may be the key upstream orchestrator. The therapeutic interventions targeting the PDGFR-α signaling may be a novel strategy to prevent thrombin-induced BBB impairment following ICH.

α7 nicotinic acetylcholine receptor agonist PNU-282987 attenuates early brain injury in a perforation model of subarachnoid hemorrhage in rats.

BACKGROUND AND PURPOSE: Early brain injury is an important pathological process after subarachnoid hemorrhage (SAH). The goal of this study was to evaluate whether the α7 nicotinic acetylcholine receptor (α7nAChR) agonist PNU-282987 attenuates early brain injury after SAH and whether α7nAChR stimulation is associated with down-regulation of caspase activity via phosphatidylinositol 3-kinase-Akt signaling. METHODS: The perforation model of SAH was performed, and neurological score, body weight loss, and brain water content were evaluated 24 and 72 hours after surgery. Western blot and immunohistochemistry were used for quantification and localization of phosphorylated Akt and cleaved caspase 3. Neuronal cell death was quantified with TUNEL staining. α7nAChR antagonist methylcaconitine and phosphatidylinositol 3-kinase inhibitor wortmannin were used to manipulate the proposed pathway, and results were quantified with Western blot. RESULTS: PNU-282987 improved neurological deficits both 24 and 72 hours after surgery and reduced brain water content in left hemispheres 24 hours after surgery. PNU-282987 significantly increased phosphorylated Akt levels and significantly decreased cleaved caspase 3 levels in ipsilateral hemispheres after SAH. Methylcaconitine and wortmannin reversed effects of treatment. Phosphorylated Akt and cleaved caspase 3 were colocalized to neurons in the ipsilateral basal cortex. Phosphorylated Akt was mainly localized in TUNEL-negative cells. PNU-282987 significantly reduced neuronal cell death in the ipsilateral basal cortex. CONCLUSIONS: α7nAChR stimulation decreased neuronal cell death and brain edema and improved neurological status in a rat perforation model of SAH. α7nAChR stimulation is associated with increasing phosphorylation of Akt and decreasing cleaved caspase 3 levels in neurons.

Isoflurane posttreatment reduces brain injury after an intracerebral hemorrhagic stroke in mice.

BACKGROUND: Intracerebral hemorrhage (ICH) is a devastating stroke subtype affecting 120,000 Americans annually. Of those affected, 40%to 50% will die within the first 30 days, whereas the survivors are left with a lifetime of neurobehavioral disabilities. Recently, it has been shown that volatile anesthetics such as isoflurane can reduce brain injury after an ischemic stroke. As a result, in this study, we investigated the effects of isoflurane as a posttreatment therapeutic modality in ICH-injured mice. Specifically, we investigated whether isoflurane posttreatment can preserve the structural integrity of the brain by reducing apoptotic damage and, in turn, improve functional outcome by amelioration of brain edema and neurobehavioral deficits. METHODS: Male CD1 mice (n = 53) were divided into the following groups: sham (n = 14), ICH (n = 14), ICH treated with 1.5% isoflurane posttreatment for 1 hour (n = 15), and ICH treated with 1.5% isoflurane posttreatment for 2 hours (n = 10). The blood injection ICH model was adapted; this involved extracting autologous blood from the mouse tail and injecting it directly into the right basal ganglia. One hour after surgery, treated mice were placed in a glass chamber maintained at 37°C and infused with 1.5% isoflurane for 1 or 2 hours. At 24 hours postinjury, mice were assessed for neurobehavioral deficits using the Modified Garcia Score and then killed and assessed for brain water content. Double immunofluorescent staining was performed using neuronal marker MAP-2 and TUNEL under a fluorescent microscope to assess for apoptosis. RESULTS: Our results indicated that after 1-hour 1.5% isoflurane posttreatment, there was a significant reduction in brain edema, a decrease in apoptotic cell death, and a significant improvement in neurobehavioral deficits. CONCLUSIONS: Our results suggest that isoflurane may be an effective posttreatment therapeutic option for ICH because of its ability to reduce structural damage and subsequently preserve functional integrity.