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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 euthanized at four 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 euthanized at 72 hours 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 hours post-surgery. At 72 hours 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 hours 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. This article is protected by copyright. All rights reserved.

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.

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 whetherα7 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 eitherα7nAChR agonist PHA-543613 alone or combined withα7nAChR 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-αand 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.

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.

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 (3.0 mg/kg). SAH was induced by endovascular perforation and all reagents were administered 1 hour after surgery. Neurological deficits, brain water content, Evans blue dye extravasation, and Western blot assays were evaluated at 24 hours 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-β1 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-β1 up-regulation and E-selectin reduction, resulting in protection of the BBB after SAH.

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.

Although there is evidence that sphingosine-1-phosphate receptor-1 (S1P1) activation occurs following experimental brain injury, there is little information about its metabolic pathway in cerebral ischemia. The purpose of this study was to evaluate the role of the sphingosine metabolic pathway including S1P1, sphingosine kinases 1 (SphK1), and 2 (SphK2) in transient middle cerebral artery occlusion (MCAO). Fifty-eight male Sprague-Dawley rats were used to asses temporal profiles of S1P1, SphK1 and 2 on neurons in infarct and periinfarct cortices at pre-infarct state, 6, and 24 hours after MCAO. The animals were then treated with vehicle and 0.25 mg/kg FTY720, which is an agonist of S1P receptors, and evaluated regarding neurological function, infarct volume, and S1P1 expression on neurons at 24 hours after MCAO. The expressions of S1P1, SphK1, and SphK2 were significantly decreased after MCAO. Labeling of all markers were reduced in the infarct cortex but remained present in the periinfarct cortex, and some were found to be on neurons. Significant improvements of neurological function and brain injury were observed in the FTY720 group compared with the vehicle and untreated groups, although S1P1 expression on neurons was reduced in the FTY720 group compared with the vehicle group. We demonstrated that S1P1, SphK1, and SphK2 were downregulated in the infarct cortex, whereas they were preserved in the periinfarct cortex where FTY720 reduced neuronal injury possibly via S1P1 activation. Our findings suggest that activation of the sphingosine metabolic pathway may be neuroprotective in cerebral ischemia.

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.

Object Primary pontine hemorrhage (PPH) represents approximately 7% of all intracerebral hemorrhages (ICHs) and is a clinical condition of which little is known. The aim of this study was to characterize the early brain injury, neurobehavioral outcome, and long-term histopathology in a novel preclinical rat model of PPH. Methods The authors stereotactically infused collagenase (Type VII) into the ventral pontine tegmentum of the rats, in accordance with the most commonly affected clinical region. Measures of cerebrovascular permeability (brain water content, hemoglobin assay, Evans blue, collagen Type IV, ZO-1, and MMP-2 and MMP-9) and neurological deficit were quantified at 24 hours postinfusion (Experiment 1). Functional outcome was measured over a 30-day period using a vertebrobasilar scale (the modified Voetsch score), open field, wire suspension, beam balance, and inclined-plane tests (Experiment 2). Neurocognitive ability was determined at Week 3 using the rotarod (motor learning), T-maze (working memory), and water maze (spatial learning and memory) (Experiment 3), followed by histopathological analysis 1 week later (Experiment 4). Results Stereotactic collagenase infusion caused dose-dependent elevations in hematoma volume, brain edema, neurological deficit, and blood-brain barrier rupture, while physiological variables remained stable. Functional outcomes mostly normalized by Week 3, whereas neurocognitive deficits paralleled the cystic cavitary lesion at 30 days. Obstructive hydrocephalus did not develop despite a clinically relevant 30-day mortality rate (approximately 54%). Conclusions These results suggest that the model can mimic several translational aspects of pontine hemorrhage in humans and can be used in the evaluation of potential preclinical therapeutic interventions.

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.

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.

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.

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.

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.

Germinal matrix hemorrhage (GMH) is the most common neurological disease of premature newborns. GMH causes neurological sequelae such as cerebral palsy, post-hemorrhagic hydrocephalus, and mental retardation. Despite this, there is no standardized animal model of spontaneous GMH using newborn rats to depict the condition. We asked whether stereotactic injection of collagenase type VII (0.3 U) into the ganglionic eminence of neonatal rats would reproduce the acute brain injury, gliosis, hydrocephalus, periventricular leukomalacia, and attendant neurological consequences found in humans. To test this hypothesis, we used our neonatal rat model of collagenase-induced GMH in P7 pups, and found that the levels of free-radical adducts (nitrotyrosine and 4-hyroxynonenal), proliferation (mammalian target of rapamycin), inflammation (COX-2), blood components (hemoglobin and thrombin), and gliosis (vitronectin and GFAP) were higher in the forebrain of GMH pups, than in controls. Neurobehavioral testing showed that pups with GMH had developmental delay, and the juvenile animals had significant cognitive and motor disability, suggesting clinical relevance of the model. There was also evidence of white-matter reduction, ventricular dilation, and brain atrophy in the GMH animals. This study highlights an instructive animal model of the neurological consequences after germinal matrix hemorrhage, with evidence of brain injuries that can be used to evaluate strategies in the prevention and treatment of post-hemorrhagic complications.

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, β-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

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.

Subarachnoid hemorrhage (SAH) results in significant long-lasting cognitive dysfunction. Therefore, evaluating acute and long-term outcomes after therapeutic intervention is important for clinical translation. The aim of this study was to use minocycline, a known neuroprotectant agent, to evaluate the long-term benefits in terms of neurobehavior and neuropathology after experimental SAH in rats, and to determine which neurobehavioral test would be effective for long-term evaluation. SAH was induced by endovascular perforation in adult male Sprague-Dawley rats (n=118). The animals were treated with intraperitoneal injection of minocycline (45 mg/kg or 135 mg/kg) or vehicle 1 h after SAH induction. In the short-term, animals were euthanized at 24 and 72 h for evaluation of neurobehavior, brain water content, and matrix metalloproteinase (MMP) activity. In the long-term, neurobehavior was evaluated at days 21–28 post-SAH, and histopathological analysis was done at day 28. High-dose but not low-dose minocycline reduced brain water content at 24 h, and therefore only the high-dose regimen was used for further evaluation, which reduced MMP-9 activity at 24 h. Further, high-dose minocycline improved spatial memory and attenuated neuronal loss in the hippocampus and cortex. The rotarod, T-maze, and water maze tests, but not the inclined plane test, detected neurobehavioral deficits in SAH rats at days 21–28. This study demonstrates that minocycline attenuates long-term functional and morphological outcomes after endovascular perforation-induced SAH. Long-term neurobehavioral assessments using the rotarod, T-maze, and water maze tests could be useful to evaluate the efficacy of therapeutic intervention after experimental SAH.

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.

The aim of this study was to evaluate whether sampling of cerebrospinal fluid (CSF) via the cisterna magna and of blood via the heart affects brain water content in a rat subarachnoid hemorrhage (SAH) model. Twenty-nine animals were divided into four groups: sham-operated group with sampling of CSF and blood (Sham S+), sham-operated group without sampling of CSF and blood (Sham S−), SAH group with sampling of CSF and blood (SAH S+), and SAH without sampling of CSF and blood (SAH S−). SAH was induced via endovascular perforation of the left internal carotid artery bifurcation. Cerebrospinal fluid via the cisterna magna and blood via cardiac puncture was collected in the Sham S+ and SAH S+ groups before killing the animals for brain water content measurements. Left hemisphere brain water content was significantly higher in the SAH S− group compared with the Sham S− group (p< 0.05) and in Sham S+ group compared with the Sham S− group (p

Background and Purpose—FTY720 is a known sphingosine 1–phosphate receptor agonist. In the present study, we investigated the neuroprotective effect of postischemic administration of FTY720 in rats with 2 hours transient middle cerebral artery occlusion (MCAO).Methods—One hundred eleven male rats were randomly assigned to sham-operated and MCAO treated with vehicle, 0.25 mg/kg and 1 mg/kg of FTY720, another selective sphingosine 1–phosphate receptor-1 agonist SEW2871 (5 mg/kg), or 0.25 mg/kg of FTY720 plus a sphingosine 1–phosphate antagonist, VPC23019 (0.5 mg/kg). Drugs were injected intraperitoneally immediately after reperfusion. Neurological score and infarct volume were assessed at 24 and 72 hours after MCAO. Western blotting, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labeling were conducted at 24 hours after MCAO.Results—FTY720 significantly reduced infarct volume and improved neurological score at 24 and 72 hours after MCAO compared with the vehicle group. SEW2871 showed similar neuroprotective effects to FTY720, whereas VPC 20319 abolished the neuroprotective effects of FTY720. FTY720 significantly retained Akt and extracellular signal-regulated kinase phosphorylation and Bcl-2 expression and decreased cleaved caspase-3 expression and terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labeling-positive neurons at 24 hours after MCAO. VPC23019 blocked the antiapoptotic effects of FTY720.Conclusions—These data suggest that activation of sphingosine 1–phosphate-1 by FTY720 reduces neuronal death after transient MCAO.

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.

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.

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.

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.

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.

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 post-hemorrhagic 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 post-hemorrhagic hydrocephalus affecting this vulnerable infant population.

Formation of brain edema after intracerebral hemorrhage (ICH) is highly associated with its poor outcome, thus it is clinically important to understand the effect brain edema has on 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 (0.0375 U or 0.075 U). At 24 and 72 hours 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

Chronic communicating hydrocephalus is a significant health problem affecting up to 20% of survivors of spontaneous subarachnoid hemorrhage (SAH). The development of new treatment strategies is hampered by the lack of well characterized disease models. This study investigated the incidence of chronic hydrocephalus by evaluating the temporal profile of intracranial pressure (ICP) elevation after SAH, induced by endovascular perforation in rats. Twenty-five adult male Sprague-Dawley rats (260-320 g) were subjected to either endovascular perforation or sham surgery. Five animals died after SAH induction. At 7, 14 and 21 days after surgery ICP was measured by stereotaxic puncture of the cisterna magna in SAH (n=10) and SHAM (n=10) animals. On day 21 T-maze test was performed and the number of alterations and latency to decision was recorded. On day 23, samples were processed for histological analyses. The relative ventricle area was evaluated in coronal Nissl stained sections. On day 7 after surgery all animals showed normal ICP. The absolute ICP values were significantly higher in SAH compared to SHAM animals on day 21 (8.26±4.53 mmHg versus 4.38±0.95 mmHg) but not on day 14. Observing an ICP of 10 mmHg as cut-off, 3 animals showed elevated ICP on day 14 and another animal on day 21. The overall incidence of ICP elevation was 40% in SAH animals. On day 21, results of T-maze testing were significantly correlated with ICP values, i.e. animals with elevated ICP showed a lower number of alterations and a delayed decision. Histology yielded a significantly higher (3.59 fold increased) relative ventricle area in SAH animals with ICP elevation compared to SAH animals without ICP elevation. In conclusion, the current study shows that experimental SAH leads to chronic hydrocephalus, which is associated with ICP elevation, behavioral alterations and ventricular dilation in about 40% of SAH animals.

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-3β, thus, stabilizing β-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-3β and β-catenin stabilization.

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 cytokines IL-1β/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 24 hours before SAH. Brilliant Blue G (BBG), a non-competitive antagonist of P2X7R, was administered intraperitoneally 30 minutes 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 24 hours following SAH. Inhibition of P2X7R/cryopyrin inflammasome axis suppressed caspase-1 activation, which subsequently decreased maturation of IL-1β/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 naïve rats, BzATP induced caspase-1 activation and mature IL-1β release was neutralized by cryopyrin siRNA. Thus, the P2X7R/cryopyrin inflammasome axis may contribute to neuroinflammation via activation of caspase-1 and thereafter mature IL-1β/IL-18 production following SAH. Therapeutic interventions targeting P2X7R/cryopyrin pathway may be a novel approach to ameliorate EBI following SAH.

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 hours following experimental ICH in CD-1 mice (n=103; p

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, 1 h 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, 1 h) was started 3 h 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 24 h after MCAo. However HBOT administered 3 h 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.

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.

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-3β (GSK-3β) 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-3β (p-GSK-3β), 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 P P Conclusions— α7nAChR stimulation improved functional and morphological outcomes after experimental ICH in mice. PHA-543613 reduced the expression of proapoptotic GSK-3β through the PI3K-Akt signaling pathway.

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.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.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.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.

Intracerebral hemorrhage (ICH) accounts for 20% of all strokes and is the most devastating form across all stroke types. Lymphocytes have been shown to potentiate cerebral inflammation and brain injury after stroke. FTY720 (Fingolimod) is an immune-modulating drug that prevents the egress of peripheral lymphocytes from peripheral stores. We hypothesized that FTY720 would reduce peripheral circulating lymphocytes, resulting in reduced brain injury and improved functional outcomes. CD-1 mice were anesthetized and then injected with collagenase into the right basal ganglia. Animals were divided into three groups: sham, ICH + Vehicle, and ICH + FTY720, by the intra-peritoneal route at 1 h after ICH induction. Brain water content was measured at 24 and 72 h. Neurobehavioral tests included corner test, forelimb use asymmetry, paw placement, wire-hang test, beam balance test, and a Neuroscore. FTY720 significantly reduced brain edema and improved neurological function at all time points tested. Lymphocyte modulation with FTY720 is an effective neuroprotective strategy to reduce brain injury and promote functional recovery after ICH.

Germinal matrix hemorrhage (GMH) is a neurological disorder associated with very low birth weight premature infants. This event can lead to post-hemorrhagic hydrocephalus, cerebral palsy, and mental retardation. This study developed a novel animal model for pre-clinical investigations.Neonatal rats underwent infusion of clostridial collagenase into the right germinal matrix (anterior caudate) region using stereotaxic techniques. Developmental milestones were evaluated over 10 days, cognitive function at 3 weeks, and sensorimotor function at 4 weeks after collagenase infusion. This was accomplished by anthropometric quantifications of cranial, cerebral, cardiac, and splenic growths.Collagenase infusion led to delays in neonatal developmental milestones, followed by cognitive and sensorimotor dysfunctions in the juvenile animals. Cranial growth was accelerated during the first week after injury, and this was followed by significant brain atrophy, splenomegaly, and cardiac hypertrophy 3 weeks later.This study characterized the developmental delays, mental retardation, and cerebral palsy features resembling the long-term clinical course after germinal matrix hemorrhage in premature infants. Pre-clinical testing of therapeutics in this experimental model could lead to improved patient outcomes while expanding upon the pathophysiological understanding of this disease.

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.

Intracerebral hemorrhage (ICH) is one of the most common causes of maternal deaths related to the postpartum period. This is a devastating form of stroke for which there is no available treatment. Although premenopausal females tend to have better outcomes after most forms of brain injury, the effects of pregnancy and child birth lead to wide maternal physiological changes that may predispose the mother to an increased risk for stroke and greater initial injury.Three different doses of collagenase were used to generate models of mild, moderate and severe cerebellar hemorrhage in postpartum female and male control rats. Brain water, blood-brain barrier rupture, hematoma size and neurological evaluations were performed 24 h later.Postpartum female rats had worsened brain water, blood-brain barrier rupture, hematoma size and neurological evaluations compared to their male counterparts.The postpartum state reverses the cytoprotective effects commonly associated with the hormonal neuroprotection of (premenopausal) female gender, and leads to greater initial injury and worsened neurological function after cerebellar hemorrhage. This experimental model can be used for the study of future treatment strategies after postpartum brain hemorrhage, to gain a better understanding of the mechanistic basis for stroke in this important patient subpopulation.

Germinal matrix hemorrhage (GMH) is a neurological disease of very low birth weight premature infants leading to post-hemorrhagic hydrocephalus, cerebral palsy, and mental retardation. Hydrogen (H2) is a potent antioxidant shown to selectively reverse cytotoxic oxygen-radical injury in the brain. This study investigated the therapeutic effect of hydrogen gas after neonatal GMH injury.Neonatal rats underwent stereotaxic infusion of clostridial collagenase into the right germinal matrix brain region. Cognitive function was assessed at 3 weeks, and then sensorimotor function, cerebral, cardiac and splenic growths were measured 1 week thereafter.Hydrogen gas inhalation markedly suppressed mental retardation and cerebral palsy outcomes in rats at the juvenile developmental stage. The administration of H2 gas, early after neonatal GMH, also normalized the brain atrophy, splenomegaly and cardiac hypertrophy 1 month after injury.This study supports the role of cytotoxic oxygen-radical injury in early neonatal GMH. Hydrogen gas inhalation is an effective strategy to help protect the infant brain from the post-hemorrhagic consequences of brain atrophy, mental retardation and cerebral palsy. Further studies are necessary to determine the mechanistic basis of these protective effects.

Cerebral hypoxia-ischemia (HI) is an important cause of mortality and disability in newborns. It is a result of insufficient oxygen and glucose circulation to the brain, initiating long-term cerebral damage and cell death. Emerging evidence suggests that endothelin receptor-A (ETA) activation can play an important role in mediating brain damage. In this study, we investigated the role of ETA receptor inhibition using ABT-627 in neonatal HI injured rats. Postnatal day 10 Sprague-Dawley rat pups (n = 91) were assigned to the following groups: sham (n = 28), HI (vehicle, n = 32), and HI with ABT-627 at 3 mg/kg (n = 31). The Rice-Vannucci model was used to induce ischemia by ligating the right common carotid artery, followed by a 2 h hypoxic episode using 8% oxygen in a 37°C chamber. Postoperative assessment was conducted at 48 h after injury and again at 4 weeks. At the acute time point, investigative markers included cerebral edema, infarction volume, and body weight change. Neurobehavioral testing was measured at 4 weeks post-injury. Our findings indicated that ABT-627 had no effect on the measured parameters. This study suggests that ETA receptor blockade using ABT-627 post-treatment fails to improve neurological outcomes in neonatal HI injured rats.

Germinal matrix hemorrhage (GMH) is a devastating neurological disorder of very low birth weight premature infants that leads to post-hemorrhagic hydrocephalus, cerebral palsy, and mental retardation. Melatonin is a potent antioxidant known to reverse free-radical mediated injury in the brain. This study investigated the effect of melatonin treatment after GMH injury.Clostridial collagenase was infused into the right germinal matrix region of neonatal rats with stereotaxic technique. Cognitive function, sensorimotor ability, cerebral, cardiac and splenic growths were measured in juvenile animals.Systemic melatonin treatment ameliorated cognitive and sensorimotor dysfunction at the juvenile developmental stage. This hormone also normalized brain atrophy, splenomegaly, and cardiac hypertrophy consequences at 1 month after injury.This study supports the role of free radicals in acute neonatal hemorrhagic brain injury. Melatonin is an effective antioxidant that can protect the infant's brain from the post-hemorrhagic consequences of mental retardation and cerebral palsy. Further mechanistic studies are warranted to determine the mechanisms behind these neuroprotective effects.