A composite neurobehavioral test to evaluate acute functional deficits after cerebellar haemorrhage in rats.

Cerebellar haemorrhage accounts for 5-10% of all intracerebral haemorrhages and leads to severe, long-lasting functional deficits. Currently, there is limited research on this stroke subtype, which may be due to the lack of a suitable composite neuroscoring system specific for cerebellar injury in rodents. The purpose of this study is to develop a comprehensive composite neuroscore test for cerebellar injury using a rat model of cerebellar haemorrhage. Sixty male Sprague-Dawley rats were subjected to either sham surgery or cerebellar haemorrhage. Twenty-four hours post-injury, neurological behaviour was evaluated using 17 cost-effective and easy-to-perform tests, and a composite neuroscore was developed. The composite neuroscore was then used to assess functional recovery over seven days after cerebellar haemorrhage. Differences in the composite neuroscore deficits for the mild and moderate cerebellar haemorrhage models were observed for up to five days post-ictus. Until now, a composite neuroscore for cerebellar injury was not available for rodent studies. Herein, using mild and moderate cerebellar haemorrhage rat models a composite neuroscore for cerebellar injury was developed and used to assess functional deficits after cerebellar haemorrhage. This composite neuroscore may also be useful for other cerebellar injury models.

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

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

Crotalus helleri venom preconditioning reduces postoperative cerebral edema and improves neurological outcomes after surgical brain injury.

INTRODUCTION: Postoperative cerebral edema is a devastating complication in neurosurgical patients. Loss of blood-brain barrier integrity has been shown to lead to the development of brain edema following neurosurgical procedures. The aim of this study was to evaluate preconditioning with Crotalus helleri venom (Cv-PC) as a potential preventive therapy for reducing postoperative brain edema in the rodent SBI model. C. helleri venom is known to contain phospholipase A2 (PLA2), an enzyme upstream to cyclooxygenase-2 (COX-2) in the inflammatory cascade, acts to increase the production of inflammatory mediators, such as prostaglandins. We hypothesize that Cv-PC will downregulate the response of the COX-2 pathway to injury, thereby reducing the inflammatory response and the development of brain edema after SBI. MATERIALS AND METHODS: 75 male Sprague Dawley rats (280-330g) were divided to the following groups-naïve+vehicle, naïve+Cv-PC, sham, vehicle, Cv-PC, Cv-PC+NS398 (COX-2 inhibitor). Vehicle preconditioned and Cv-PC animals received either three daily subcutaneous doses of saline or C. helleri venom at 72h, 48h, and 24h prior to surgery. In Cv-PC+NS398 animals, NS398 was administered intraperitoneally 1h prior to each Cv-PC injection. Sham-operated animals received craniotomy only, whereas SBI animals received a partial right frontal lobectomy. Neurological testing and brain water content were assessed at 24h and 72h after SBI; COX-2 and PGE2 expression was assessed at 24h postoperatively by Western blot and immunohistochemistry, respectively. RESULTS: At 24h after SBI, the vehicle-treated animals were observed to have increased brain water content (83.1±0.2%) compared to that of sham animals (80.2±0.1%). The brain water content of vehicle-treated animals at 72h post-SBI was elevated at 83.3±0.2%. Cv-PC-treated animals with doses of 10% LD50 had significantly reduced brain water content of 81.92±0.7% and 81.82±0.3% at 24h and 72h, respectively, after SBI compared to that of vehicle-treated animals, while Cv-PC with 5% LD50 doses showed brain water content that trended lower but did not reach statistical significance. At 24h and 72h post-SBI, Cv-PC-treated animals had significantly higher neurological score than vehicle-treated animals. The COX-2 over-expression characterized in SBI was attenuated in Cv-PC-treated animals; NS398 reversed the protective effect of Cv-PC on COX-2 expression. Cv-PC tempered the over-expression of the inflammatory marker PGE2. CONCLUSION: Our findings indicate that Cv-PC may provide a promising therapy for reducing postoperative edema and improving neurological function after neurosurgical procedures.

Crotalus atrox venom preconditioning increases plasma fibrinogen and reduces perioperative hemorrhage in a rat model of surgical brain injury.

Perioperative bleeding is a potentially devastating complication in neurosurgical patients, and plasma fibrinogen concentration has been identified as a potential modifiable risk factor for perioperative bleeding. The aim of this study was to evaluate preconditioning with Crotalus atrox venom (Cv-PC) as potential preventive therapy for reducing perioperative hemorrhage in the rodent model of surgical brain injury (SBI). C. atrox venom contains snake venom metalloproteinases that cleave fibrinogen into fibrin split products without inducing clotting. Separately, fibrinogen split products induce fibrinogen production, thereby elevating plasma fibrinogen levels. Thus, the hypothesis was that preconditioning with C. atrox venom will produce fibrinogen spilt products, thereby upregulating fibrinogen levels, ultimately improving perioperative hemostasis during SBI. We observed that Cv-PC SBI animals had significantly reduced intraoperative hemorrhage and postoperative hematoma volumes compared to those of vehicle preconditioned SBI animals. Cv-PC animals were also found to have higher levels of plasma fibrinogen at the time of surgery, with unchanged prothrombin time. Cv-PC studies with fractions of C. atrox venom suggest that snake venom metalloproteinases are largely responsible for the improved hemostasis by Cv-PC. Our findings indicate that Cv-PC increases plasma fibrinogen levels and may provide a promising therapy for reducing perioperative hemorrhage in elective surgeries.

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.

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.

Imatinib preserves blood-brain barrier integrity following experimental subarachnoid hemorrhage in rats.

Blood-brain barrier (BBB) disruption and consequent edema formation contribute to the development of early brain injury following subarachnoid hemorrhage (SAH). Various cerebrovascular insults result in increased platelet-derived growth factor receptor (PDGFR)-α stimulation, which has been linked to BBB breakdown and edema formation. This study examines whether imatinib, a PDGFR inhibitor, can preserve BBB integrity in a rat endovascular perforation SAH model. Imatinib (40 or 120 mg/kg) or a vehicle was administered intraperitoneally at 1 hr after SAH induction. BBB leakage, brain edema, and neurological deficits were evaluated. Total and phosphorylated protein expressions of PDGFR-α, c-Src, c-Jun N-terminal kinase (JNK), and c-Jun were measured, and enzymatic activities of matrix metalloproteinase (MMP)-2 and MMP-9 were determined in the injured brain. Imatinib treatment significantly ameliorated BBB leakage and edema formation 24 hr after SAH, which was paralleled by improved neurological functions. Decreased brain expressions of phosphorylated PDGFR-α, c-Src, JNK, and c-Jun as well as reduced MMP-9 activities were found in treated animals. PDGFR-α inhibition preserved BBB integrity following experimental SAH; however, the protective mechanisms remain to be elucidated. Targeting PDGFR-α signaling might be advantageous to ameliorate early brain injury following SAH.

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

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

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

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

Splenic immune cells in experimental neonatal hypoxia-ischemia.

Neuroimmune processes contribute to hypoxic-ischemic damage in the immature brain and may play a role in the progression of particular variants of neonatal encephalopathy. The present study was designed to elucidate molecular mediators of interactions between astrocytes, neurons and infiltrating peripheral immune cells after experimental neonatal hypoxia-ischemia (HI). Splenectomy was performed on postnatal day-7 Sprague-Dawley rats 3 days prior to HI surgery; in which the right common carotid artery was permanently ligated followed by 2 hours of hypoxia (8% O2). Quantitative analysis showed that natural killer (NK) and T cell expression was reduced in spleen but increased in the brain following HI. Elevations in cyclooxygenase-2 (COX-2) expression after HI by immune cells promoted interleukin-15 expression in astrocytes and infiltration of inflammatory cells to site of injury; additionally, down-regulated the pro-survival protein, phosphoinositide-3-kinase, resulting in caspase-3 mediated neuronal death. The removal of the largest pool of peripheral immune cells in the body by splenectomy, COX-2 inhibitors, as well as rendering NK cells inactive by CD161 knockdown, significantly ameliorated cerebral infarct volume at 72 hours, diminished body weight loss and brain and systemic organ atrophy, and reduced neurobehavioral deficits at 3 weeks. Herein we demonstrate with the use of surgical approach (splenectomy), with pharmacological loss-gain function approach using COX-2 inhibitors/agonists, as well as with NK cell-type specific siRNA that after neonatal HI, the infiltrating peripheral immune cells may modulate downstream targets of cell death and neuroinflammation by COX-2 regulated signals.

Evaluation of the hematoma consequences, neurobehavioral profiles, and histopathology in a rat model of pontine hemorrhage.

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.

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

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

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.

Post-treatment with SR49059 improves outcomes following an intracerebral hemorrhagic stroke in mice.

Intracerebral hemorrhage (ICH) is a devastating stroke subtype characterized by severe brain edema formation leading to cerebral blood flow compromise and parenchymal damage. Arginine vasopressin (AVP), a non-peptide antidiuretic hormone, has recently been implicated as a modulator of brain edema following injury. In this study, we investigated the effects of SR49059, a highly specific AVP V1a receptor antagonist, on brain injury outcomes following ICH, specifically assessing the ability of SR49059 in reducing brain edema and improving neurobehavioral deficits. Male CD1 mice (n=35) were randomly assigned to the following groups: sham, ICH, ICH with SR49059 at 0.5 mg/kg, and ICH with SR49059 at 2 mg/kg. ICH was induced by using the collagenase injection model, and treatment was given 1 h after surgery. Post-assessment was conducted at 24 and 72 h after surgery, and included brain water content and neurobehavioral testing. The study found that SR49059 significantly reduced cerebral edema at 24 and 72 h post-ICH injury and improved neurobehavioral deficits at 72 h. Our study suggests that blockage of the AVP V1a receptor is a promising treatment target for improving ICH-induced brain injury. Further studies will be needed to confirm this relationship and determine future clinical direction.

Protective effect of hydrogen gas therapy after germinal matrix hemorrhage in neonatal rats.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Tissue inhibitor of matrix metalloproteinase-1 mediates erythropoietin-induced neuroprotection in hypoxia ischemia.

Previous studies have shown that erythropoietin (EPO) is neuroprotective in both in vivo and in vitro models of hypoxia ischemia. However these studies hold limited clinical translations because the underlying mechanism remains unclear and the key molecules involved in EPO-induced neuroprotection are still to be determined. This study investigated if tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and its upstream regulator signaling molecule Janus kinase-2 (JAK-2) are critical in EPO-induced neuroprotection. Hypoxia ischemia (HI) was modeled in-vitro by oxygen and glucose deprivation (OGD) and in-vivo by a modified version of Rice-Vannucci model of HI in 10-day-old rat pups. EPO treated cells were exposed to AG490, an inhibitor of JAK-2 or TIMP-1 neutralizing antibody for 2h with OGD. Cell death, phosphorylation of JAK-2 and signal transducers and activators of transcription protein-3 (STAT-3), TIMP-1 expression, and matrix metalloproteinase-9 (MMP-9) activity were measured and compared with normoxic group. Hypoxic ischemic animals were treated one hour following HI and evaluated 48 h after. Our data showed that EPO significantly increased cell survival, associated with increased TIMP-1 activity, phosphorylation of JAK-2 and STAT-3, and decreased MMP-9 activity in vivo and in vitro. EPO's protective effects were reversed by inhibition of JAK-2 or TIMP-1 in both models. We concluded that JAK-2, STAT-3 and TIMP-1 are key mediators of EPO-induced neuroprotection during hypoxia ischemia injury.

Arginine-vasopressin V1a receptor inhibition improves neurologic outcomes following an intracerebral hemorrhagic brain injury.

Cerebral edema is a devastating consequence of brain injury leading to cerebral blood flow compromise and worsening parenchyma damage. In the present study, we investigated the effects of arginine-vasopressin (AVP) V(1a) receptor inhibition following an intracerebral hemorrhagic (ICH) brain injury in mice and closely assessed the role it played in cerebral edema formation, neurobehavioral functioning, and blood-brain-barrier (BBB) disruption. To support our investigation, SR49059, an AVP V(1a) receptor competitive antagonist, and NC1900, an arginine-vasopressin analogue, were used. Male CD1 mice (n=205) were randomly assigned to the following groups: naïve, sham, ICH, ICH with SR49059 at 0.5 mg/kg, ICH with SR49059 at 2mg/kg, ICH with NC1900 at 1 ng/kg, ICH with NC1900 at 10 ng/kg, and ICH with a combination of SR49059 at 2 mg/kg and NC1900 at 10 ng/kg. ICH was induced by using the collagenase injection model and treatment was given 1h after surgery. Post assessment was conducted at 6, 12, 24, and 72 h after surgery and included brain water content, neurobehavioral testing, Evans Blue assay, western blotting, and hemoglobin assay. The study found that inhibition of the AVP V(1a) receptor significantly reduced cerebral edema at 24 and 72 h post-ICH injury and improved neurobehavioral function while reducing BBB disruption at 72 h. Western blot analysis demonstrated increased protein expression of aquaporin 4 (AQP4) in vehicle, which was reduced with AVP V(1a) receptor inhibition. Our study suggests that blockage of the AVP V(1a) receptor, is a promising treatment target for improving ICH-induced brain injury. Further studies will be needed to confirm this relationship and determine future clinical direction.

Characterization of the brain injury, neurobehavioral profiles, and histopathology in a rat model of cerebellar hemorrhage.

Spontaneous cerebellar hemorrhage (SCH) represents approximately 10% of all intracerebral hemorrhage (ICH) and is an important clinical problem of which little is known. This study stereotaxically infused collagenase (type VII) into the deep cerebellar paramedian white matter, which corresponds to the most common clinical injury region. Measures of hemostasis (brain water, hemoglobin assay, Evans blue, collagen-IV, ZO-1, and MMP-2 and MMP-9) and neurodeficit were quantified 24 hours later (Experiment 1). Long-term functional outcomes were measured over 30 days using the ataxia scale (modified Luciani), open field, wire suspension, beam balance, and inclined plane (Experiment 2). Neurocognitive ability was assessed on the third week using the rotarod (motor learning), T maze (working memory), and water maze (spatial learning and memory) (Experiment 3), followed by a histopathological analysis 1 week later (Experiment 4). Stereotaxic collagenase infusion caused dose-dependent elevations in brain edema, neurodeficit, hematoma volume, and blood-brain barrier rupture, while physiological variables remained stable. Most functional outcomes normalized by the third week, while neurocognitive testing showed deficits parallel to the cystic-cavitary lesion at 30 days. All animals survived until sacrifice, and obstructive hydrocephalus did not develop. These results suggest that the model can generate important translational information about this subtype of ICH and could be used for future investigations of therapeutic mechanisms after cerebellar hemorrhage.

Remote limb ischemic postconditioning protects against neonatal hypoxic-ischemic brain injury in rat pups by the opioid receptor/Akt pathway.

BACKGROUND AND PURPOSE: Remote ischemic postconditoning, a phenomenon in which brief ischemic stimuli of 1 organ protect another organ against an ischemic insult, has been demonstrated to protect the myocardium and adult brain in animal models. However, mediators of the protection and underlying mechanisms remain to be elucidated. In the present study, we tested the hypothesis that remote limb ischemic postconditioning applied immediately after hypoxia provides neuroprotection in a rat model of neonatal hypoxia-ischemia (HI) by mechanisms involving activation of the opioid receptor/phosphatidylinositol-3-kinase/Akt signaling pathway. METHODS: HI was induced in postnatal Day 10 rat pups by unilateral carotid ligation and 2 hours of hypoxia. Limb ischemic postconditioning was induced by 4 conditioning cycles of 10 minutes of ischemia and reperfusion on both hind limbs immediately after HI. The opioid antagonist naloxone, phosphatidylinositol-3-kinase inhibitor wortmannin, or opioid agonist morphine was administered to determine underlying mechanisms. Infarct volume, brain atrophy, and neurological outcomes after HI were evaluated. Expression of phosphorylated Akt, Bax, and phosphorylated ERK1/2 was determined by Western blotting. RESULTS: Limb ischemic postconditioning significantly reduced infarct volume at 48 hours and improved functional outcomes at 4 weeks after HI. Naloxone and wortmannin abrogated the postconditioning-mediated infarct-limiting effect. Morphine given immediately after hypoxia also decreased infarct volume. Furthermore, limb ischemic postconditioning recovered Akt activity and decreased Bax expression, whereas no differences in phosphorylated ERK1/2expression were observed. CONCLUSIONS: Limb ischemic postconditioning protects against neonatal HI brain injury in rats by activating the opioid receptor/phosphatidylinositol-3-kinase/Akt signaling pathway.

Isoflurane posttreatment reduces neonatal hypoxic-ischemic brain injury in rats by the sphingosine-1-phosphate/phosphatidylinositol-3-kinase/Akt pathway.

BACKGROUND AND PURPOSE: Isoflurane, administered before or during cerebral ischemia, has been shown to exhibit neuroprotection in animal models of ischemic stroke. However, the underlying mechanism remains to be elucidated. In the present study, we determined whether isoflurane posttreatment provides neuroprotection after neonatal hypoxia-ischemia (HI) in rats and evaluated the role of the sphingosine-1-phosphate/phosphatidylinositol-3-kinase/Akt pathway in this volatile anesthetic-mediated neuroprotection. METHODS: HI was induced in postnatal day 10 (P10) rat pups by unilateral carotid ligation and 2 hours of hypoxia. For treatment, 2% isoflurane was administered immediately after HI for 1 hour. As pharmacological interventions, the sphingosine-1-phosphate antagonist VPC23019, phosphatidylinositol-3-kinase inhibitor wortmannin, or opioid antagonist naloxone was administered before HI. Isoflurane posttreatment was evaluated for effects on infarct volume at 48 hours after HI and brain atrophy and neurological outcomes at 4 weeks after HI. The expression of phosphorylated Akt and cleaved caspase-3 was determined by Western blotting and immunofluorescence analysis. RESULTS: Isoflurane posttreatment significantly reduced infarct volume at 48 hours after HI. VPC23019 or wortmannin abrogated the neuroprotective effect of isoflurane, whereas naloxone did not inhibit the isoflurane-induced neuroprotection. Isoflurane posttreatment significantly preserved phosphorylated Akt expression and decreased cleaved caspase-3 levels. These effects were reversed by VPC23019 and wortmannin, respectively. Isoflurane also confers long-term neuroprotective effects against brain atrophy and neurological deficits at 4 weeks after HI. CONCLUSIONS: Isoflurane posttreatment provides lasting neuroprotection against hypoxic-ischemic brain injury in neonatal rats. Activation of the sphingosine-1-phosphate/phosphatidylinositol-3-kinase/Akt pathway may play a key role in isoflurane posttreatment-induced neuroprotection.