Astrocytic transforming growth factor-beta signaling reduces subacute neuroinflammation after stroke in mice.

Astrocytes limit inflammation after CNS injury, at least partially by physically containing it within an astrocytic scar at the injury border. We report here that astrocytic transforming growth factor-beta (TGFß) signaling is a second, distinct mechanism that astrocytes utilize to limit neuroinflammation. TGFßs are anti-inflammatory and neuroprotective cytokines that are upregulated subacutely after stroke, during a clinically accessible time window. We have previously demonstrated that TGFßs signal to astrocytes, neurons and microglia in the stroke border days after stroke. To investigate whether TGFß affects astrocyte immunoregulatory functions, we engineered "Ast-Tbr2DN" mice where TGFß signaling is inhibited specifically in astrocytes. Despite having a similar infarct size to wildtype controls, Ast-Tbr2DN mice exhibited significantly more neuroinflammation during the subacute period after distal middle cerebral occlusion (dMCAO) stroke. The peri-infarct cortex of Ast-Tbr2DN mice contained over 60% more activated CD11b(+) monocytic cells and twice as much immunostaining for the activated microglia and macrophage marker CD68 than controls. Astrocytic scarring was not altered in Ast-Tbr2DN mice. However, Ast-Tbr2DN mice were unable to upregulate TGF-ß1 and its activator thrombospondin-1 2 days after dMCAO. As a result, the normal upregulation of peri-infarct TGFß signaling was blunted in Ast-Tbr2DN mice. In this setting of lower TGFß signaling and excessive neuroinflammation, we observed worse motor outcomes and late infarct expansion after photothrombotic motor cortex stroke. Taken together, these data demonstrate that TGFß signaling is a molecular mechanism by which astrocytes limit neuroinflammation, activate TGFß in the peri-infarct cortex and preserve brain function during the subacute period after stroke.

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.

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.

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

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

The evolving landscape of neuroinflammation after neonatal hypoxia-ischemia.

Hypoxic-ischemic brain injury remains a leading cause of mortality and morbidity in neonates. The inflammatory response, which is characterized in part by activation of local immune cells, has been implicated as a core component for the progression of damage to the immature brain following hypoxia-ischemia (HI). However, mounting evidence implicates circulating immune cells recruited to the site of damage as orchestrators of neuron-glial interactions and perpetuators of secondary brain injury. This suggests that re-directing our attention from the local inflammatory response toward the molecular mediators believed to link brain-immune cell interactions may be a more effective approach to mitigating the inflammatory sequelae of perinatal HI. In this review, we focus our attention on cyclooxygenase-2, a mediator by which peripheral immune cells may modulate signaling pathways in the brain that lead to a worsened outcome. Additionally, we present an overview of emerging therapeutic modalities that target mechanisms of neuroinflammation in the hypoxic-ischemic neonate.

Geldanamycin reduced brain injury in mouse model of intracerebral hemorrhage.

We investigated the effect of the heat shock protein inducer geldanamycin on the development of secondary brain injury after ICH in mice. The effect of the drug at two different concentrations was evaluated at two time points: 24 and 72 h after ICH induction. In the first part of this study, a total of 30 male CD-1 mice were randomly divided into four groups: one sham group and three ICH groups. ICH animals received either an intraperitoneal injection of vehicle or geldanamycin (1 or 10 mg/kg). Neurological deficits and brain water content were evaluated 24 h after ICH. In the second part of this study, the effect of a high concentration of geldanamycin was evaluated 72 h after ICH. Neurological deficits were evaluated by the Garcia neuroscoring, wire hanging and beam balance tests. For estimation of brain water content, the "wet/dry weight" method was used. We demonstrated that administration of geldanamycin (10 mg/kg) ameliorated ICH-induced increase of brain water content significantly in both parts of the study. Geldanamycin improved the neurological outcome according to performance on Garcia and beam balance tests in the 72 h part of this study. Geldanamycin-induced induction of heat shock protein after ICH has a neuroprotective effect and may be a therapeutic target for ICH.

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.

The postpartum period of pregnancy worsens brain injury and functional outcome after cerebellar hemorrhage in rats.

BACKGROUND: 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. METHODS: 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. RESULTS: Postpartum female rats had worsened brain water, blood-brain barrier rupture, hematoma size and neurological evaluations compared to their male counterparts. CONCLUSION: 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.

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.

Prostaglandin E2 EP1 receptor inhibition fails to provide neuroprotection in surgically induced brain-injured mice.

Recent trials have shown that the prostaglandin E2 EP1 receptor is responsible for NMDA excitotoxicity in the brain after injury. Consequently, in this study, we investigated the use of SC-51089, a selective prostaglandin E2 EP1 receptor antagonist, as a pre-treatment modality to decrease cell death, reduce brain edema, and improve neurobehavioral function after surgically induced brain injury (SBI) in mice. Eleven-week-old C57 black mice (n=82) were randomly assigned to four groups: sham (n=31), SBI (n=27), SBI treated with SC51089 at 10 µg/kg (n=7), and SBI treated with SC51089 at 100 µg/kg (n=17). Treated groups received a single dose of SC51089 intrapertioneally at 12 and 1 h pre-surgery. SBI was performed by resecting the right frontal lobe using a frontal craniotomy. Postoperative assessment occurred at 24 and 72 h, and included neurobehavioral testing and measurement of brain water content and cell death. Results indicated that neither low- nor high-dose EP1 receptor inhibition protected against the SBI-related effects on brain edema formation or cell death. There was however a significant improvement in neurobehavioral function 24 h post-SBI with both dosing regimens. Further studies will be needed to assess the potential therapeutic role of EP1 receptor targeting in SBI.

Endothelin receptor-A (ETa) inhibition fails to improve neonatal hypoxic-ischemic brain injury in rats.

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.

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.

Cyclooxygenase-2 inhibition provides lasting protection against neonatal hypoxic-ischemic brain injury.

OBJECTIVE: To investigate whether inhibition of cyclooxygenase-2, a critical component of the inflammatory pathway, is neuroprotective in a neonatal rat model of cerebral hypoxia-ischemia. The development of brain inflammation largely contributes to neonatal brain injury that may lead to a lifetime of neurologic deficits. DESIGN: Laboratory investigation. SETTING: University research laboratory. SUBJECTS: Postnatal day ten Sprague-Dawley rats. INTERVENTIONS: Neonatal hypoxia-ischemia was induced by ligation of the right common carotid artery followed by 2 hrs of hypoxia (8% oxygen). The pups in treatment groups were administered 10 mg/kg (low dose) or 30 mg/kg (high dose) of a known selective cyclooxygenase-2 inhibitor (NS398). Animals were euthanized at three time points: 72 hrs, 2 wks, or 6 wks. Inflammation outcomes were assessed at 72 hrs; brain damage was assessed at 2 wks and 6 wks along with other organs (heart, spleen). Detailed neurobehavioral examination was performed at 6 wks. MEASUREMENTS AND MAIN RESULTS: Pharmacologic inhibition of cyclooxygenase-2 markedly increased survivability within the first 72 hrs compared with untreated rats (100% vs. 72%). Low- and high-dose NS398 significantly attenuated the loss of brain and body weights observed after hypoxia-ischemia. Neurobehavioral outcomes were significantly improved in some parameters with low-dose treatment, whereas high-dose treatment consistently improved all neurologic deficits. Immunohistochemical results showed a marked decrease in macrophage, microglial, and neutrophil abundance in ipsilateral hemisphere of the NS398-treated group along with a reduction in interleukin-6 expression. CONCLUSIONS: Selective cyclooxygenase-2 inhibition protected neonatal rats against death, progression of brain injury, growth retardation, and neurobehavioral deficits after a hypoxic-ischemic insult.

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.

Distal hypoxic stroke: a new mouse model of stroke with high throughput, low variability and a quantifiable functional deficit.

C57BL/6J are the most commonly used strain of mouse for stroke experiments but vascular anatomy of the Circle of Willis within this strain is extremely variable and the cortex has extensive collateralization. This causes large variability in stroke models that target the middle cerebral artery proximally and confers resistance to ischemia in those that target it distally. We tested the hypothesis that by combining distal middle cerebral artery occlusion with 1h of hypoxia, we could generate a large lesion that causes a behavioral deficit with low variability. We found that this new distal hypoxic (DH) model of stroke generates a lesion with a volume of 25% of the ipsilateral hemisphere, extends to the motor cortex and causes a behavioral deficit. It also has a very clear border, exceptionally low variability, and can be performed by a single surgeon on up to 30 animals a day. Moreover, survivability is 100% in young adult animals, the model can be performed on old animals, and therapeutic intervention can reduce infarct volume. Therefore DH stroke is an excellent complement to existing stroke models and could be used for preclinical studies in C57BL/6J mice.

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.

Long-term evaluation of granulocyte-colony stimulating factor on hypoxic-ischemic brain damage in infant rats.

PURPOSE: Hypoxia-ischemia (HI), as a major cause of fetal brain damage, has long-lasting neurological implications. Therefore, therapeutic interventions that attenuate the neuropathological outcome of HI while also improving the neurofunctional outcome are of paramount clinical importance. The aim of this study was to investigate the long-term functional and protective actions of granulocyte-colony stimulating factor (G-CSF) treatment in an experimental model of cerebral HI. METHODS: Postnatal day-7 Sprague-Dawley rats were subjected to HI surgery, which entailed ligation of the right common carotid artery followed by 2 h of hypoxia (8% O(2)). Treatment consisted of subcutaneous injection of G-CSF at 1 h after hypoxia followed by an additional one injection per day for 5 days (6 total injections) or for 10 days (11 total injections). Animals were euthanized 5 weeks post-insult for extensive evaluation of neurological deficits and assessment of brain, spleen, heart, and liver damage. RESULTS: G-CSF treatment promoted somatic growth and prevented brain atrophy and underdevelopment of the heart. Moreover, reflexes, limb placing, muscle strength, motor coordination, short-term memory, and exploratory behavior were all significantly improved by both G-CSF dosing regimens. CONCLUSIONS: Long-term neuroprotection afforded by G-CSF in both morphological and functional parameters after a hypoxic-ischemic event in the neonate provides a rationale for exploring clinical translation.

Heat shock protein 70 upregulation by geldanamycin reduces brain injury in a mouse model of intracerebral hemorrhage.

UNLABELLED: This study investigated the effect of geldanamycin post-treatment on the development of secondary brain injury and neurological deficits in a mouse model of intracerebral hemorrhage. CD-1 mice received stereotactic injection of collagenase type VII into the right basal ganglia. Treatment groups were administered 1 mg/kg (low dose) or 10 mg/kg (high dose) of geldanamycin. Mice were euthanized at two time-points: 24 h or 72 h. Blood-brain-barrier permeability, brain edema, and neurobehavioral deficits were assessed. Additionally, the effects of geldanamycin on heat shock protein 27 and 72; tumor necrosis factor-alpha and interleukin 1 beta expressions were evaluated. High dose geldanamycin significantly attenuated blood-brain barrier disruption and brain edema after intracerebral hemorrhage. Neurobehavioral outcomes were significantly improved in some parameters by high dose treatment. Molecular results showed a marked increase in heat shock protein 72 expression in ipsilateral brain of geldanamycin treated groups with a reduction in the pro-inflammatory tumor necrosis factor-alpha. CONCLUSION: Geldanamycin post-treatment is neuroprotective in the mouse model of intracerebral hemorrhage. Geldanamycin administration results in reduction of inflammation, preservation of blood-brain-barrier and amelioration of neurobehavioral deficits after an insult possibly by upregulation of heat shock protein 72.

Glibenclamide improves neurological function in neonatal hypoxia-ischemia in rats.

Recent studies demonstrated that sulfonylurea receptor 1 (SUR 1) regulated nonselective cation channel, the NC(Ca-ATP) channel, is involved in brain injury in rodent models of stroke. Block of SUR 1 with sulfonylurea such as glibenclamide has been shown to be highly effective in reducing cerebral edema, infarct volume and mortality in adult rat models of ischemic stroke. In this study, we tested glibenclamide in both severe and moderate models of neonatal hypoxia-ischemia (HI) in postnatal day 10 Sprague-Dawley rat pups. A total of 150 pups were used in the present study. Pups were subjected to unilateral carotid artery ligation followed by 2.5 or 2 h of hypoxia in the severe and moderate HI models, respectively. In the severe HI model, glibenclamide, administered immediately after HI and on postoperative Day 1, was not effective in attenuating short-term effects (brain edema and infarct volume) or long-term effects (brain weight and neurological function) of neonatal HI. In the moderate HI model, when injected immediately after HI and on postoperative Day 1, glibenclamide at 0.01 mg/kg improved several neurological parameters at 3 weeks after HI. We conclude that glibenclamide provided some long-term neuroprotective effect after neonatal HI.

Hydrogen gas is ineffective in moderate and severe neonatal hypoxia-ischemia rat models.

Hydrogen gas (H(2)) has been shown to ameliorate brain injury in experimental adult rat focal ischemia and in a mild neonatal hypoxia-ischemia (HI, 90 min hypoxia) rat model. In this study we tested H(2) in moderate (120 min hypoxia) and severe (150 min hypoxia) neonatal HI rat models. We hypothesized that H(2) would improve outcomes after neonatal HI by scavenging free radicals. Two hundred (200) unsexed Sprague-Dawley rats at day 10 of life (p10) underwent neonatal HI with the Rice-Vannucci model. Multiple treatment protocols were studied, including pre-ischemic treatment, intra-ischemic treatment, and post-ischemic treatment (Sham n=32, HI n=82, HI+H(2)n=86). We also tested H(2) in middle cerebral artery occlusion (MCAO) in adult rats (MCAO n=9, MCAO+H(2)n=7) for comparison. Analysis at 24 h included infarction volume, measurement of brain concentration of malondialdehyde (MDA) (an end-product of lipid peroxidation), daily weight, Nissl histology, and mortality. In moderate and severe neonatal HI models, hydrogen gas therapy (2.9% concentration H(2)) was not associated with decreased volume of infarction or decreased concentration of MDA. H(2) gas pretreatment (2.9%) was associated with increased infarction volume in neonatal HI. In MCAO in adult rats, H(2) gas therapy demonstrated a trend of beneficial effect. Exposure of H(2) gas to non-ischemic neonates resulted in a significant increase in brain concentration of MDA. We conclude that 2.9% H(2) gas therapy does not ameliorate moderate to severe ischemic damage in neonatal hypoxia-ischemia.