The insights into molecular pathways of hypoxia-inducible factor in the brain.

The objectives of this present work were to review recent developments on the role of hypoxia-inducible factor (HIF) in the survival of cells under normoxic versus hypoxic and inflammatory brain conditions. The dual nature of HIF effects appears well established, based on the accumulated evidence of HIF playing both the role of adaptive factor and mediator of cell demise. Cellular HIF responses depend on pathophysiological conditions, developmental phase, comorbidities, and administered medications. In addition, HIF-1α and HIF-2α actions may vary in the same tissues. The multiple roles of HIF in stem cells are emerging. HIF not only regulates expression of target genes and thereby influences resultant protein levels but also contributes to epigenetic changes that may reciprocally provide feedback regulations loops. These HIF-dependent alterations in neurological diseases and its responses to treatments in vivo need to be examined alongside with a functional status of subjects involved in such studies. The knowledge of HIF pathways might be helpful in devising HIF-mimetics and modulating drugs, acting on the molecular level to improve clinical outcomes, as exemplified here by clinical and experimental data of selected brain diseases, occasionally corroborated by the data from disorders of other organs. Because of complex role of HIF in brain injuries, prospective therapeutic interventions need to differentially target HIF responses depending on their roles in the molecular mechanisms of neurologic diseases.

Isoflurane on brain inflammation.

Brain inflammation may play an important role in the pathophysiology of early brain injury after subarachnoid hemorrhage (SAH). Our aim was to demonstrate brain inflammation development and to determine whether isoflurane, a clinically available volatile anesthetic agent, prevents brain inflammation after SAH. This study used 162 8-week-old male CD-1 mice. We induced SAH with endovascular perforation in mice and randomly assigned animals to sham-operated (n=21), SAH+vehicle-air (n=35) and SAH+2% isoflurane (n=31). In addition to the evaluation of brain injury (neurological scores, brain edema and Evans blue dye extravasation), brain inflammation was evaluated by means of expression changes in markers of inflammatory cells (ionized calcium binding adaptor molecule-1, myeloperoxidase), cytokines (tumor necrosis factor [TNF]-α, interleukin-1ß), adhesion molecules (intercellular adhesion molecule [ICAM]-1, P-selectin), inducers of inflammation (cyclooxygenase-2, phosphorylated c-Jun N-terminal kinase [p-JNK]) and endothelial cell activation (von Willebrand factor) at 24h post-SAH. Sphingosine kinase inhibitor (N, N-dimethylsphingosine [DMS]) and sphingosine-1-phosphate receptor-1/3 antagonist (VPC23019) were used to block isoflurane's effects (n=22, each). SAH caused early brain injury, which was associated with inflammation so that all evaluated markers of inflammation were increased. Isoflurane significantly inhibited both brain injury (P<0.001, respectively) and inflammation (myeloperoxidase, P=0.022; interleukin-1ß, P=0.002; TNF-α, P=0.015; P-selectin, P=0.010; ICAM-1, P=0.016; p-JNK, P<0.001; cyclooxygenase-2, P=0.003, respectively). This beneficial effect of isoflurane was abolished with DMS and VPC23019. Isoflurane may suppress post-SAH brain inflammation possibly via the sphingosine-related pathway.

TDP-43 pathology in subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis (SSPE) is a fatal, slowly progressive brain disorder caused by a mutated measles virus. Both subacute inflammatory and neurodegenerative mechanisms appear to play significant roles in the pathogenesis. TAR DNA-binding protein 43 (TDP-43) inclusions are a common co-pathology in several neurodegenerative disorders with diverse pathogenesis. In the present study, we examined brains of 16 autopsied SSPE patients for the presence of TDP-43 pathology and possible associations with tau pathology. Immunohistochemical staining identified TDP-43 inclusions in 31% of SSPE cases. TDP-43 pathology was widely distributed in the brains, most severely in the atrophied cerebral cortex (temporal and parietal), and most frequently as tangle- and thread-like neuronal cytoplasmic inclusions. It was associated with longer disease duration (>4 years) and tau pathology (all TDP-43-positive cases had tau-positive neurofibrillary tangles). This study demonstrates for the first time an association between TDP-43 pathology and SSPE. The co-occurrence of TDP-43 and tau aggregates and correlation with the disease duration suggest that both pathological proteins are involved in the neurodegenerative process induced by viral inflammation.

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

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

Statin-induced T-lymphocyte modulation and neuroprotection following experimental subarachnoid hemorrhage.

INTRODUCTION: Statins influence immune system activities through mechanisms independent of their lipid-lowering properties. T cells can be subdivided based on cytokine secretion patterns into two subsets: T-helper cells type 1 (Th1) and type 2 (Th2). Independent laboratory studies have shown statins to be potent inducers of a Th2 switch in immune cell response and be neuroprotective in several models of central nervous system (CNS) disease. This study was the first to evaluate the immune modulating effects of statins in subarachnoid hemorrhage (SAH). METHODS: Simvastatin was administered to rats intraperitoneally in two dosages (1 and 20 mg/kg) 30 min after the induction of SAH using endovascular perforation. Neurological scores were assessed 24 h later. Animals were then sacrificed, and samples of cortex and brain stem were tested for expression of the T-regulatory cell cytokine transforming growth factor (TGF) ß1, as well as interleukin (IL) 1ß, a proinflammatory cytokine associated with Th1 immune responses. The presence of TGF-ß1 secreting T cells was evaluated with the use of brain slices. RESULTS: SAH significantly impaired neurological function in all SAH groups (treated and untreated) versus sham. Animals treated with high-dose simvastatin had less neurological impairment than both untreated and low-dose groups. Cortical and brain-stem levels of TGF-ß1 were significantly elevated following SAH in the high-dose group. IL-1ß was significantly elevated following the induction of SAH but was inhibited by high-dose simvastatin. Double-labeled fluorescent immunohistochemical data demonstrated the presence of lymphocytes in the subarachnoid and perivascular spaces following SAH. Expression of TGF-ß1 by lymphocytes was markedly increased following treatment with high-dose simvastatin. CONCLUSION: The present study elucidated the potential role of a Th2 immune switch in statin provided neuroprotection following SAH.

CHOP silencing reduces acute brain injury in the rat model of subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Endoplasmic reticulum stress triggers apoptotic cascades in neurons of the central nervous system after subarachnoid hemorrhage. The aim of this work was to study the mechanism of neuroprotection conferred by targeting cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) in the acute brain injury following subarachnoid hemorrhage. METHODS: A total of 172 rats were used. Endovascular perforation induced subarachnoid hemorrhage. Two small interfering RNAs for CHOP were injected 24 hours before hemorrhage induction. At 24 or 72 hours, rats were neurologically evaluated and euthanized. The brains were recovered for molecular biology and histology studies. RESULTS: Western blot analysis revealed effective silencing of CHOP associated with suppression of Bim-Caspase-3 apoptotic pathway. Moreover, the antiapoptotic Bcl2 was found upregulated with CHOP siRNA treatment. A reduced number of TUNEL-positive cells in the subcortex and in the hippocampus reflected histological protection. CHOP siRNA treatment ameliorated intracranial sequelae of and improved functional performance. CONCLUSIONS: We conclude that CHOP silencing alleviates early brain injury following subarachnoid hemorrhage via inhibiting apoptosis and that CHOP siRNA treatment has a clinical potential for patients with this type of hemorrhagic stroke.

Role of the pituitary­adrenal axis in granulocyte-colony stimulating factor-induced neuroprotection against hypoxia­ischemia in neonatal rats.

Several reports indicate that the activity of the hypothalamic­pituitary­adrenal axis (HPA) is increased after a brain insult and that its down-regulation can improve detrimental outcomes associated with ischemic brain injuries.Granulocyte-colony stimulating factor (G-CSF) is a neuroprotective drug shown in the naïve rat to regulate hormones of the HPA axis. In this study we investigate whether G-CSF confers its neuroprotective properties by influencing the HPA response after neonatal hypoxia­ischemia (HI). Following the Rice­Vannucci model, seven day old rats (P7)were subjected to unilateral carotid ligation followed by 2.5 h of hypoxia. To test our hypothesis,metyrapone was administered to inhibit the release of rodent specific glucocorticoid, corticosterone, at the adrenal level. Dexamethasone, a synthetic glucocorticoid, was administered to agonize the effects of corticosterone.Our results show that both G-CSF and metyrapone significantly reduced infarct volume while dexamethasone treatment did not reduce infarct size even when combined with G-CSF. The protective effects of G-CSF do not include blood brain barrier preservation as suggested by the brain edema results. G-CSF did not affect the pituitary released adrenocorticotropic hormone (ACTH) levels in the blood plasma at 4 h, but suppressed the increase of corticosterone in the blood. The administration of G-CSF and metyrapone increased weight gain, and significantly reduced the Bax/Bcl-2 ratio in the brain while dexamethasone reversed the effects of G-CSF. The combination of G-CSF and metyrapone significantly decreased caspase-3 protein levels in the brain, and the effect was antagonized by dexamethasone.We report that G-CSF is neuroprotective in neonatal HI by reducing infarct volume, by suppressing the HI-induced increase of the Bax/Bcl-2 ratio, and by decreasing corticosterone in the blood. Metyrapone was able to confer similar neuroprotection as G-CSF while dexamethasone reversed the effects of G-CSF. In conclusion, we show that decreasing HPA axis activity is neuroprotective after neonatal HI, which can be conferred by administering G-CSF.

Harnessing oxidative stress for anti-glioma therapy.

Glioma cells use intermediate levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) for growth and invasion, and suppressing these reactive molecules thus may compromise processes that are vital for glioma survival. Increased oxidative stress has been identified in glioma cells, in particular in glioma stem-like cells. Studies have shown that these cells harbor potent antioxidant defenses, although endogenous protection against nitrosative stress remains understudied. The enhancement of oxidative or nitrosative stress offers a potential target for triggering glioma cell death, but whether oxidative and nitrosative stresses can be combined for therapeutic effects requires further research. The optimal approach of harnessing oxidative stress for anti-glioma therapy should include the induction of free radical-induced oxidative damage and the suppression of antioxidant defense mechanisms selectively in glioma cells. However, selective induction of oxidative/nitrosative stress in glioma cells remains a therapeutic challenge, and research into selective drug delivery systems is ongoing. Because of multifactorial mechanisms of glioma growth, progression, and invasion, prospective oncological therapies may include not only therapeutic oxidative/nitrosative stress but also inhibition of oncogenic kinases, antioxidant molecules, and programmed cell death mediators.

Inhibiting CK2 among Promising Therapeutic Strategies for Gliomas and Several Other Neoplasms.

In gliomas, casein kinase 2 (CK2) plays a dominant role in cell survival and tumour invasiveness and is upregulated in many brain tumours. Among CK2 inhibitors, benzimidazole and isothiourea derivatives hold a dominant position. While targeting glioma tumour cells, they show limited toxicity towards normal cells. Research in recent years has shown that these compounds can be suitable as components of combined therapies with hyperbaric oxygenation. Such a combination increases the susceptibility of glioma tumour cells to cell death via apoptosis. Moreover, researchers planning on using any other antiglioma investigational pharmaceutics may want to consider using these agents in combination with CK2 inhibitors. However, different compounds are not equally effective when in such combination. More research is needed to elucidate the mechanism of treatment and optimize the treatment regimen. In addition, the role of CK2 in gliomagenesis and maintenance seems to have been challenged recently, as some compounds structurally similar to CK2 inhibitors do not inhibit CK2 while still being effective at reducing glioma viability and invasion. Furthermore, some newly developed inhibitors specific for CK2 do not appear to have strong anticancer properties. Further experimental and clinical studies of these inhibitors and combined therapies are warranted.

Proteasome and Neuroprotective Effect of Hyperbaric Oxygen Preconditioning in Experimental Global Cerebral Ischemia in Rats.

OBJECTIVES: We investigated the involvement of the proteasome in the mechanism of preconditioning with hyperbaric oxygen (HBO-PC). METHODS: The experiments were performed on male Wistar rats subjected to a transient global cerebral ischemia of 5 min duration (2-vessel occlusion model) and preconditioned or not with HBO for 5 preceding days (1 h HBO at 2.5 atmosphere absolute [ATA] daily). In subgroups of preconditioned rats, the proteasome inhibitor MG132 was administered 30 min prior to each preconditioning session. Twenty-four hours and 7 days post-ischemia, after neurobehavioral assessment, the brains were collected and evaluated for morphological changes and quantitative immunohistochemistry of cell markers and apoptosis-related proteins. RESULTS: We observed reduced damage of CA1 pyramidal cells in the HBO preconditioned group only at 7 days post-ischemia. However, both at early (24 h) and later (7 days) time points, HBO-PC enhanced the tissue expression of 20S core particle of the proteasome and of the nestin, diminished astroglial reactivity, and reduced p53, rabbit anti-p53 upregulated modulator of apoptosis (PUMA), and rabbit anti-B cell lymphoma-2 interacting mediator of cell death (Bim) expressions in the hippocampus and cerebral cortex. HBO-PC also improved T-maze performance at 7 days. Proteasome inhibitor abolished the beneficial effects of HBO-PC on post-ischemic neuronal injury and functional impairment and reduced the ischemic alterations in the expression of investigated proteins. SIGNIFICANCE: Preconditioning with hyperbaric oxygen-induced brain protection against severe ischemic brain insult appears to involve the proteasome, which can be linked to a depletion of apoptotic proteins and improved regenerative potential.

Dexras1 Induces Dysdifferentiation of Oligodendrocytes and Myelin Injury by Inhibiting the cAMP-CREB Pathway after Subarachnoid Hemorrhage.

White matter damage (WMD), one of the research hotspots of subarachnoid hemorrhage (SAH), mainly manifests itself as myelin injury and oligodendrocyte differentiation disorder after SAH, although the specific mechanism remains unclear. Dexamethasone-induced Ras-related protein 1(Dexras1) has been reported to be involved in nervous system damage in autoimmune encephalitis and multiple sclerosis. However, whether Dexras1 participates in dysdifferentiation of oligodendrocytes and myelin injury after SAH has yet to be examined, which is the reason for creating the research content of this article. Here, intracerebroventricular lentiviral administration was used to modulate Dexras1 levels in order to determine its functional influence on neurological injury after SAH. Immunofluorescence, transmission electron microscopy, and Western blotting methods, were used to investigate the effects of Dexras1 on demyelination, glial cell activation, and differentiation of oligodendrocyte progenitor cells (OPCs) after SAH. Primary rat brain neurons were treated with oxyhemoglobin to verify the association between Dexras1 and cAMP-CREB. The results showed that Dexras1 levels were significantly increased upon in vivo SAH model, accompanied by OPC differentiation disturbances and myelin injury. Dexras1 overexpression significantly worsened OPC dysdifferentiation and myelin injury after SAH. In contrast, Dexras1 knockdown ameliorated myelin injury, OPC dysdifferentiation, and glial cell activation. Further research of the underlying mechanism discovered that the cAMP-CREB pathway was inhibited after Dexras1 overexpression in the in vitro model of SAH. This study is the first to confirm that Dexras1 induced oligodendrocyte dysdifferentiation and myelin injury after SAH by inhibiting the cAMP-CREB pathway. This present research may reveal novel therapeutic targets for the amelioration of brain injury and neurological dysfunction after SAH.

Cyclooxygenase-2 mediates hyperbaric oxygen preconditioning in the rat model of transient global cerebral ischemia.

BACKGROUND AND PURPOSE: Hyperbaric oxygen (HBO) preconditioning (PC) allows brain protection against transient global ischemia. In the present study, we hypothesize that the mechanism of HBO-PC involves the induction of cyclooxygenase-2 (COX-2) in cerebral tissues before ischemia, which leads to a suppression of COX-2 and its downstream targets after global ischemic insult. METHODS: One hundred twenty-nine male Sprague Dawley rats (body weight 280-300 grams) were allocated to the naive control group and the sham operation group, and 3 groups of animals were subjected to 15-minute 4-vessel occlusion: untreated, preconditioned with HBO 2.5 atmospheres absolutes for 1 hour daily for 5 days, preconditioned as mentioned and administered with COX-2 inhibitor NS-398 (1 mg/kg body weight intraperitoneal) before each preconditioning session, and normal rats preconditioned with HBO without ischemia. The mortality, the incidence of seizures, and T-maze scores were recorded. The quantitative cell count in Nissl stain and TUNEL was conducted on day 7 after ischemia. The brain expression of COX-2 was analyzed with Western blotting and immunofluorescence staining. RESULTS: HBO-PC increased the number of surviving neurons in the Cornu Ammonis area 1, which was associated with the reduced COX-2 expression in the hippocampus and in the cerebral cortex at 1 and 3 days after ischemia. HBO-PC improved functional performance and tended to decrease mortality and the frequency of seizures. These beneficial effects of HBO-PC were abolished by the COX-2 selective inhibitor NS-398. CONCLUSIONS: HBO-PC reduced COX-2 expression and provided brain protection after global ischemia. Administration of COX-2 inhibitor with HBO before ischemia abolished preconditioning effect, thereby implicating COX-2 as a mediator of HBO-PC in the ischemic brain.

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.

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.

Hydrogen inhalation is neuroprotective and improves functional outcomes in mice after intracerebral hemorrhage.

OBJECTIVE: Oxidative stress contributes significantly to the development of secondary brain injury after intracerebral hemorrhage (ICH). It has been previously demonstrated that hydrogen gas can decrease oxidative stress by scavenging reactive oxygen species. We hypothesized that hydrogen therapy will reduce brain oxidative stress in mice after ICH and thereby will lead to reduced brain edema and improved neurological outcomes. MATERIALS AND METHODS: CD1 male mice (weight 30-35 g) were divided into the following groups: sham, ICH+vehicle (room air), ICH+1-h hydrogen treatment, and ICH+2-h hydrogen treatment. ICH was induced by injection of bacterial collagenase into the right basal ganglia. The evaluation of outcomes was done at two time points: 24 and 72 h post-ICH. Brain water content was measured for assessment of brain edema (wet/dry weight method), and three neurological tests were performed pre- and postoperatively. RESULTS: Collagenase injection was found to induce brain edema and impair functional performance of rats. The hydrogen inhalation reduced these effects acutely (24 h); however it exhibited only a tendency to improvement in the delayed study (72 h). CONCLUSIONS: Our results suggest that hydrogen inhalation exerts an acute brain-protective effect in the mouse ICH model. However, the acute hydrogen therapy alone is not sufficient to improve delayed ICH outcomes in this model.

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.

Beneficial effect of hyperbaric oxygenation after neonatal germinal matrix hemorrhage.

BACKGROUND: Germinal matrix hemorrhage (GMH) is a potentially devastating neurological disease of very low birth weight premature infants. This leads to post-hemorrhagic hydrocephalus, cerebral palsy, and mental retardation. Hyperbaric oxygen (HBO) treatment is a broad neuroprotectant after brain injury. This study investigated the therapeutic effect of HBO after neonatal GMH. METHODS: Neonatal rats underwent stereotaxic infusion of clostridial collagenase into the right germinal matrix (anterior caudate) brain region. Cognitive function was assessed at 3 weeks, and then sensorimotor, cerebral, cardiac, and splenic growths were measured 1 week thereafter. RESULTS: Hyperbaric oxygen (HBO) treatment markedly improved upon the mental retardation and cerebral palsy outcome measurements in rats at the juvenile developmental stage. The administration of HBO early after neonatal GMH also normalized brain atrophy, splenomegaly, and cardiac hypertrophy 1 month after injury. CONCLUSION: This study supports the role of hyperbaric oxygen (HBO) treatment in the early period after neonatal GMH. HBO is an effective strategy to help protect the infant's 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 neuroprotective effects.

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.

Suppression of hypoxia-inducible factor-1alpha and its downstream genes reduces acute hyperglycemia-enhanced hemorrhagic transformation in a rat model of cerebral ischemia.

We evaluated a role of hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream genes in acute hyperglycemia-induced hemorrhagic transformation in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats weighing 280-300 g (n = 105) were divided into sham, 90 min middle cerebral artery occlusion (MCAO), MCAO plus HIF-1alpha inhibitors, 2-methoxyestradiol (2ME2) or 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), groups. Rats received an injection of 50% dextrose (6 ml/kg intraperitoneally) at 15 min before MCAO. HIF-1alpha inhibitors were administered at the onset of reperfusion. The animals were examined for neurological deficits and sacrificed at 6, 12, 24, and 72 hr following MCAO. The cerebral tissues were collected for histology, zymography, and Western blot analysis. The expression of HIF-1alpha was increased in ischemic brain tissues after MCAO and reduced by HIF-1alpha inhibitors. In addition, 2ME2 reduced the expression of vascular endothelial growth factor (VEGF) and the elevation of active matrix metalloproteinase-2 and -9 (MMP-2/MMP-9) in the ipsilateral hemisphere. Both 2ME2 and YC-1 reduced infarct volume and ameliorated neurological deficits. However, only 2ME2 attenuated hemorrhagic transformation in the ischemic territory. In conclusion, the inhibition of HIF-1alpha and its downstream genes attenuates hemorrhagic conversion of cerebral infarction and ameliorates neurological deficits after focal cerebral ischemia.