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.

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.

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.

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.

Hyperbaric oxygen increases glioma cell sensitivity to antitumor treatment with a novel isothiourea derivative in vitro.

Glioblastoma (GBM) is the most common primary brain tumor. Tumor hypoxia is a pivotal factor responsible for the progression of this malignant glioma, and its resistance to radiation and chemotherapy. Thus, improved tumor tissue oxygenation may promote greater sensitivity to anticancer treatment. Protein kinase D1 (PKD1) protects cells from oxidative stress, and its abnormal activity serves an important role in multiple malignancies. The present study examined the effects of various oxygen conditions on the cytotoxic potential of the novel isothiourea derivate N,N'­dimethyl­S­(2,3,4,5,6­pentabromobenzyl)­â€‹isothiouronium bromide (ZKK­3) against the T98G GBM cell line. ZKK­3 was applied at concentrations of 10, 25 and 50 µM, and cells were maintained under conditions of normoxia, anoxia, hypoxia, hyperbaric oxygen (HBO), hypoxia/hypoxia and hypoxia/HBO. The proliferation and viability of neoplastic cells, and protein expression levels of hypoxia­inducible factor 1α (HIF­1α), PKD1, phosphorylated (p)PKD1 (Ser 916) and pPKD1 (Ser 744/748) kinases were evaluated. Oxygen deficiency, particularly regarding hypoxia, could diminish the cytotoxic effect of ZKK­3 at 25 and 50 µM and improve T98G cell survival compared with normoxia. HBO significantly reduced cell proliferation and increased T98G cell sensitivity to ZKK­3 when compared with normoxia. HIF­1α expression levels were increased under hypoxia compared with normoxia and decreased under HBO compared with hypoxia/hypoxia at 0, 10 and 50 µM ZKK­3, suggesting that HBO improved oxygenation of the cells. ZKK­3 exhibited inhibitory activity against pPKD1 (Ser 916) kinase; however, the examined oxygen conditions did not appear to significantly influence the expression of this phosphorylated form in cells treated with the tested compound. Regarding pPKD1 (Ser 744/748), a significant difference in expression was observed only for cells treated with 10 µM ZKK­3 and hypoxia/hypoxia compared with normoxia. However, there were significant differences in the expression levels of both phosphorylated forms of PKD1 under different oxygen conditions in the controls. In conclusion, the combination of isothiourea derivatives and hyperbaric oxygenation appears to be a promising therapeutic approach for malignant glioma treatment.

Potent Antitumour Effects of Novel Pentabromobenzylisothioureas Studied on Human Glial-derived Tumour Cell Lines.

BACKGROUND/AIM: Tumours of astroglial origin are the most common primary brain malignancy characterized by infiltrative growth and resistance to standard antitumour therapy. Glioma progression is thought to be related to various intracellular signal transduction pathways that involve the activation of protein kinases. Protein kinases play important roles in cell differentiation, proliferation, and survival. Recently, novel, specific inhibitors of constitutively active serine/threonine kinases and structurally similar isothiourea derivatives were suggested to induce apoptosis and inhibit proliferation in several types of human cancer cells. MATERIALS AND METHODS: In this study, we examined the cytotoxic and proapoptotic activities of selected modified pentabromobenzyl isothioureas (ZKKs) in an adult human glioblastoma (T98G) and a subependymal giant cell astrocytoma cell (SEGA) line. We evaluated cell proliferation, viability, and apoptosis. RESULTS: Two pentabromobenzyl isothiourea bromide derivatives, ZKK-13 and N,N,N'-trimethyl-ZKK1 (TRIM), exhibited the most potent cytotoxic and proapoptotic efficacies against human glioma-derived cells, even at a very low concentration (1 µM). ZKK-13 (25-50 µM) inhibited cell growth by approximately 80-90% in 24 and 48 h of treatment. We showed that selected ZKKs exerted antiproliferative activity against astroglial neoplastic cells of both low- and high-grade tumour malignancy classes. No synergistic effects were detected when ZKKs were combined with serine/threonine kinase inhibitors. CONCLUSION: Our findings indicated that modified ZKKs show promise for the treatment of glioma-derived brain tumours.

Hyperbaric oxygen as an adjunctive therapy in treatment of malignancies, including brain tumours.

Hyperbaric oxygen (HBO) therapy is widely used as an adjunctive treatment for various pathological states, predominantly related to hypoxic and/or ischaemic conditions. It also holds promise as an approach to overcoming the problem of oxygen deficiency in the poorly oxygenated regions of the neoplastic tissue. Occurrence of local hypoxia within the central areas of solid tumours is one of the major issues contributing to ineffective medical treatment. However, in anti-cancer therapy, HBO alone gives a limited curative effect and is typically not applied by itself. More often, HBO is used as an adjuvant treatment along with other therapeutic modalities, such as radio- and chemotherapy. This review outlines the existing data regarding the medical use of HBO in cancer treatment, with a particular focus on the use of HBO in the treatment of brain tumours. We conclude that the administration of HBO can provide many clinical benefits in the treatment of tumours, including management of highly malignant gliomas. Applied immediately before irradiation, it is safe and well tolerated by patients, causing rare and limited side effects. The results obtained with a combination of HBO/radiotherapy protocol proved to be especially favourable compared to radiation treatment alone. HBO can also increase the cytostatic effect of certain drugs, which may render standard chemotherapy more effective. The currently available data support the legitimacy of conducting further research on the use of HBO in the treatment of malignancies.

Erythropoietin inhibits HIF-1α expression via upregulation of PHD-2 transcription and translation in an in vitro model of hypoxia-ischemia.

Hypoxia inducible factor (HIF)-1α is the central transcriptional factor for the regulation of oxygen-associated genes in response to hypoxia. Erythropoietin (EPO), a hematopoietic growth factor, increases oxygen availability during hypoxia/ischemia and is associated with neuroprotection following hypoxia-ischemia in laboratory models of stroke. However, EPO has failed to translate in a clinical setting. Thus, it is critical to elucidate the key players in EPO-induced neuroprotection. Our preliminary studies have shown that EPO, as a downstream gene of HIF, inhibits HIF-1α in a dose-dependent manner in an in vitro model of hypoxia-ischemia. This study is designed to elucidate the primary mediator of EPO-induced HIF-1α inhibition and subsequent cell survival/neuroprotection. Oxygen and glucose deprivation (OGD) of nerve growth factor-differentiated rat pheochromocytoma (PC-12) cells were used to model hypoxia-ischemia in an in vitro environment. The profile of HIF-1α, HIF-2α and prolyl hydroxylase domain 2 (PHD-2) expression; HIF-1α and prolyl hydroxylase (PHD-2) mRNA levels; matrix metalloproteinase (MMP)-9; and cell death was evaluated in the presence and absence of either EPO or PHD-2 inhibitor during OGD. Our findings showed that EPO treatment resulted in an increase in PHD-2 transcription and translation, inhibition of HIF-1α expression, reactive oxygen species formation, and MMP-9 activity, resulting in increased cell survival after OGD. We also observed that EPO-induced cell survival/neuroprotection was reversed by siRNA silencing of PHD-2. This led to the conclusion that PHD-2 is a key mediator of EPO-induced HIF-1α inhibition and subsequent neuroprotection in an in vitro model of hypoxia-ischemia.

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.

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.

Targeting C/EBP homologous protein with siRNA attenuates cerebral vasospasm after experimental subarachnoid hemorrhage.

Endothelial apoptosis plays a major role in the development of cerebral vascular spasm after subarachnoid hemorrhage (SAH). C/EBP homologous protein (CHOP) orchestrates apoptosis in a variety of cell types in response to endoplasmic reticulum (ER) stress, implicated in the brain injury after SAH. However, the role of CHOP in the mechanism of cerebral vasospasm (CVS) after SAH remains unexplored. The aim of this study was to evaluate the effect of CHOP silencing on endothelial apoptosis and CVS following subarachnoid hemorrhage in the rat. The study was conducted on 65 rats and employed endovascular perforation model of SAH. CHOP siRNAs were injected 24 h prior to the hemorrhage. At 72 h after SAH brains with basilar arteries (BA) were collected from euthanized rats for laboratory investigations. Triple fluorescence stain revealed expression of CHOP in cerebral vascular endothelia after SAH. Marked reduction of CHOP protein and the reduction of its downstream signaling effectors, bim and caspase-3, were found in BA with Western blot analysis. CHOP silencing reduced number of apoptotic endothelial cells in BA, and increased BA diameter after SAH. The amelioration of CVS was associated with reduced neuronal injury in cerebral tissues. In conclusion, CHOP siRNA treatment can effectively combat apoptotic mechanisms of cerebral vasospasm set in motion by subarachnoid bleeding.

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.

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.

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.

Brain irradiation improves focal cerebral ischemia recovery in aged rats.

BACKGROUND: Studies have shown that aging is a significant factor in worsening stroke outcomes. While many mechanisms may aggravate brain injury in the elderly, one such potential system may involve increased glial proliferation in the aged stroke patient that could result in increased scar formation. We hypothesized that in aged rats a single brain-only exposure to a low radiation dose prior to focal brain ischemia would reduce glial proliferation and confer a long-term neuroprotective effect. METHODS: Brain-only proton irradiation (8 Gy) was performed ten days prior to middle cerebral artery occlusion (MCAO) in aged male rats. Magnetic resonance imaging (MRI) was undertaken in naive, radiation-only (Rad), MCAO, and MCAO+Rad groups at 2, 14 and 28 days post-stroke followed by immunohistochemistry (day 28). RESULTS: Ischemic lesion volume in MCAO+Rad group was decreased by 50.7% with an accelerated temporal reduction in peri-lesional brain edema and increased water mobility within the ischemic core (39.8%) compared to MCAO-only rats. In the peri-lesional brain region of MCAO+Rad rats there was a decreased scar formation (49%, glial fibrillary acidic protein), brain tissue sclerosis (30%, aquaporin-4) and necrosis/apoptosis (58%, TUNEL positive cells) compared to those in MCAO animals. CONCLUSION: In aged animals a single exposure to brain-only radiation prior to focal cerebral ischemia is neuroprotective as it prevents glial hyperproliferation, progressive brain tissue sclerosis and reduces the apoptosis/necrosis in the peri-lesional region. Decreased lesion volume is in agreement with accelerated reduction of brain edema in these animals.

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.

Hyperbaric oxygen therapy improves early posttransplant islet function.

OBJECTIVE: This study investigates the therapeutic potential of hyperbaric oxygen therapy (HBO) in reducing hypoxia and improving engraftment of intraportal islet transplants by promoting angiogenesis. METHODS: Diabetic BALB/c mice were transplanted with 500 syngeneic islets intraportally and received six consecutive twice-daily HBO treatments (n = 9; 100% oxygen for 1 h at 2.5 atmospheres absolute) after transplantation. Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) was used to assess new vessel formation at postoperative days (POD) 3, 7, and 14. Liver tissue was recovered at the same time points for correlative histology, including: hematoxylin and eosin, hypoxia-inducible factor (HIF1α), Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL), vascular endothelial growth factor (VEGF), and von Willebrand Factor immunohistochemistry. RESULTS: HBO therapy significantly reduced HIF-1α, TUNEL and VEGF expression in islets at POD 7. In the non-HBO transplants, liver enhancement on DCE MRI peaked at POD 7 consistent with less mature vasculature but this enhancement was suppressed at POD 7 in the HBO-treated group. The number of new peri-islet vessels at POD 7 was not significantly different between HBO and control groups. CONCLUSION: These results are consistent with a hyperbaric oxygen-mediated decrease in hypoxia that appeared to enhance vessel maturation in the critical days following intraportal islet transplantation.

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.

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.