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In the present study, we found HIE induced serious brain damage including cerebral infarct and swelling, and neuron loss, then differentially expressed proteins were detected by the protein chip. According to venny2.1 and biometric analysis, PDGF-AA was selected ultimately. Then, IF, IHC and WB results verified the localization and expression of PDGF in vivo. To further investigate the role of PDGF in vitro , functional analysis of PDGF knockdown and Tuj1 staining showed that suppression of PDGF induced neuronal apoptosis and inhibited neurite regeneration in the OGD neurons. Furthermore, the potential mechanism may be associated with downregulation of P-PI3K and P-AKT. HI: hypoxia ischemia. IF: immunofluorescent staining. IHC: Immunohistochemistry. PCR: quantitative polymerase chain reaction. WB: Western Blot. Tuj1: Neuronal Class III β-Tubulin. OGD: Oxygen Glucose Deprivation. • The expression of PDGF is increased by stress after HIE. • Inhibition of PDGF leads to neuronal loss and shorten neurite length. • The mechanism may be associated with phosphorylation of P-PI3K and P-AKT pathways. Neonatal hypoxic-ischemic encephalopathy (HIE) always results in severe neurologic dysfunction, nevertheless effective treatments are limited and the underlying mechanism also remains unclear. In this study, we firstly established the neonatal HIE model in the postnatal day 7 SD rats, Zea-Longa score and TTC staining were employed to assess the neurological behavior and infarct volume of the brain after cerebral hypoxia-ischemia (HI). Afterwards, protein chip was adopted to detect the differential proteins in the right cortex, hippocampus and lung, ultimately, PDGF was noticed. Then, immunohistochemistry, immunofluorescence double staining of NeuN/PDGF, and western blot were used to validate the expression level of PDGF in the cortex and hippocampus at 6 hours (h), 12 h and 24 h after HI. To determine the role of PDGF, the primary cortical neurons were prepared and performed PDGF shRNA administration. The results showed that HIE induced a severe behavioral dysfunction and brain infarction in neonatal rats, and the expression of PDGF in right cortex and hippocampus was remarkably increased after HI. Whereas, suppressing PDGF resulted in a significant loss of neurons and inhibition of neurite growth. Moreover, the protein level of P-PI3K and P-AKT signaling pathways were largely decreased following PDGF-shRNA application in the cortical neurons. In conclusion, PDGF suppression aggravated neuronal dysfunction, and the underlying mechanism is associated with inhibiting the phosphorylation of P-PI3K and P-AKT. Together, PDGF regulating PI3K and AKT may be an important panel in HIE events and therefore may provide possible strategy for the treatment of HIE in future clinic trail. [ABSTRACT FROM AUTHOR]

Glial cell line-derived neurotrophic factor (GDNF) is regarded as a potent neuroprotector and a corrector of neural network activity in stress conditions. This work aimed to investigate the effect of GDNF on primary hippocampal cultures during acute normobaric hypoxia. Hypoxia induction was performed using day 14 in vitro cultures derived from mouse embryos (E18) with the preventive addition of GDNF (1 ng/ml) to the culture medium 10 min before oxygen deprivation. An analysis of spontaneous bioelectrical activity that included defining the internal neural network structure, morphological studies, and viability tests was performed during the post-hypoxic period. This study revealed that GDNF does not influence spontaneous network activity during normoxia but protects cultures from cell death and maintains the network activity during hypoxia. GDNF created unique conditions that supported the viability of cells even in cases of cellular mitochondrial damage. GDNF partially negated the consequences of hypoxia by influencing synaptic plasticity. Intravital mRNA detection identified fewer GluR2 mRNA-positive cells, whereas GDNF preserved the number of these cells in the post-hypoxic period. Activation of the synthesis of GluR2 subunits of AMPA-receptors is one possible mechanism of the neuroprotective action of GDNF. [ABSTRACT FROM AUTHOR]

[Display omitted] • Ketamine-xylazine mixture modestly decreases brain and body temperature. • This drug mixture induces metabolic brain activation and skin vasodilation. • Ketamine increases temperature due to metabolic activation and skin vasoconstriction. • Xylazine alone decreases temperature due to strong skin vasodilation. • Ketamine-xylazine mixture induces modest but prolonged brain hypoxia. Among the numerous general anesthetics utilized in rodent surgical procedures, the co-administration of ketamine and xylazine is the current standard for induction and maintenance of surgical planes of anesthesia and pain control. In contrast to classical GABAergic anesthetics, which act to inhibit CNS activity, inducing muscle relaxation, sedation, hypothermia, and brain hypoxia, ketamine and xylazine act through different mechanisms to induce similar effects while also providing potent analgesia. By using three-point thermorecording in freely moving rats, we show that the ketamine-xylazine mixture induces modest brain hyperthermia, resulting from increased intra-cerebral heat production due to metabolic brain activation and increased heat loss due to skin vasodilation. The first effect derives from ketamine, which alone increases brain and body temperatures due to brain metabolic activation and skin vasoconstriction. The second effect derives from xylazine, which increases heat loss due to potent skin vasodilation. By using oxygen sensors coupled with amperometry, we show that the ketamine-xylazine mixture modestly decreases brain oxygen levels that results from relatively weak respiratory depression. This tonic pharmacological effect was preceded by a strong but transient oxygen increase that may result from a stressful injection or unknown, possibly peripheral action of this drug combination. This pattern of physiological effects elicited by the ketamine-xylazine mixture differs from the effects of other general anesthetic drugs, particularly barbiturates. [ABSTRACT FROM AUTHOR]

The incidence of preterm birth is rising worldwide. Among preterm infants, many face a lifetime of neurologic impairments. Recent studies have revealed that systemic inflammation can sensitize the immature brain to hypoxic-ischemic (HI) injury. Therefore, it is important to identify the mechanisms involved in inflammation-sensitized HI injury in immature brains. PTEN-induced putative kinase 1 (PINK1) is a regulatory protein that is highly expressed in the brain. We have previously found that PINK1 gene knockout can protect matured brains from HI injury in postnatal day 10 mice. However, the mechanisms are unknown. In this study, we employed an inflammation-sensitized HI injury model using postnatal day 3 mice to study the roles and mechanisms that PINK1 plays in the immature brains. Lipopolysaccharide (LPS) was injected intraperitoneally into the mice before HI treatment to set up the model. We found that PINK1-knockout mice had fewer brain infarcts and less cell apoptosis than did the wild-type mice. Furthermore, we found that α-synuclein was markedly higher in the PINK1-knockout mice than in the wild-type mice, and inhibition of α-synuclein through small interfering RNA (siRNA) reversed the protective effect in the PINK1-knockout mice. Collectively, these findings indicate that loss of PINK1 plays a novel role in the protection of inflammation-sensitized HI brain damage. [ABSTRACT FROM AUTHOR]

[Display omitted] • Abnormal autonomic activity correlated with outcomes in cerebral ischemia. • Unstable sleep patterns correlated with outcomes in cerebral ischemia. • The sympathetic activity decreased under cerebral ischemia with sleep apnea. • The time of paradoxical sleep decreased under cerebral ischemia with sleep apnea. Sleep apnea (SA) is characterized by intermittent hypoxia (IH), which increases sympathetic activity and sleep fragmentation, thus increasing the risk of stroke. SA is a highly prevalent disease and can worsen prognosis in patients with stroke. However, the correlation of changes in the cardiac autonomic nervous system and sleep patterns under IH with sensorimotor behavior and cerebral infarction after stroke remains unclear. We hypothesized that dysregulated autonomic activity and unstable sleep patterns induced by IH and correlated with cerebral infarction and abnormal sensorimotor behavior after middle cerebral artery occlusion (MCAO). Wistar–Kyoto rats (WKY) were divided into IH (hypoxia: 5 % O 2, 8 h/day) and RA group (room air) for 2 weeks and both groups were subjected to MCAO. After MCAO, the IH group was continuously exposed to IH for 1 week. The 24-h physiological signals, blood pressure, and sensorimotor behavior were recorded at baseline (Bas), the first and second weeks during IH (RA/IH1W and RA/IH2W, respectively), and poststroke. Before MCAO, IH caused sympathetic activity during sleep and parasympathetic activity of active waking (AW) to increase. Moreover, IH reduced the accumulated time and duration of paradoxical sleep (PS) and increased the interruption during sleep. After MCAO, IH increased blood pressure, more severe brain damage, and poor sensorimotor performance. Moreover, IH reduced autonomic activity after MCAO and decreased sympathetic activity was associated with poor sensorimotor performance. Autonomic activity and sleep patterns affected by IH were correlated with increased cerebral infarction and poor sensorimotor behavior after MCAO. [ABSTRACT FROM AUTHOR]

The aim of the present study was to determine the effect of high altitude (HA) immigration on cerebral electrical activity. Electro-EncephaloGraphic (EEG) activity were recorded from 25 lowland soldiers during eyes-closed resting conditions under the following conditions: 7 days at lowland before ascending to altitude (Test 1), during the first 7 days (Test 2) and 30 days (Test 3) at 3800 m altitude, and 7 days after return to lowland (Test 4). The EEG was Fourier transformed to provide absolute and relative power estimates for the alpha, beta, delta, and theta bands. HA immigrants showed changes of EEG power confined in the posterior parietal cortex, right posterior temporal cortex, and occipital cortex. Compared with baseline Test 1, acute acclimatization (Test 2) only decreased theta power; chronic acclimatization (Test 3) discriminately increased alpha and beta powers but decreased delta power; after descending to lowland (Test 4), alpha power decreased, beta power remained increase, but delta and theta power recovered to the baseline level. Our findings demonstrated different EEG patterns during hypoxia exposure at HA as time goes on and after following reoxygenation at lowland, showing hypoxia decreased lower EEG frequencies while hypoxia/reoxygenation increased higher EEG frequencies. Our findings supports for the hypothesis that certain behavioral and physiological changes induced by sojourn at altitude could be caused by alterations in central nervous system function. [ABSTRACT FROM AUTHOR]

Hypertension is associated with cerebrovascular disease, white matter lesion and cognitive deficit, both in experimental models and clinical observations. Furthermore, in non-clinical models it is shown that hippocampus is affected by hypertension and hypoxia. Herein, two distinct hypertension models were used to study neurogenic response in hippocampus. Dahl salt sensitive (DSS) rat model is a genetic based idiopathic model, while chronic intermittent hypoxia (CIH) mimics the hypertension observed in patients with obstructive sleep apnea (OSA). Both models are chronic and trigger hypertension. No macroscopic alterations based on histological analysis were found in hippocampus derived from DSS and CIH exposure rats. Nevertheless, in hippocampus derived from CIH-induced hypertensive rats, there was a decrease on neuronal population (MAP2 and NeuN positive cells) and an increase on astrocytic marker GFAP. Accordingly, a higher increase on Ki67 expressing cells was found in dentate gyrus (DG) region, suggesting an enhancement of cell proliferation, concomitantly with an increase of Nestin staining, which indicates the presence of immature neurons under differentiation. While, in hippocampus of DSS rats with or without high salt diet, there was no remarkable difference indicating potential neuronal loss, astrocytic activation or neurogenesis. Furthermore, in both models hypertension did not alter the levels of expression of the stress response enzyme heme oxygenase-1 in DG. These data indicate that intermittent hypoxia might be the key factor involved in neurogenesis modulation in hippocampus. Furthermore, two hypotheses can be explored: (i) activation of neurogenesis is a response against neuronal loss induced by hypertension and/or hypoxia or (ii) neurogenesis can be directly stimulated by hypoxia as a neuroprotective mechanism. [ABSTRACT FROM AUTHOR]

Background Hypoxia is a critical secondary injury mechanism in traumatic brain injury (TBI), and early intervention to alleviate post-TBI hypoxia may be beneficial. NVX-108, a dodecafluoropentane perfluorocarbon, was screened for its ability to increase brain tissue oxygen tension (PbtO 2 ) when administered soon after TBI. Methods Ketamine-acepromazine anesthetized rats ventilated with 40% oxygen underwent moderate controlled cortical impact (CCI)-TBI at time 0 (T0). Rats received either no treatment (NON, n =8) or 0.5 ml/kg intravenous (IV) NVX-108 (NVX, n =9) at T15 (15 min after TBI) and T75. Results Baseline cortical PbtO 2 was 28±3 mm Hg and CCI-TBI resulted in a 46±6% reduction in PbtO 2 at T15 ( P <0.001). Significant differences in time-group interactions ( P =0.013) were found when comparing either absolute or percentage change of PbtO 2 to post-injury (mixed-model ANOVA) suggesting that administration of NVX-108 increased PbtO 2 above injury levels while it remained depressed in the NON group. Specifically in the NVX group, PbtO 2 increased to a peak 143% of T15 ( P =0.02) 60 min after completion of NVX-108 injection (T135). Systemic blood pressure was not different between the groups. Conclusion NVX-108 caused an increase in PbtO 2 following CCI-TBI in rats and should be evaluated further as a possible immediate treatment for TBI. [ABSTRACT FROM AUTHOR]

Abstract: Dynamic microvascular remodeling maintains an optimal continuous supply of oxygen and nutrients to the brain to account for prolonged environmental variations. The objective of this study was to determine the relative time course of capillary regression during re-oxygenation after exposure to prolonged moderate hypoxia and expression of the primary signaling factors involved in the process. Four-month old male C57BL/6 mice were housed and maintained in a hypobaric chamber at 290Torr (0.4atm) for 21 days and allowed to recover at normoxia (room air) for up to 21 days. The mice were either decapitated or perfused in-situ and brain samples collected were either homogenized for Western blot analysis or fixed and embedded in paraffin for immunohistochemistry. Hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and erythropoietin (EPO) expression were increased during hypoxic exposure and diminished during subsequent re-oxygenation. However, cyclooxygenase-2 (COX-2) and angiopoietin-2 (Ang-2) were both elevated during hypoxia as well as subsequent re-oxygenation. Significantly increased capillary density at the end of the 3rd week of hypoxia regressed back toward normoxic baseline as the duration of re-oxygenation continued. In conclusion, elevated COX-2 and Ang-2 expression during hypoxia where angiogenesis occurs and re-oxygenation, when micro-vessels regress, identifies these proteins as vascular remodeling molecules crucial for angioplasticity. [Copyright &y& Elsevier]

Abstract: Despite efforts to reduce mortality caused by stroke and perinatal asphyxia, these are still the 2nd largest cause of death worldwide in the age groups they affect. Furthermore, survivors of cerebral hypoxia-ischemia often suffer neurological morbidities. A better understanding of pathophysiological mechanisms in focal and global brain ischemia will contribute to the development of tailored therapeutic strategies. Similarly, insight into molecular pathways involved in preconditioning-induced brain protection will provide possibilities for future treatment. Microarray technology is a great tool for investigating large scale gene expression, and has been used in many experimental studies of cerebral ischemia and preconditioning to unravel molecular (patho-) physiology. However, the amount of data across microarray studies can be daunting and hard to interpret which is why we aim to provide a clear overview of available data in experimental rodent models. Findings for both injurious ischemia and preconditioning are reviewed under separate subtopics such as cellular stress, inflammation, cytoskeleton and cell signaling. Finally, we investigated the transcriptome signature of brain protection across preconditioning studies in search of transcripts that were expressed similarly across studies. Strikingly, when comparing genes discovered by single-gene analysis we observed only 15 genes present in two studies or more. We subjected these 15 transcripts to DAVID Annotation Clustering analysis to derive their shared biological meaning. Interestingly, the MAPK signaling pathway and more specifically the ERK1/2 pathway geared toward cell survival/proliferation was significantly enriched. To conclude, we advocate incorporating pathway analysis into all microarray data analysis in order to improve the detection of similarities between independently derived datasets. [Copyright &y& Elsevier]

Abstract: Umbilical cord blood mononuclear cells (UCBMC) transplantation may improve hypoxia-induced brain injury in neonatal rats, but the mechanism is unclear. This study examines whether UCBMC promote neural stem cell (NSC) proliferation via the Sonic hedgehog (Shh) signaling pathway. The rats underwent left carotid ligation followed by hypoxic stress. UCBMC were transplanted 24h after hypoxia ischemia (HI), and immunohistochemistry, immmunoblotting, and morphology analyses were performed at different time points after transplantation. Increased numbers of NSCs were observed in the subventrical zone (SVZ) of the HI+UCBMC group, but these increases were attenuated by cyclopamine treatment. There were significant increases in Shh and Gli1 protein levels after transplantation in the HI group treated with UCBMC compared to HI rats treated with phosphate-buffered solution (PBS). Significantly more Gli1+DAPI+ cells were observed in the SVZ of the HI+UCBMC group compared to the HI+PBS and N+UCBMC groups, but few Gli1+DAPI+ cells were found in the SVZ of the HI+cyclopamine+UCBMC group. The HI+UCBMC group had significantly less neuronal loss in the cortex and CA1 sector of the hippocampus compared to the HI+PBS group, but more neuron loss was observed in the HI+cyclopamine+UCBMC group compared to HI+UCBMC. These results indicate that UCBMC may promote NSC proliferation and alleviate brain injury in HI neonatal rats via Shh signaling. [Copyright &y& Elsevier]

Abstract: Recent studies suggest that neuron–glia interactions are involved in multiple aspects of neuronal activity regulation. In the nucleus tractus solitarius (NTS) neuron–glia interactions are thought to participate in the integration of autonomic responses to physiological challenges. However, it remains to be shown whether NTS glial cells might influence breathing and cardiovascular control, and also if they could be integral to the autonomic and respiratory responses to hypoxic challenges. Here, we investigated whether NTS glia play a tonic role in the modulation of central respiratory and sympathetic activities as well as in the changes in respiratory–sympathetic coupling induced by exposure to chronic intermittent hypoxia (CIH), a model of central autonomic and respiratory plasticity. We show that bilateral microinjections of fluorocitrate (FCt), a glial cell inhibitor, into the caudal and intermediate subnuclei of the NTS did not alter baseline respiratory and sympathetic parameters in in situ preparations of juvenile rats. Similar results were observed in rats previously exposed to CIH. Likewise, CIH-induced changes in respiratory–sympathetic coupling were unaffected by FCt-mediated inhibition. However, microinjection of FCt into the ventral medulla produced changes in respiratory frequency. Our results show that acute glial inhibition in the NTS does not affect baseline respiratory and sympathetic control. Additionally, we conclude that NTS glial cells may not be necessary for the continuous manifestation of sympathetic and respiratory adaptations to CIH. Our work provides evidence that neuron–glia interactions in the NTS do not participate in baseline respiratory and sympathetic control. [Copyright &y& Elsevier]

Abstract: Traumatic brain injury (TBI) leads to a rapid and excessive glutamate elevation in the extracellular milieu, resulting in neuronal degeneration and astrocyte damage. Posttraumatic hypoxia is a clinically relevant secondary insult that increases the magnitude and duration of glutamate release following TBI. N-acetyl-aspartyl glutamate (NAAG), a prevalent neuropeptide in the CNS, suppresses presynaptic glutamate release by its action at the mGluR3 (a group II metabotropic glutamate receptor). However, extracellular NAAG is rapidly converted into NAA and glutamate by the catalytic enzyme glutamate carboxypeptidase II (GCPII) reducing presynaptic inhibition. We previously reported that the GCPII inhibitor ZJ-43 and its prodrug di-ester PGI-02776 reduce the deleterious effects of excessive extracellular glutamate when injected systemically within the first 30min following injury. We now report that PGI-02776 (10mg/kg) is neuroprotective when administered 30min post-injury in a model of TBI plus 30min of hypoxia (FiO2=11%). 24h following TBI with hypoxia, significant increases in neuronal cell death in the CA1, CA2/3, CA3c, hilus and dentate gyrus were observed in the ipsilateral hippocampus. Additionally, there was a significant reduction in the number of astrocytes in the ipsilateral CA1, CA2/3 and in the CA3c/hilus/dentate gyrus. Administration of PGI-02776 immediately following the cessation of hypoxia significantly reduced neuronal and astrocytic cell death across all regions of the hippocampus. These findings indicate that NAAG peptidase inhibitors administered post-injury can significantly reduce the deleterious effects of TBI combined with a secondary hypoxic insult. [Copyright &y& Elsevier]

Abstract: Prolonged, moderate cerebral hypothermia is consistently neuroprotective after experimental hypoxia–ischemia. We have previously shown that hypothermia is also protective after profound asphyxia in the preterm brain. However, there is a concern whether hypothermia could suppress the proliferative response to injury in the white matter or subventricular zone (SVZ). Preterm (0.7 gestation) fetal sheep received complete umbilical cord occlusion for 25min followed by cerebral hypothermia (extradural temperature reduced from 39.4±0.3 to 29.5±2.6°C) from 90min to 70h after the end of occlusion or sham cooling. Occlusion-normothermia was associated with no effect on CNPase+ cells, but loss of O4+ oligodendrocytes, induction of cleaved caspase-3, and IB4+ microglia in the gyral and periventricular white matter compared to sham-occlusion (p < 0.05), with a significant increase in KI67+ cells in the periventricular white matter (p < 0.05). Hypothermia was associated with significant protection of O4+ cells, with suppression of IB4+ microglia and KI67+ cells in the periventricular white matter. There was no significant change in astrocytes, microglia, KI67+, or caspase-3+ cells in the SVZ after asphyxia. In conclusion, this study provides strong support for the selective vulnerability of immature oligodendrocytes to a highly relevant insult in the fetal sheep. Although white matter protection with cerebral hypothermia was associated with reduced proliferation in the white matter tracts, it did not impair proliferation in the SVZ. [Copyright &y& Elsevier]

Abstract: Mounting evidence suggests that the injury of oligodendrocyte progenitor cells (OPCs) caused by hypoxia plays a pivotal role in periventricular white matter injury (PWMI) causation. We investigated the potential role of active extracellular domain of Neuregulin1 isotypeβ1 (NRG1β1)/ErbB signaling in protecting OPCs from oxygen glucose deprivation (OGD) induced apoptosis. At different time points, endogenous NRG1β1 protein was analyzed after OGD. Escalating dosages of NRG1β1 were used to treat OPCs with OGD, and the apoptosis was measured, as well as the expression of ErbB receptors, Akt and Erk phosphorylation and caspase3 activation. OGD damage resulted in decreased expression of endogenous NRG1β1. In parallel, NRG1β1 treatment promoted the expression of p-ErbB4 receptor, phosphorylated Akt and inhibited caspase3 activation. Furthermore, the activation of PI3-kinase/Akt by NRG1β1 was ErbB4 dependent. Our data demonstrated that NRG1β1 protected OPCs from OGD induced apoptosis and the possible protective mechanism is linking with ErbB4-dependent activation of PI3-kinase/Akt pathway. [Copyright &y& Elsevier]

Abstract: The response of differentiating mouse neural progenitor cells, migrating out from neurospheres, to conditions simulating ischemia (hypoxia and extracellular or intracellular acidosis) was studied. We show here, by using BCECF and single cell imaging to monitor intracellular pH (pHi), that two main populations can be distinguished by exposing migrating neural progenitor cells to low extracellular pH or by performing an acidifying ammonium prepulse. The cells dominating at the periphery of the neurosphere culture, which were positive for neuron specific markers MAP-2, calbindin and NeuN had lower initial resting pHi and could also easily be further acidified by lowering the extracellular pH. Moreover, in this population, a more profound acidification was seen when the cells were acidified using the ammonium prepulse technique. However, when the cell population was exposed to depolarizing potassium concentrations no alterations in pHi took place in this population. In contrast, depolarization caused an increase in pHi (by 0.5 pH units) in the cell population closer to the neurosphere body, which region was positive for the radial cell marker (GLAST). This cell population, having higher resting pHi (pH 6.9–7.1) also responded to acute hypoxia. During hypoxic treatment the resting pHi decreased by 0.1 pH units and recovered rapidly after reoxygenation. Our results show that migrating neural progenitor cells are highly sensitive to extracellular acidosis and that irreversible damage becomes evident at pH 6.2. Moreover, our results show that a response to acidosis clearly distinguishes two individual cell populations probably representing neuronal and radial cells. [Copyright &y& Elsevier]

Abstract: Glomus cells within the carotid body are known to respond to hypoxic stimuli. Recently, these cells have been shown to express the long form of the leptin receptor (Ob-Rb). However, whether these glomus cells expressing the Ob-Rb are activated by hypoxic stimuli is not known. Therefore, in this study we investigated whether intermittent hypoxia (IH) or changes in circulating levels of leptin induced phosphorylated signal transducer and activator of transcription 3 (pSTAT3), the immediate early gene c-fos protein, or fos-related antigen-1 protein (Fra-1) within carotid body glomus cells that expressed the Ob-Rb, and within neurons of the petrosal (PG) and nodose (NG) ganglia. Rats were subjected to IH (120s normoxia, 80s hypoxia for 8h) or normoxia (8h), or intravenous injections of leptin (50 or 200ng/0.1mL) or the vehicle saline. Plasma leptin levels were measured in animals exposed to IH and normoxia. Exposure to 8h of IH increased plasma leptin levels greater than 2-fold compared to normoxic controls. Animals were then perfused with Zamboni''s fixative, and the region of the carotid bifurcation containing the carotid body and PG/NG complex was removed, paraffin embedded and sectioned at 6μm for immunohistochemical processing. Carotid body glomus cells were identified by their expression of tyrosine hydroxylase immunoreactivity. These glomus cells also expressed the OB-Rb and were found to express pSTAT3-, fos-, and Fra-1-like immunoreactivity in response to both IH and systemic leptin injections. IH and leptin injections also increased fos and Fra-1 like expression in the PG, NG and jugular ganglion. Taken together, these data suggest IH alters circulating leptin which in turn activates directly carotid body glomus cells to exert a modulatory effect on the peripheral chemoreceptor reflex. [Copyright &y& Elsevier]

Abstract: Neonatal hypoxic–ischemic encephalopathy (HI) is a major cause of nervous system damage and neurological morbidity. Perinatal malnutrition affects morphological, biochemical and behavioral aspects of neural development, including pathophysiological cascades of cell death triggered by ischemic events, so modifying resulting brain damage. Female Wistar rats were subjected to protein restriction during pregnancy and lactation (control group: 25% soybean protein; malnourished group: 7%). Seven days after delivery (PND7), their offspring were submitted to unilateral cerebral HI; rats were then tested for sensorimotor (PND7 and PND60) and memory (PND60) functions. Offspring of malnourished mothers showed marked reduction in body weight starting in lactation and persisting during the entire period of observation. There was a greater sensorimotor deficit after HI in malnourished (M) animals, in righting reflex and in home bedding task, indicating an interaction between diet and hypoxia–ischemia. At PND60, HI rats showed impaired performance when compared to controls in training and test sessions of rota-rod task, however there was no effect of malnutrition per se. In the open field, nourished HI (HI-N) presented an increase in crossings number; this effect was not present in HI-M group. Surprisingly, HI-M rats presented a better performance in inhibitory avoidance task and a smaller hemispheric brain damage as compared to HI-N animals. Our data points to a possible metabolic adaptation in hypoxic–ischemic animals receiving protein malnutrition during pregnancy and lactation; apparently we observed a neuroprotective effect of diet, possibly decreasing the brain energy demand, under a hypoxic–ischemic situation. [Copyright &y& Elsevier]

Abstract: Hypoxia–ischemia (HI) induced injury of the neonatal brain accounts for behavioral deficits concerning mainly neurological reflexes, sensorimotor functions and learning/memory disabilities that may evolve throughout development. The positive biological effects of resveratrol, a natural compound with anti-oxidant/anti-inflammatory properties found mainly in red wine have been indicated recently. Aim of this study was to investigate the delayed outcome of early administration of resveratrol in an experimental model of hypoxic–ischemic encephalopathy, by means of behavioral analysis and late neuropathological examination. Seven-day-old (P7) rats were separated into 3 groups: Group 1 underwent HI and treated with resveratrol. Group 2 (HI-treated) was subjected to HI and received same volume of saline. Group 3 (sham-operated) was the control group. A battery of behavioral tests was performed from days P8–P66, during which early reflexes (righting reflex, gait, geotaxis), sensorimotor (rope suspension, beam walking, rotarod) and learning/memory function (passive avoidance, Morris water-maze) were examined. Significant difference among the groups was observed in righting reflex, rotarod and water maze tests in which the resveratrol group almost reached the performance of the control animals. The other behavioral tests showed that control and resveratrol groups were better compared to HI, although not significant. Neuropathology study revealed a remarkable reduction of the infarct and preservation of myelination after resveratrol treatment, which was in most cases correlated with the better performance of the resveratrol group. These findings indicate that long-term neuroprotective effect of resveratrol on neonatal HI-induced gray and white matter damage might be associated with the preservation of behavioral functions. [Copyright &y& Elsevier]

Abstract: The enhanced carotid body (CB) chemosensory response to hypoxia induced by chronic intermittent hypoxia (CIH) has been attributed to oxidative stress, which is expected to increase the expression of chemosensory modulators including chemoexcitatory pro-inflammatory cytokines in the CB. Accordingly, we studied the time-course of the changes in the immunohistological expression of TNF-α, IL-1β, IL-6, ET-1, iNOS, eNOS and 3-nitrotyrosine in the CB, along with the progression of enhanced CB chemosensory responses to acute hypoxia in male Sprague-Dawley rats exposed to CIH (5%O2, 12times/h per 8h) for 7, 14 and 21days. Exposure to CIH for 7days resulted in a sustained potentiation of CB chemosensory responses to acute hypoxia, which persisted until 21days of CIH. The chemosensory potentiation was paralleled by an increased 3-nitrotyrosine expression in the CB. On the contrary, CIH produced a transient 2-fold increase of ET-1 immunoreactivity at 7days, a decrease in eNOS immunoreactivity, and a delayed but progressive increase of TNF-α, IL-1β and iNOS immunoreactivity, which was not associated with changes in systemic plasma levels or immune cell invasion within the CB. Thus, present results suggest that the local expression of chemosensory modulators and pro-inflammatory cytokines in the CB may have different temporal contribution to the CB chemosensory potentiation induced by CIH. [Copyright &y& Elsevier]

Abstract: Brain damage from neonatal hypoxia–ischemia (HI) plays a major role in neonatal mortality and morbidity. Using the Rice-Vannucci model of HI in rats, we verified that 8days after HI injury, adenosine deaminase (ADA), N-acetyl-glucosaminidase (NAG) and myeloperoxidase (MPO) activities increased in the left hemisphere hippocampus (HI group); however, the activity of 5′-nucleotidase (5′NT) remained unchanged. In the hematoxylin–eosin analysis (HE), we detected selective and delayed degeneration of hippocampal pyramidal neurons and astroglial reaction accompanied by glial fibrillary acidic protein (GFAP)-positive and vimentin-positive in the immunohistochemistry analysis in the HI group compared with the control group. We observed the selective necrosis of neurons, vascular endothelial proliferation and inflammatory response accompanied by the increase of the key enzyme of adenosine metabolism in the HI group. The increase of ADA activity, despite the 5′NT activity was not altered, indicates the predominance of ADA activity in the postischemic homeostasis of extra cellular adenosine. The presence of leukocytes into the ischemic areas displays the possible importance of the neutrophil-macrophages associated with the increase of MPO and NAG activities 8days after HI. These findings may contribute to the evaluation of some consequences of the damage caused by neonatal HI. [Copyright &y& Elsevier]

Abstract: To investigate whether whole body hypothermia after neonatal cerebral hypoxia–ischemia (HI) could broaden the therapeutic window of intranasal treatment of IGF-1 (iN-IGF-1), postnatal day 7 rat pups were subjected to right common carotid artery ligation, followed by 8% oxygen inhalation for 2h. After HI, one group of pups were returned to their dams and kept at room temperature (24.5±0.2°C). A second group of pups were subjected to whole body hypothermia in a cool environment (21.5±0.3°C) for 2 or 4h before being returned to their dams. Two doses of 50μg recombinant human IGF-1 were administered intranasally at a 1h interval starting at 0, 2 or 4h after hypothermia. Hypothermia decreased the rectal temperature of pups by 4.5°C as compared to those kept at room temperature. While hypothermia or iN-IGF-1 administered 2h after HI alone did not provide neuroprotection, the combined treatment of hypothermia with iN-IGF-1 significantly protected the neonatal rat brain from HI injury. Hypothermia treatment extended the therapeutic window of IGF-1 to 6h after HI. The extended IGF-1 therapeutic window by hypothermia was associated with decreases in infiltration of polymorphonuclear leukocytes and activation of microglia/macrophages and with attenuation of NF-κB activation in the ipsilateral hemisphere following HI. [Copyright &y& Elsevier]

Abstract: Vascular endothelial growth factor (VEGF) is an angiogenic inducer and neurotrophic factor both in adult and neonatal animal models. In the destructive lesions of the developing human brain, the role and expression of VEGF and of its mitogenic receptor VEGFR-2 have been hardly studied. The aim of the present work was to determine the immunohistochemical distribution of VEGF and VEGFR-2 in premature and full-term infants presenting with hypoxic/ischemic lesions, and to compare results with normal infant brains at similar developmental stages. Paraffin embedded brain tissue samples were assessed using anti-human VEGF and VEGFR-2 antibodies. In all undamaged forebrain areas, VEGF and VEGFR-2 displayed same expression patterns in control and pathologic brains, whatever the destructive lesion occurrence''s time (before 25weeks of gestation (WG), between 25 and 34WG, perinatal period and infancy). In the destructive lesions, VEGF was always expressed in neurons, astrocytes and in neovessel walls, contrary to VEGFR-2 which was only expressed in dispersed astrocytes. VEGF was expressed in oligodendrocytes of prenatally damaged brains, whereas VEGF was expressed in these cells in undamaged areas from birth only, similarly to control brains. These data suggest that VEGF plays specific roles and may not be mediated by VEGFR-2 in human forebrain structures exposed to ischemia. [Copyright &y& Elsevier]

Abstract: We have designed a clinically relevant model of perinatal asphyxia providing intrapartum hypoxia in rats. On gestation day 22 SD rats were anesthetized and the uterine horns were exteriorized and placed in a water bath at 37°C for up to 20min. After this, pups were delivered from the uterus and manually stimulated to initiate breathing in an incubator at 37°C for 1 h in air. Brains were harvested and stained with cresyl violet, caspase-3, and TUNEL to detect morphological and apoptotic changes on postnatal days (PND) 1, 3, and 7. Separate cohorts were maintained until PND 50 and tested for learning and memory using Morris water maze (WM). Survival rate was decreased with longer hypoxic time, and 100% mortality was noted when hypoxia time was beyond 18min. Apoptosis was increased with the duration of hypoxia with neuronal loss and cell shrinkage in the CA1 of hippocampus. The time taken for the juveniles to locate the hidden platform during WM was increased in animals subjected to hypoxia. These data demonstrate that perinatal ischemic injury leads to neuronal death in the hippocampus and long-lasting cognitive dysfunction. This model mimics hypoxic ischemic encephalopathy in humans and may be appropriate for investigating therapeutic interventions. [Copyright &y& Elsevier]

Abstract: The present study investigated the role of neuronal nitric oxide synthase (nNOS)/nitric oxide (NO) system in the pathophysiologic regulation of hypoxic–ischemic brain damage (HIBD) in baby rats subjected to electrical stimulation (ES). The motor function, NO concentration in cortex, and protein expressions of nNOS were examined after 14 sessions of ES. Results showed that NO levels in cortex significantly increased 24h after hypoxia–ischemia than sham. ES could improve motor functions in HIBD rats and spontaneously decrease nNOS/NO system. In conclusion, the nNOS/NO pathway might play a critical role as mediator of neuronal recovery in HIBD rats after undergoing ES treatment. [Copyright &y& Elsevier]

Abstract: N-myc downstream regulated gene 2 (NDRG2) was reported to be widely expressed in the nervous system. However, the expression and potential role of NDRG2 in focal cerebral ischemia brain remain unclear. Herein, we investigated spatial–temporal expression of NDRG2 in the rat brain following transient focal cerebral ischemia. Male Sprague–Dawley rats underwent a 120-min transient occlusion of middle cerebral artery. Rats were killed and brain samples were harvested at 4, 12, 24, and 72h after reperfusion. Expression of NDRG2 in the brain was determined by reverse transcriptase–polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemical staining. Cellular apoptosis was assessed by TUNEL staining. The results showed that NDRG2 was expressed on cells with an astrocytes-like morphology in ischemic penumbra. NDRG2 mRNA and protein expression began to increase at 4h after reperfusion and peaked at 24h in the ischemic penumbra. By using immunofluorescence, NDRG2 signals were co-localized with GFAP-positive astrocytes, and NDRG2 expression in astrocytes translocated from a cytoplasm to a nuclear localization at 24h after reperfusion. Double immunofluorescent staining for TUNEL and NDRG2 showed that some NDRG2 signals co-localized with TUNEL-positive cells, and that the apoptotic cells increased with enhancement of NDRG2-positive signals. In conclusion, NDRG2 expression is up-regulated in ischemic penumbra following transient focal cerebral ischemia. NDRG2 expression in astrocytes may play important pathological roles in cell apoptosis after stroke. [Copyright &y& Elsevier]

Abstract: The cyclin dependent kinase 5 (Cdk5)/p35 complex is essential for regulation of cell survival during development and in models of neuronal excitotoxicity. Dysregulation of Cdk5, by cleavage of its neuronal specific activators p35 and p39, has been implicated in various neurodegenerative disorders such as Alzheimer''s disease, however targets of the complex that regulate neuronal survival physiologically and/or during pathogenesis are largely unknown. Since hypoxia is a key feature in the pathogenesis of several neuronal disorders we investigated a role for Cdk5/p35 in the neuronal hypoxic response. Our data show that hypoxia modulates the p35/Cdk5 complex in primary cortical neurons at the transcriptional and protein level. Furthermore hypoxic induction of Cdk5 activity correlates with Hif-1α stabilisation, and direct interaction between these proteins can occur. Importantly, we demonstrate that Cdk5-mediated signaling is involved in Hif-1α stabilisation since inhibition of Cdk5 by roscovitine abrogates Hif-1α accumulation and induces cell death. Taken together our results show that the Cdk5/p35 complex may significantly contribute to modulation of Hif-1α stabilisation and impact neuronal survival during oxygen deprivation. Thus this study highlights a new hypoxia-mediated signaling pathway and implicates the cytoskeleton as a potential regulator of Hif-1α. [Copyright &y& Elsevier]

Abstract: Effects of mild (preconditioning) and severe injurious hypobaric hypoxia (SH), as well as of their combination on hippocampal expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors and HPA axis activity have been examined in rats. As revealed by quantitative immunocytochemistry, three-trial exposure to mild hypoxia produced robust GR and MR overexpression located mainly in the neuronal nuclei in the dentate gyrus (DG) but only MR overexpression was observed in the CA1. SH induced sharp reduction of MR levels and enhanced GR expression in the CA1, suggesting that the unbalance of GR and MR observed might be at the bottom of the extensive neuronal loss seen in this area in response to SH. Contrastingly, SH in tolerant (preconditioned) rats failed to imbalance GR and MR expression in CA1 and up-regulated GR levels in DG. Radioimmunoassay of serum corticosterone showed that both preconditioning hypoxia itself and SH in tolerant rats produced moderate activation of HPA axis followed by its proper inactivation. In the non-preconditioned rats, HPA axis response to SH was impaired. Taken together, these novel results suggest that modifications of the hippocampal expression of GR and MR produced by preconditioning may contribute to the molecular and neuroendocrine mechanisms of tolerance to severe hypoxic stress. [Copyright &y& Elsevier]

Abstract: In the present study, we investigated the effects of sabiporide, a specific NHE-1 inhibitor, on blood–brain barrier (BBB) endothelial dysfunction during ischemia/hypoxia in rat cerebral ischemia in vivo and bEnd.3 cells in vitro. Sabiporide significantly attenuated ischemia/hypoxia-induced BBB hyperpermeability and disruption of occludin and zonula occludens-1 both in vivo and in vitro. Sabiporide also inhibited the hypoxia-induced increase in [Ca2+]i in bEnd.3 cells. Taken together, the protective effect of sabiporide on BBB permeability may be mediated through maintaining tight junction integrity and associated with its inhibitory effect on [Ca2+]i overload under hypoxia. These results suggest that the BBB protective effect of sabiporide may be of therapeutical benefit in brain injury after an ischemic stroke. [ABSTRACT FROM AUTHOR]

Abstract: Angiogenesis is a critical component of mammalian brain adaptation to prolonged hypoxia. Hypoxia-induced angiogenesis is mediated by hypoxia-inducible factor-1 (HIF-1)-dependent transcriptional activation of growth factors, such as vascular endothelial growth factor (VEGF). Microvascular angiogenesis occurs over a 3-week period in the rodent brain. We have recently reported that HIF-1α accumulation and transcriptional activation of HIF target genes in the aged cortex of 24-month-old F344 rats is significantly attenuated during acute hypoxic exposure. In the present study, we show that cortical HIF-1α accumulation and HIF-1 activation remain absent during chronic hypoxic exposure in the aged rat brain (24-month-old F344). Despite this lack of HIF-1 activation, there is no significant difference in baseline or post-hypoxic brain capillary density counts between the young (3-month-old F344) and old age groups. VEGF mRNA and protein levels are significantly elevated in the aged cortex despite the lack of HIF-1 activation. Other HIF-independent mediators of hypoxia-inducible genes could be involved during chronic hypoxia in the aged brain. PPAR-γ coactivator (PGC)-1α, a known regulator of VEGF gene transcription, is elevated in the young and aged cortex during the chronic hypoxic exposure. Overall, our results suggest a compensatory HIF-1-independent preservation of hypoxic-induced microvascular angiogenesis in the aged rat brain. [ABSTRACT FROM AUTHOR]

Abstract: The current study obtained multi-modal MRI data from 28 immigrant high altitude (HA) young adults who were born and grew up at Qinghai-Tibetan plateau matched with 28 matched sea level (SL) controls. We compared their regional gray matter volumes (VBM) and white matter quality (DAI FA values) as well as resting state brain activity (Regional Homogeneity (ReHo) of BOLD-fMRI). We found that HA residents showed decreased gray matter volume at bilateral anterior insula, bilateral prefrontal cortex, the left precentral, the left cingulate and the right lingual cortex; accompanied by changed FA and ReHo values in relevant and other regions. The resting state activity at the hippocampus and the right insula were increasing with SL relocation. The HA subjects performed worse on a series of working memory tasks, with the ReHo values of several regions as significant predictors of their performance. This study demonstrated the cerebral and functional modifications with chronic high altitude hypoxia. [ABSTRACT FROM AUTHOR]

Abstract: Neonatal hypoxia–ischemia (HI) brain injury involves reactive oxygen species (ROS) and inflammatory responses. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, has cytoprotective effects against oxidative stress and its effect was mediated by NF-E2-related factor-2 (Nrf2), a transcription factor, and heme oxygenase 1 (HO-1) which is one of Nrf2 downstream target genes. This study was undertaken to investigate the neuroprotective mechanisms of SFN in a neonatal HI rat model. Seven-day-old rat pups were subjected to left common carotid artery ligation and hypoxia (8% oxygen at 37°C) for 90min. SFN (5mg/kg) was systemically administered 30min before HI insult. Brain injury was assessed by 2,3,5-triphenyltetrazoliumchloride (TTC), Nissl, TUNEL staining, malondialdehyde (MDA), 8OH-dG level, and caspase-3 activity in the cortex and hippocampus. SFN pretreatment increased the expression of Nrf2 and HO-1 in the brain and reduced infarct ratio at 24h after HI. The number of TUNEL-positive neurons as well as activated macroglia and the amount of 8OH-dG, were markedly reduced after SFN treatment, accompanied by suppressed caspase-3 activity and reduced lipid peroxidation (MDA) level. These results demonstrated that SFN could exert neuroprotective effects through increasing Nrf2 and HO-1 expression. [Copyright &y& Elsevier]

Abstract: Vascular endothelial growth factor A (VEGF) likely plays a role in the hypoxic preconditioning (PC) induced tolerance to subsequent hypoxic–ischemic (HI) injury to the brain. However, limited data is available concerning VEGF in the developing brain after HI following PC. Neuroprotection by VEGF involves activation of Akt which inhibits apoptotic processes that contribute significantly to the brain injury in neonatal HI. We evaluated whether PC provides neuroprotection and affects VEGF, Akt and caspase-3 following HI in the developing rat brain. Newborn rats (6days) were subjected to normoxia (21% O2) or PC (8% O2) for 3h followed by 24h of reoxygenation. The rats then had the right carotid artery permanently ligated followed by 140min of hypoxia (8% O2) (HI or PC+HI). Brains from rats at the corresponding age without any exposure to PC or HI were examined for comparison (Sham). PC significantly reduced brain damage as measured by weight loss of the right hemisphere at 22days after HI and by gross and microscopic morphology. PC amplified and prolonged the induction of mRNA of VEGF splice variants measured by real-time RT-PCR and enhanced the increase in VEGF protein measured by ELISA in brain following HI. PC preserved the phosphorylation of Akt-Ser-473 and diminished the increase in caspase-3 activity in brain following HI. We conclude that PC provides neuroprotection and augments and preserves the increase in VEGF following HI in the newborn rat brain which may play an important role in neuroprotection. [Copyright &y& Elsevier]

Abstract: Serotonergic fibres have a pervasive innervation of hypoxic–ischemic (HI)-affected areas in the neonatal brain and serotonin (5-HT) is pivotal in numerous neurobehaviours that match many HI-induced deficits. However, little is known about how neonatal HI affects the serotonergic system. We therefore examined whether neonatal HI can alter numbers of serotonergic raphe neurons in specific sub-divisions of the midbrain and brainstem since these nuclei are the primary sources of serotonin throughout the central nervous system (CNS). We utilised an established neonatal HI model in the postnatal day 3 (P3) rat pup (right common carotid artery ligation+30min 6% O2) and determined the effects of P3 HI on 5-HT counts in 5 raphe sub-divisions in the midbrain and brainstem one and six weeks later. After P3 HI, numbers of 5-HT-positive neurons were significantly decreased in the dorsal raphe dorsal, dorsal raphe ventrolateral and dorsal raphe caudal nuclei on P10 but only in the dorsal raphe dorsal and dorsal raphe ventrolateral nuclei on P45. In contrast, P3 HI did not alter counts in the dorsal raphe interfascicular and raphe magnus nuclei. We also discovered that P3 HI significantly reduces brainstem SERT protein expression; the key regulator of 5-HT in the CNS. In conclusion, neonatal HI injury caused significant disruption of the brainstem serotonergic system that can persist for up to six weeks after the insult. The different vulnerabilities of serotonergic populations in specific raphe nuclei suggest that certain raphe nuclei may underpin neurological deficits in HI-affected neonates through to adulthood. [Copyright &y& Elsevier]

Abstract: White matter damage is a significant problem in the human pre-term baby. Damage to white matter is usually associated with injury or insults to babies born prematurely, typically before 32weeks'' gestation, however there is increasing evidence of both grey and white matter damage occurring after 32weeks'' gestation. Astrocytes play a vital role in white matter, regulating molecules such as glutamate in the extracellular space and preventing excitotoxic damage to neighbouring oligodendrocytes and axons. We have previously described dramatic changes in grey matter astrocytes in response to a hypoxic/ischemic (H/I) insult around the time of birth. In this study, we have used GFAP immunohistochemistry and Golgi–Kopsch staining to examine the morphology of white matter astrocytes in control neonatal pig brains, and in the brains of animals exposed to the same (perinatal) H/I insult. We demonstrate that the areal percentage of the section occupied by GFAP-immunoreactive processes and cell bodies is significantly decreased (by 46%, P <0.0001) in subcortical white matter from H/I brains. This loss of GFAP was accompanied by alterations in astrocyte morphology and an overall decrease in the size (field of section occupied by an individual astrocyte) of white matter astrocytes from 649μm2 to 426μm2, as revealed by Golgi–Kopsch staining and image analysis. These data suggest that astrocytes may contribute to the pathology of white matter damage following an H/I insult around the time of birth, and suggest that astrocytes may offer a novel target for therapies to improve outcomes after H/I. [Copyright &y& Elsevier]

Abstract: Human seizure disorders represent a heterogeneous collection of neuropathies, many of which are poorly understood. To investigate the etiology of seizure disorders, we have used a group of Drosophila mutants known as the bang-sensitive (BS) paralytics. The BS mutants exhibit seizure-like activity (SLA) following a wide variety of insults including mechanical shock, electrical shock, high frequency light and cold temperatures. In this study, we show that two novel insults, hypoxia and hypercapnia (elevated CO2 levels) are potent triggers of SLA in a number of the BS mutants. We also show that both of these insults, hypoxia and hypercapnia, can trigger SLA in wild-type flies as well. However, we find that the BS mutants are more susceptible than wild-type flies to these insults as they exhibit more SLA following these insults and, in the case of hypercapnia, they exhibit SLA at a lower threshold. In addition, we demonstrate that the BS mutants are more susceptible to the anesthetizing effects of CO2 as compared to wild-type flies. The increased sensitivity to both hypoxia and hypercapnia in these BS mutants suggests possible physiological defects that may underlie seizure susceptibility. [Copyright &y& Elsevier]

Abstract: Nitric oxide (NO) production increases during hypoxia/ischemia–reperfusion in the immature brain and is associated with neurotoxicity. NO at physiologic concentrations has been shown to modulate GABAergic (gamma-aminobutyric acid) synaptic transmission in the adult brain. However, the effects of neurotoxic concentrations of NO (relevant to hypoxia–ischemia) on GABAergic synaptic transmission remain unknown. The present study tests the hypothesis that nNOS is expressed at GABAergic synapses and that exposure to neurotoxic concentrations of NO results in enhanced GABAergic synaptic transmission in cultured hippocampal neurons (days-in-vitro 10–14) prepared from fetal rats. Using double immunocytochemistry techniques, we were able to demonstrate that nNOS is co-localized to both presynaptic and postsynaptic markers of GABAergic synapses. The effects of NO on GABAergic synaptic transmission were then studied using whole cell patch-clamp electrophysiology. Spontaneous and miniature inhibitory postsynaptic currents (sIPSCS and mIPSCs) were recorded prior to and after exposure to 250 μM of the NO donor diethyleneamine/nitric oxide adduct (DETA-NO). Exposure to DETA-NO resulted in increased sIPSCs and mIPSCs frequency, indicating that neurotoxic concentrations of NO enhance GABAergic synaptic transmission in cultured hippocampal neurons. Because GABA synapses appear to be excitatory in the immature brain, this effect may contribute to overall enhanced synaptic transmission and hyperexcitability. We speculate that NO represents one of the mechanisms by which hypoxia–ischemia increases seizure susceptibility in the immature brain. [Copyright &y& Elsevier]

Abstract: Astroglial cells clear extracellular glutamate through the glutamate transporters, GLT-1 and GLAST, and subsequently convert the incorporated glutamate into glutamine by the enzyme, glutamine synthetase (GS). Several forms of acute brain injury are associated with the increased expression of GS and the decreased expression of GLT-1 and/or GLAST, eventually leading to the accumulation of excitotoxic extracellular glutamate concentrations. Although of clinical interest, the actual trigger of these injury-related changes of glial glutamate turnover remains unknown. Our present studies provide evidence that increases in extracellular glutamate, as present in many brain injuries, are sufficient to modulate the expression of glutamate transporters and GS. Subjecting cultured cortical astrocytes to glutamate concentrations of 0.5–20 mM resulted in a 25% loss of GLT-1 and GLAST protein levels after 24 h; GLT-1 and GLAST levels maximally decreased by 40% and 75%, respectively, after 72 h. This decline was not due to astroglial cell death, since glutamate up to 50 mM did not affect the survival of cultured astrocytes within 72 h. Major astrocytic cell death, however, occurred in cultures maintained under severe (4% O2), but not mild (9% O2), hypoxia, as well as in the presence of aspartate (≥20 mM). Glutamate at ≥1 mM induced a prolonged increase of GS expression in contrast to glutamate transporters. Neither the decline of glutamate transporter expression nor the increase in GS expression induced by high extracellular glutamate was further modulated by mild hypoxia. Whereas the stimulatory influences of glutamate on GS expression were prevented by the non-competitive NMDA receptor antagonist, MK801, the inhibitory influences on glutamate transporter expression were neither sensitive to MK801, the non-competitive mGluR5 antagonist, MTEP, nor the non-competitive AMPA receptor antagonist, GYKI52466, implying that glutamate controls glial glutamate transport by a glutamate receptor-independent mechanism. [Copyright &y& Elsevier]

Abstract: Previous work had demonstrated that there was elevated regional cerebral O2 consumption in the brains of a tuberous sclerosis model (Eker rat). We tested the hypothesis that the increased cerebral O2 consumption was related to an increased contribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors to the control of cerebral metabolism. Young (4weeks) male control Long Evans (N =14) and Eker (N =14) rats (70–100g) were divided into control and NBQX (50mg/kg, AMPA antagonist) treated animals. Cerebral regional blood flow (14C-iodoantipyrine) and O2 consumption (cryomicrospectrophotometry) were determined in isoflurane anesthetized rats. We found significantly increased basal O2 consumption (+85%) in the cortex, hippocampus, cerebellum and pons of the Eker rats. Regional cerebral blood flow was also elevated in Eker rats at baseline (+53%) as was cerebral O2 extraction (+22%). NBQX significantly lowered O2 consumption in all regions of the Long Evans rats (−41%). Cerebral O2 consumption was lowered in the cortex to a lesser extent (−24%) and there were no changes in other regions of the Eker rats after NBQX. Cerebral blood flow was reduced in the Long Evans, but not the Eker rats, after NBQX. In conclusion, Eker rats had significantly elevated cerebral O2 consumption, O2 extraction and blood flow, but this was not related to AMPA receptor activation. In fact, the importance of AMPA receptors in the control of basal cerebral O2 consumption was reduced. [Copyright &y& Elsevier]

Abstract: Obstructive sleep apnea is primarily characterized by hypoxemia due to frequent apneic episodes and fragmentation of sleep due to the brief arousals that terminate the apneic episodes. Though neurobehavioral deficits frequently accompany sleep apnea, the relative roles of hypoxia versus sleep fragmentation are difficult to separate in apneic patients. Here, we assessed cognitive function as measured by water maze in the Fischer/Brown Norway (FBN) rat, comparing 24 h of sleep interruption (SI) to 24 h of intermittent hypoxia (IH), in order to dissociate their relative contributions to cognitive impairment. For SI, automated treadmills were used to induce brief ambulation in rats every 2 min, either prior to, or after, initial water maze acquisition training. IH was simulated by cycling environmental oxygen levels between 6% and 19% every 2 min, again either prior to, or after, acquisition. Twenty-four hours of IH exposure had no significant effect on either acquisition or retention, irrespective of whether IH occurred prior to, or after, acquisition. To replicate previous work, another group of rats, exposed to 3 days of IH (10 h/day) prior to acquisition, had impaired performance during acquisition. A comparison of the 24 h IH and 3 day IH findings suggests that a minimum amount of IH exposure is necessary to produce detectable spatial memory impairments. Although SI before acquisition had no effect on acquisition or later retention of the hidden platform location, SI after acquisition robustly impaired retention, indicating that spatial memory consolidation is more susceptible to the effects of sleep disruption than is the acquisition (learning) of spatial information. [Copyright &y& Elsevier]

Abstract: Activation of phospholipase A2, degradation of membrane phospholipids resulting in tissue accumulation of arachidonic acid, and the activation of cyclooxygenase that leads to the formation of prostaglandin and free radicals may occur after hypoxic-ischemic damage. The aim of this study was to investigate the effects of indomethacin, a nonselective cyclooxygenase inhibitor, on caspase activity, glutathione levels and lipid peroxidation in newborn rats with hypoxic-ischemic encephalopathy. The effects of indomethacin were evaluated by measuring caspase-3 and caspase-8 activities and glutathione levels. Lipid peroxidation was evaluated by measuring concentrations of malondialdehyde in rat brains. Seven-day-old rat pups with the Levine-Rice model of hypoxic-ischemic cerebral injury were randomly divided into three study groups. In the indomethacin-treated group, rats were administered three doses of indomethacin, at a dose of 2 mg/kg every 12 h. Sham and the hypoxic-ischemic group of rats were given physiologic saline. The sham group underwent all surgical procedures except for arterial ligation. After 72 hours, the rats were decapitated and brain tissues were evaluated. Caspase-3 and caspase-8 activities and glutathione and malondialdehyde levels were evaluated in all groups. There was an obvious decrease in caspase-3 and caspase-8 activities and depleted glutathione levels were reversed in the indomethacin-treated group compared to the hypoxic-ischemia group (p <0.001). As indomethacin was unable to prevent lipid peroxidation, malondialdehyde concentrations increased to ischemia-induced levels. In conclusion, indomethacin administration after hypoxic-ischemic encephalopathy injury has a neuroprotective effect since it inhibits caspase activity and reverses the depletion of glutathione. However, it also aggravates lipid peroxidation-induced ischemia. [Copyright &y& Elsevier]

Abstract: Hilar basal dendrites form on dentate granule cells following seizures. To determine whether other brain insults cause the formation of hilar basal dendrites, a model of global cerebral hypoxia/ischemia was used. Rats underwent a transient induction of ischemia by occlusion of both common carotid arteries followed by reperfusion. Hippocampal slices were prepared from these animals 1 month after the ischemic insult, and granule cells were labeled with a retrograde tracing technique after biocytin injections into stratum lucidum of CA3b. Ischemic rats had numerous biocytin-labeled granule cells with hilar basal dendrites located at the hilar border of the granule cell layer. Quantitative analysis of ischemic rats compared to controls showed a significant increase in the percentage of biocytin-labeled granule cells with hilar basal dendrites. These data demonstrate that other brain insults in addition to epilepsy may result in the formation of hilar basal dendrites on granule cells. [Copyright &y& Elsevier]

Abstract: Phosphocreatine–Mg-complex acetate (PCr-Mg-CPLX) is a creatine-derived compound that in previous in vitro research was able to increase neuronal creatine independently of the creatine transporter, thus providing hope to cure the hereditary syndrome of creatine transporter deficiency. In previous research we showed that it reproduces in vitro the known neuroprotective effect of creatine against anoxic damage. In the present paper we investigated if PCr-Mg-CPLX reproduces this neuroprotective effect in vivo, too. We used a mouse model of transient middle cerebral artery occlusion. Mice received PCr-Mg-CPLX or a mixture of the two separate compounds phosphocreatine (PCr) and MgSO4, or vehicle. The injections were done 60 min and 30 min before ischemia. Forty-eight hours after ischemia neurological damage was evaluated with Clark''s behavioural tests, then the infarct volume was measured. PCr-Mg-CPLX reduced the infarct volume by 48%, an effect that was not duplicated by the separate administration of PCr and MgSO4 and the neurological damage was decreased in a statistically significant way. We conclude that PCr-Mg-CPLX affords in vivo neuroprotection when administered before ischemia. These results are comparable to previous research on creatine administration in experimental stroke. PCr-Mg-CPLX maintains creatine-like neuroprotective effects in vivo as well as in vitro. Our study suggests that PCr-Mg-CPLX might have a therapeutic role in the treatment of hereditary creatine transporter deficiency and of conditions where there is a high risk of impending stroke or cerebral ischemic damage, like high-risk transient ischemic attacks, open heart surgery, and carotid surgery. [Copyright &y& Elsevier]

Abstract: Glutamate receptor-mediated neurotoxicity is a major mechanism contributing to hypoxic–ischemic brain injury (HIBI). Memantine is a safe non-competitive NMDA receptor blocker characterized by its low affinity and fast unblocking kinetics. Topiramate is an AMPA/KA receptor blocker and use-dependent sodium channel blocker with several other neuroprotective actions and little neurotoxicity. We hypothesized that the coadministration of memantine and topiramate would be highly effective to attenuate HIBI in neonatal rats. Seven-day-old Sprague–Dawley rat pups were subjected to right common carotid artery ligation and hypoxia for 2 h, and then were randomly and blindly assigned to one of four groups: vehicle, memantine, topiramate and combination group. Brain injury was evaluated by gross damage and weight deficit of the right hemisphere at 22d after hypoxic-ischemia (HI) and by neurofunctional assessment (foot-fault test) at 21d post-HI. Acute neuronal injury was also evaluated by microscopic damage grading at 72 h post-HI. Results showed the combination of memantine and topiramate improved both pathological outcome and performance significantly. The drug-induced apoptotic neurodegeneration was assessed by TUNEL staining at 48 h post-HI and the result showed no elevated apoptosis in all observed areas. The result of the experiment indicates the combination therapy is safe and highly effective to reduce brain damage after HIBI. [Copyright &y& Elsevier]

Abstract: The carotid body (CB) is a polymodal sensor which increases its neural output to the nucleus tractus solitarii with a subsequent activation of several reflex cardiopulmonary responses. Current reports identify acetylcholine (ACh) and adenosine triphosphate (ATP) as two essential excitatory neurotransmitters in the cat and rat CBs. This study explored the impact of hypoxia, low glucose, and the two together on the release of both ACh and ATP from two incubated cat CBs. The CBs were prepared with standard procedures in accordance with the policies and regulations of the Institutional Animal Care and Use Committee. When normalized to their controls, a significant increase of ACh in the incubation medium was measured in response to hypoxia, low glucose, and the combined stimuli. When normalized to their controls, a significant increase in ATP in the incubation medium was measured in response to hypoxia and to the combined stimuli. Low glucose generated an increase in ATP which was not statistically significant (P >0.05). Second, normalizing the initial 3–4 or 2–3 min Time Segment of the challenge Stage to the final 3–4 or 2–3 min Time Segment of the control Stage for both ACh and ATP generated significant increases in response to hypoxia, low glucose (ACh only), and the combined stimuli. The data suggested the possibility that in the cat the increased CB neural output in response to low glucose might be due primarily to ACh. [Copyright &y& Elsevier]

Abstract: Hydrogen gas (H2) 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 H2 in moderate (120 min hypoxia) and severe (150 min hypoxia) neonatal HI rat models. We hypothesized that H2 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+H2 n =86). We also tested H2 in middle cerebral artery occlusion (MCAO) in adult rats (MCAO n =9, MCAO+H2 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 H2) was not associated with decreased volume of infarction or decreased concentration of MDA. H2 gas pretreatment (2.9%) was associated with increased infarction volume in neonatal HI. In MCAO in adult rats, H2 gas therapy demonstrated a trend of beneficial effect. Exposure of H2 gas to non-ischemic neonates resulted in a significant increase in brain concentration of MDA. We conclude that 2.9% H2 gas therapy does not ameliorate moderate to severe ischemic damage in neonatal hypoxia–ischemia. [Copyright &y& Elsevier]

Abstract: Reperfusion injury induced by cardiac arrest and resuscitation leads to secondary challenges to the brainstem. A 12-minute cardiac arrest results in about a 50% survival rate in resuscitated rats over a 4-day recovery period. We investigated hypoxic ventilatory response (HVR) to mild hypoxia by measuring the minute volume before and during a brief exposure to 10% oxygen before and following cardiac arrest and resuscitation. Our results indicate that after cardiac arrest and resuscitation the baseline spontaneous ventilation was elevated significantly in all rats due to both increased frequency and tidal volume; HVR in the non-survivor group was essentially absent while the brainstem responsiveness to hypoxia is fully maintained in the survivor group. Thus, the HVR was shown in this study to be a reliable indicator of survival vs. non-survival during early days of recovery following cardiac arrest and resuscitation. [Copyright &y& Elsevier]

Abstract: Cerebral hypoxia–ischemia (HI) represents a major cause of brain damage in the term newborn. This study aimed to examine the short and long-term neuroprotective effect of hydrogen saline (H2 saline) using an established neonatal HI rat pup model. Seven-day-old rat pups were subjected to left common carotid artery ligation and then 90 min hypoxia (8% oxygen at 37 °C). H2 saturated saline was administered by peritoneal injection (5 ml/kg) immediately and again at 8 h after HI insult. At 24 h after HI, the pups were decapitated and brain morphological injury was assessed by 2,3,5-triphenyltetrazolium chloride (TTC), Nissl, and TUNEL staining. Acute cell death, inflammation and oxidative stress were evaluated at 24 h by studying caspase-3 activity, MDA measurement as well as Iba-1 immunochemistry in the brain. At 5 weeks after HI, spontaneous activity test and Morris water maze test were conducted. We observed that H2 saline treatment reduced the caspase activity, MDA, Iba-1 levels, the infarct ratio, and improved the long-term neurological and neurobehavioral functions. H2 saline has potentials in the clinical treatment of HI and other ischemia-related cerebral diseases. [Copyright &y& Elsevier]

Abstract: It is recognized in recent years that activation of δ-opioid receptor (DOR) elicits neuroprotection against hypoxia and ischemia. However, the underlying mechanisms are not well understood yet. Mitochondrial dysfunction plays a key role in hypoxic neuronal injury, but the effect of DOR activation on neurons with a mitochondrial respiratory chain deficiency is poorly elucidated. In this study we tested the effects of DOR activation and inhibition on cultured cortical neurons after inhibiting mitochondrial respiratory chain with sodium azide (NaN3) in days 8 cultures. Neuronal injury was assessed by lactate dehydrogenase release. Changes in DOR proteins were investigated using an antibody against the N-terminus of the DOR, which recognizes the 60, 48, and 32 kDa proteins. Our main findings are that 1) δ- but not μ-opioid receptor activation reduces NaN3-induced neuronal damage, and this neuroprotective effect is abolished by DOR antagonist (naltrindole, NTI); 2) prolonged DOR inhibition with NTI further increases NaN3-induced neuronal damage; 3) NaN3 treatment down-regulates DOR protein levels in neurons, and the 60 and 32 kDa proteins are particularly sensitive; 4) DADLE, besides activating DOR directly, also reverses the decrease of neuronal DOR protein levels induced by NaN3, which may contribute greatly to its neuroprotective effect; 5) NTI reverses NaN3-induced down-regulation of DOR proteins as well, the effect of NTI amplifying NaN3-induced neuronal damage therefore is probably due to its inhibition on DOR activity only. In conclusion, these data suggest that DOR activation plays an important role in neuroprotection against mitochondrial respiratory chain injury. [Copyright &y& Elsevier]

Abstract: Mutations in tau proteins are associated with a group of neurodegenerative diseases, termed tauopathies. To investigate whether over-expressing human tau with P301L mutation also affects stroke-induced brain damage, we performed hypoxia/ischemia (H/I) in young adult P301L tau transgenic mice. Surprisingly, brain infarct volume was significantly smaller in transgenic mice compared to wild-type mice 24 h after H/I induction. TUNEL staining also revealed less brain apoptosis in transgenic mice following H/I. H/I resulted in a significant increase in tau fragments generated by caspase activation and a marked decrease in tau phosphorylation at residue T231 in cortex of wild-type but not transgenic mice. Activation of calpain and caspase-3 following H/I was also reduced in transgenic compared to wild-type mice, as reflected by lower levels of the specific spectrin breakdown products generated by calpain or caspase-3. Finally, basal levels of the glial glutamate transporter, GLT-1, were higher in brains of transgenic as compared to wild-type mice. These results support the idea that enhanced levels of GLT-1 in transgenic mice are responsible for reducing H/I-induced brain damage by decreasing extracellular glutamate accumulation and subsequent calpain and caspase activation. [Copyright &y& Elsevier]