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Ascend to high altitude results in a drastic change in the environmental conditions an individual is exposed to. As the altitude increases there is a decrease in partial pressure of oxygen leading to a unique condition known as hypobaric hypoxia (HH). Brain is highly vulnerable to hypoxia and it has been well established that hypobaric hypoxia leads to neurodegeneration in different brain regions. However, the response of glial cells during hypobaric hypoxia needs to be explored yet. The present study was aimed to understand the role of glial cells viz. astrocytes, microglia and oligodendrocytes in HH-induced neuronal death. The study aims to understand the effect of HH exposure on glial physiology in a time-dependent (0, 1, 3, 7 and 14 days of HH exposure) and region-dependent (CA1, CA3 and DG regions of hippocampus) manner. We examined the morphological changes and activation of glial cells along with pro-inflammatory cytokine levels. Results indicated that chronic hypobaric hypoxia (7 and 14 days exposure) causes activation of both astrocytes (GFAP-positive cells) and microglia (IBA-1-positive cells) but oligodendrocytes (M-PLP-positive cells) expression was found to be significantly reduced. The results obtained were found to be more profound in the CA1 region of hippocampus, indicating its vulnerability to hypoxia. Neuroinflammation was suggested by elevated IL-β1, IL-6 and TNF-α cytokine levels upon chronic HH. The study also explored the role of microglia and A1 astrocyte interplay in HH-induced neurodegeneration and demyelination. This study explores the shift in role of glial cell toward neurodegeneration under chronic hypobaric hypoxia stress. Chronic stress results in glial activation which leads to neuroinflammation a plausible factor in HH-induced neurodegeneration. [ABSTRACT FROM AUTHOR]

12/15 Lipoxygenase has recently been described as potent propagator of oxidative stress and is closely associated with cognitive decline in neurodegenerative diseases. The mechanism/s behind 12/15 LOX involvement in cognitive deficits remain obscure. The current study has been designed to investigate the underlying role of 12/15LOX and effect of 12/15 LOX inhibition on hypobaric hypoxia-induced memory impairment and cholinergic deficits. Male Balb/c mice subjected to simulated hypobaric hypoxia/reoxygenation condition for 3 days showed marked working memory impairment concomitant with hippocampal neuronal damage and malondialdehyde production which were significantly attenuated by baicalein, a specific inhibitor of 12/15LOX. Hypobaric hypoxia-exposed mice had consistently increased expression of 12/15LOX and elevated 12(S) HETE levels in the hippocampus as well as plasma which were significantly mitigated following baicalein treatment. 12/15LOX inhibition also reduced hypobaric hypoxia-mediated upregulation of hippocampal HIF-1α protein expression along with reduction in expression of inflammatory genes. The inhibition of 12/15 LOX resulted in a significant decrease in NO levels in the hippocampal homogenate associated with downregulated iNOS, nNOS transcription but not eNOS speculating that 12/15 LOX is critically involved in HIF-1α, mediated by nitric oxide-induced neurotoxicity. We also observed a similar effect of 12/15 LOX inhibition on hippocampal COX2 expression. 12/15LOX inhibition could effectively modulate central cholinergic indices during hypobaric hypoxia by restoring mAChR-1, α7NAChR expression and AChE, ChAT activity in the hippocampus comparable to normal mice. We report here the mechanistic involvement of 12/15LOX in orchestrating hypoxia-associated neuronal damage and HIF-1α-dependent neuroinflammation resulting in cognitive decline. [ABSTRACT FROM AUTHOR]

The stimuli that commonly activate the catecholaminergic C1 neurons (nociception, hypotension, and hypoxia) also increase breathing. Pharmacogenetic evidence suggests that catecholaminergic neurons regulate breathing. Therefore, we evaluated whether the loss of C1 cells affects cardiorespiratory control during resting, hypoxic (8% O 2 ) and hypercapnic (7% CO 2 ) conditions. A bilateral injection of the immunotoxin anti-dopamine β-hydroxylase-saporin (anti-DβH-SAP; 2.4 ng/100 nl) or saline was performed in adult male Wistar rats (270–300 g, N = 5–8/group). Histology revealed a 60–75% loss of C1 neurons in anti-DβH-SAP-treated rats, but no significant changes or C1 cell loss was observed in sham-treated rats or those with off-target injection sites. Bilateral depletion of C1 neurons did not alter cardiorespiratory variables during rest and hypercapnia (7% CO 2 ), but it did affect the response to hypoxia. Specifically, the increase in ventilation, the number of sighs, and the tachycardia were reduced, but unexpectedly, the mean arterial pressure increased during hypoxia (8% O 2 ). The present study indicates that C1 neurons contribute to cardiorespiratory control during hypoxia rather than at rest or during hypercapnia. [ABSTRACT FROM AUTHOR]

Males are more susceptible than females to long-term cognitive deficits following neonatal hypoxic-ischemic encephalopathy (HIE). Mitochondrial dysfunction is implicated in the pathophysiology of cerebral hypoxia–ischemia (HI), but the influence of sex on mitochondrial quality control (MQC) after HI is unknown. Therefore, we tested the hypothesis that mitophagy is sexually dimorphic and neuroprotective 20–24 h following the Rice–Vannucci model of rat neonatal HI at postnatal day 7 (PN7). Mitochondrial and lysosomal morphology and degree of co-localization were determined by immunofluorescence in the cerebral cortex. No difference in mitochondrial abundance was detected in the cortex after HI. However, net mitochondrial fission increased in both hemispheres of female brain, but was most extensive in the ipsilateral hemisphere of male brain following HI. Basal autophagy, assessed by immunoblot for the autophagosome marker LC3BI/II, was greater in males suggesting less intrinsic reserve capacity for autophagy following HI. Autophagosome formation, lysosome size, and TOM20/LAMP2 co-localization were increased in the contralateral hemisphere following HI in female, but not male brain. An accumulation of ubiquitinated mitochondrial protein was observed in male, but not female brain following HI. Moreover, neuronal cell death with NeuN/TUNEL co-staining occurred in both hemispheres of male brain, but only in the ipsilateral hemisphere of female brain after HI. In summary, mitophagy induction and neuronal cell death are sex dependent following HI. The deficit in elimination of damaged/dysfunctional mitochondria in the male brain following HI may contribute to male vulnerability to neuronal death and long-term neurobehavioral deficits following HIE. [ABSTRACT FROM AUTHOR]

Repetitive acute intermittent hypoxia (rAIH) increases growth/trophic factor expression in respiratory motor neurons, thereby eliciting spinal respiratory motor plasticity and/or neuroprotection. Here we demonstrate that rAIH effects are not unique to respiratory motor neurons, but are also expressed in non-respiratory, spinal alpha motor neurons and upper motor neurons of the motor cortex. In specific, we used immunohistochemistry and immunofluorescence to assess growth/trophic factor protein expression in spinal sections from rats exposed to AIH three times per week for 10 weeks (3 × wAIH). 3 × wAIH increased brain-derived neurotrophic factor (BDNF), its high-affinity receptor, tropomyosin receptor kinase B (TrkB), and phosphorylated TrkB (pTrkB) immunoreactivity in putative alpha motor neurons of spinal cervical 7 (C 7 ) and lumbar 3 (L 3 ) segments, as well as in upper motor neurons of the primary motor cortex (M 1 ). 3 × wAIH also increased immunoreactivity of vascular endothelial growth factor A (VEGFA), the high-affinity VEGFA receptor (VEGFR-2) and an important VEGF gene regulator, hypoxia-inducible factor-1α (HIF-1α). Thus, rAIH effects on growth/trophic factors are characteristic of non-respiratory as well as respiratory motor neurons. rAIH may be a useful tool in the treatment of disorders causing paralysis, such as spinal injury and motor neuron disease, as a pretreatment to enhance motor neuron survival during disease, or as preconditioning for cell-transplant therapies. [ABSTRACT FROM AUTHOR]

Genetic background is known to influence the outcome in mouse models of human disease, and previous experimental studies have shown strain variability in the neonatal mouse model of hypoxia–ischemia. To further map out this variability, we compared five commonly used mouse strains: C57BL/6, 129SVJ, BALB/c, CD1 and FVB in a pure hypoxic–ischemic setup and following pre-sensitization with lipopolysaccharide (LPS). Postnatal day 7 pups were subjected to unilateral carotid artery occlusion followed by continuous 30 min 8% oxygen exposure at 36 °C. Twelve hours prior, a third of the pups received a single intraperitoneal LPS (0.6 μg/g) or a saline (vehicle) administration, respectively; a further third underwent hypoxia–ischemia alone without preceding injection. Both C57BL/6 and 129SVJ strains showed minimal response to 30 min hypoxia–ischemia alone, BALB/c demonstrated a moderate response, and both CD1 and FVB revealed the highest brain damage. LPS pre-sensitization led to substantial increase in overall brain infarction, microglial and astrocyte response and cell death in four of the five strains, with exception of BALB/c that only showed a significant effect with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Saline administration prior to hypoxia–ischemia resulted in an increase in inflammatory-associated markers, particularly in the astroglial activation of C57BL/6 mice, and in combined microglial activation and neuronal cell loss in FVB mice. Finally, two of the four strongly affected strains – C57BL/6 and CD1 – revealed pronounced contralateral astrogliosis with a neuroanatomical localization similar to that observed on the occluded hemisphere. Overall, the current findings demonstrate strain differences in response to hypoxia–ischemia alone, to stress associated with vehicle injection, and to LPS-mediated pre-sensitization, which partially explains the high variability seen in the neonatal mouse models of hypoxia–ischemia. These results can be useful in future studies of fetal/neonatal response to inflammation and reduced oxygen–blood supply. [ABSTRACT FROM AUTHOR]

Transient global ischemia selectively damages neurons in specific brain areas. A reproducible pattern of selective vulnerability is observed in the dorsal hippocampus of rodents where ischemic damage typically affects neurons in the CA1 area while sparing neurons in CA3 and granule cells. The “neuronal factors” underlying the differential vulnerability of CA1 versus CA3 have been of great interest. This review first provides on overview of the histological pattern of ischemic–hypoxic damage, the phenomenon of delayed neuronal death, the necrosis–apoptosis discussion, and multiple molecular mechanisms studied in the hippocampus. Subsequently, genomic studies of basal gene expression in CA1 and CA3 are summarized and changes in gene expression in response to global brain ischemia are surveyed. A formal analysis is presented for the overlap between genes expressed under basal conditions in the hippocampus and genes responding to ischemia–hypoxia in general. A possible role of the elusive vascular factors in selective vulnerability is reviewed, and a gene set for angiogenesis is then shown to be enriched in the CA3 gene set. A survey of selective vulnerability in the human hippocampus in relation to genomic studies in ischemia–hypoxia is presented, and neurodegeneration genes with high expression in CA1 are highlighted (e.g. WFS1). It is concluded that neuronal factors dominate the selective vulnerability of CA1 but that vascular factors also deserve more systematic studies. [ABSTRACT FROM AUTHOR]

In animal models, environmental enrichment (EE) has been found to be an efficient treatment for alleviating the consequences of neonatal hypoxia-ischemia (HI). However the potential for this therapeutic strategy and the mechanisms involved are not yet clear. The aim of present study is to investigate behavioral performance in the ox-maze test and Na + ,K + -ATPase, catalase (CAT) and glutathione peroxidase (GPx) activities in the hippocampus of rats that suffered neonatal HI and were stimulated in an enriched environment. Seven-day-old rats were submitted to the HI procedure and divided into four groups: control maintained in standard environment (CTSE), control submitted to EE (CTEE), HI in standard environment (HISE) and HI in EE (HIEE). Animals were stimulated with EE for 9 weeks (1 h/day for 6 days/week) and then behavioral and biochemical parameters were evaluated. Present results indicate learning and memory in the ox-maze task were impaired in HI rats and this effect was recovered after EE. Hypoxic–ischemic event did not alter the Na + ,K + -ATPase activity in the right hippocampus (ipsilateral to arterial occlusion). However, on the contralateral hemisphere, HI caused a decrease in this enzyme activity that was recovered by EE. The activities of GPx and CAT were not changed by HI in any group evaluated. In conclusion, EE was effective in recovering learning and memory impairment in the ox-maze task and Na + ,K + -ATPase activity in the hippocampus caused by HI. The present data provide further support for the therapeutic potential of environmental stimulation after neonatal HI in rats. [ABSTRACT FROM AUTHOR]

Neonatal anoxia in rodents has been used to understand brain changes and cognitive dysfunction following asphyxia. This study investigated the time-course of cellular and subcellular changes and hippocampal cell death in a non-invasive model of anoxia in neonatal rats, using Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) to reveal DNA fragmentation, Fluoro-Jade® B (FJB) to show degenerating neurons, cleaved caspase-3 immunohistochemistry (IHC) to detect cells undergoing apoptosis, and transmission electron microscopy (TEM) to reveal fine ultrastructural changes related to cell death. Anoxia was induced by exposing postnatal day 1 (P1) pups to a flow of 100% gaseous nitrogen for 25 min in a chamber maintained at 37 °C. Control rats were similarly exposed to this chamber but with air flow instead of nitrogen. Brain changes following anoxia were evaluated at postnatal days 2, 14, 21 and 60 (P2, P14, P21 and P60). In addition, spatial reference memory following anoxia and control treatments was evaluated in the Morris water maze, starting at P60. Compared to their respective controls, P2 anoxic rats exhibited (1) higher TUNEL labeling in cornus ammonis (CA) 1 and the dentate gyrus (DG), (2) higher FJB-positive cells in the CA2–3, and (3) somato-dendritic swelling, mitochondrial injury and chromatin condensation in irregular bodies, as well as other subcellular features indicating apoptosis, necrosis, autophagy and excitotoxicity in the CA1, CA2–3 and DG, as revealed by TEM. At P14, P21 and P60, both groups showed small numbers of TUNEL-positive and FJB-positive cells. Stereological analysis at P2, P14, P21 and P60 revealed a lack of significant differences in cleaved caspase-3 IHC between anoxic and control subjects. These results suggest that the type of hippocampal cell death following neonatal anoxia is likely independent of caspase-3 activation. Neonatal anoxia induced deficits in acquisition and performance of spatial reference memory in the Morris water maze task. Compared to control subjects, anoxic animals exhibited increased latencies and path lengths to reach the platform, as well as decreased searching specifically for the platform location. In contrast, no significant differences were observed for swimming speeds and frequency within the target quadrant. Together, these behavioral results indicate that the poorer performance by anoxic subjects is related to spatial memory deficits and not to sensory or motor deficits. Therefore, this model of neonatal anoxia in rats induces hippocampal changes that result in cell losses and impaired hippocampal function, and these changes are likely related to spatial memory deficits in adulthood. [ABSTRACT FROM AUTHOR]

Eider duck ( Somateria mollissima ) cerebellar neurons are highly tolerant toward hypoxia in vitro , which in part is due to a hypoxia-induced depression of their spontaneous activity. We have studied whether this response involves ATP-sensitive potassium (K ATP ) channels, which are known to be involved in the hypoxic/ischemic defense of mammalian neural and muscular tissues, by causing hyperpolarization and reduced ATP demand. Extracellular recordings in the Purkinje layer of isolated normoxic eider duck cerebellar slices showed that their spontaneous neuronal activity decreased significantly compared to in control slices when the K ATP channel opener diazoxide (600 μM) was added ( F 1,70 = 92.781, p < 0.001). Adding the K ATP channel blocker tolbutamide (400 μM) 5 min prior to diazoxide completely abolished its effect ( F 1,55 = 39.639, p < 0.001), strongly suggesting that these drugs have a similar mode of action in this avian species as in mammals. The spontaneous activity of slices treated with tolbutamide in combined hypoxia/chemical anoxia (95% N 2 –5% CO 2 and 2 mM NaCN) was not significantly different from that of control slices ( F 1,203 = 0.071, p = 0.791). Recovery from hypoxia/anoxia was, however, slightly but significantly weaker in tolbutamide-treated slices than in control slices ( F 1,137 = 15.539, p < 0.001). We conclude that K ATP channels are present in eider duck cerebellar neurons and are activated in hypoxia/anoxia, but that they do not play a key role in the protective shut-down response to hypoxia/anoxia. [ABSTRACT FROM AUTHOR]

Highlights: [•] Autophagy and apoptosis were studied in the brain following hypoxia–ischemia (HI). [•] The function of lysosomes decreased in female but not in the male cortex following HI. [•] Activation of autophagy occurred in the cortex and the hippocampus after HI. [•] Females had greater caspase-3 activation than males in the cortex/hippocampus following HI. [•] Autophagy and apoptosis activation by HI differed by sex and brain region. [ABSTRACT FROM AUTHOR]

Abstract: Cholinergic system is important for respiratory control from the first days of life. Disturbances in cholinergic pathway due to early life stress like hypoxic shock can adversely affect the ventilatory response. The present study evaluates neonatal hypoxic insult mediated cholinergic disturbances and the role of glucose, oxygen and epinephrine resuscitation. The changes in total muscarinic, muscarinic M1, M2, M3 receptors and the enzymes involved in acetylcholine metabolism – cholineacetyl transferase and acetylcholine easterase in the cerebellum were analyzed. Hypoxic stress decreased cerebellar muscarinic receptor density with a decreased muscarinic M1, M2 and M3 receptor gene expression. The metabolic shift in the acetylcholine synthesis and release is indicated by the decreased cholineacetyl transferase mRNA expression and increased acetylcholine esterase gene expression. Glucose, acting as a precursor for acetyl choline synthesis and an immediate energy source, helps in reversing the cholinergic disturbances in hypoxic neonates. The limitation of immediate oxygenation and epinephrine administration in ameliorating cholinergic disturbances in hypoxic neonates was also reported. This will help in devising a better resuscitation program for the management of neonatal hypoxia. [Copyright &y& Elsevier]

Abstract: Experimental preconditioning provides beneficial outcomes in conditions such as cardiac surgery, brain surgery and stroke. Here we evaluated the protective effects of low-dose subcutaneous GYKI-52466 preconditioning in a rat model of hypoxic–ischaemic (HI) brain injury. Male Sprague–Dawley rats (postnatal day 26) were administered saline or GYKI-52466 (GYKI; 3-mg/kg, 90min; 1-mg/kg, twice in 120min; or 0.5-mg/kg, thrice in 180min) prior to left common carotid artery occlusion. Animals were allowed to recover for 2h, and then placed in a hypoxia chamber (8% O2/92% N2; 33±1°C) for 1h. A sham surgery group received saline without HI. Seizure activity was scored during hypoxia and sensorimotor tests performed before surgery and at 1, 7, 14 and 90days post-HI. On days 14 and 90 brains were fixed and sectioned for the assessment of infarct size and ventricular enlargement. Low-dose GYKI-52466 preconditioning significantly reduced infarct volume and ventricular enlargement relative to saline-treated controls at day 14 after HI. On day 90, tissue loss was significantly reduced by GYKI 3-mg/kg compared to saline. Foot-faults, paw use asymmetry, and postural reflex scores were significantly improved in all GYKI treatment groups. Our results show that GYKI-52466 is effective at doses well-below, and at pre-administration intervals well-beyond previous studies, and suggest that a classical blockade of ionotropic AMPA receptors does not underlie its neuroprotective effects. Low-dose GYKI-52466 preconditioning represents a novel, prophylactic strategy for neuroprotection in a field almost devoid of effective pharmaceuticals. [Copyright &y& Elsevier]

Abstract: Previously we showed that 1-(4′-aminophenyl)-4-methyl-7,8-methylene-dioxy-2,3-benzodiazepine (GYKI-52466), an ionotropic AMPA receptor antagonist, can trigger strong, presumably metabotropic, protection against seizures and stroke at very low doses. To date, no study has determined brain and plasma concentrations of GYKI-52466 following subcutaneous administration in animals with or without brain damage. Here we developed and validated a rapid method of high-performance liquid chromatography with diode array detection. Chromatographic separation was achieved by a Luna C18 column using a mixture of 25mM phosphate buffer (pH 7.0)–methanol–acetonitrile (40:37.5:22.5, v/v/v) as the mobile phase at a flow rate of 1.2mL/min. The method showed acceptable precision and accuracy and allowed a precise quantification of 25ng/mL GYKI-52466 in the plasma and brain. Recovery of GYKI-52466 from the plasma and brain was >87%, and GYKI was stable at room temperature and during prolonged storage at −20°C. The method was successfully applied in measuring levels of GYKI-52466 following administration of 3 and 20mg/kg of GYKI-52466 in control and brain damaged rats. A low brain concentration of 0.56μM GYKI-52466 was observed with 3mg/kg compared to 10.7μM with 20mg/kg at 90min post drug administration. Severe ataxia was observed with the 20mg/kg dose for up to 90min. Furthermore, in ischaemic animals, there was no evidence of a ‘surge’ in brain GYKI concentrations at the injury site, confirming the integrity of the blood–brain barrier in the region of infarct. Taken together, our findings support a metabotropic mode of action underlying the low-dose neuroprotective efficacy of GYKI-52466. [Copyright &y& Elsevier]

Abstract: The angiotensin II receptor subtype 2 (AT2-R) has been proposed to mediate protective vascular actions after brain injury. In this study we investigated the participation of this peptide in the tolerance to cellular damage induced by preconditioning in a rat model of neonatal hypoxia–ischemia (HI). We found that injured animals present a decreased number of microvessels in the ipsilateral (IPLT) side of the brain while in the contralateral (CNLT) side the microvessel number is increased. On the contrary, in the preconditioned animals the microvessels maintained the same number as in control animals. However these vessels show a remarkable increase of the fluorescent signal when they are labeled with antiFlk-1 (VEGFR2), while the Flt-1 (VEGFR1) signal faded in both the injured and the preconditioned animals. The pharmacological blockade of the AT2-R by the drug PD123319 (1.69mM in the lateral ventricle) diminished the resilience of the microvasculature to HI injury provided by preconditioning and also the Flk-1 increase that occurred in these animals. In conclusion these results suggest an interaction of the AT2-R with VEGFR2 in the neonatal brain microvasculature that produces protective effects which are associated with injury tolerance. [Copyright &y& Elsevier]

Abstract: Despite the beneficial effects of cell-based therapies on brain repair shown in most studies, there has not been a consensus regarding the optimal dose of human umbilical cord blood cells (HUCBC) for neonatal hypoxia-ischemia (HI). In this study, we compared the long-term effects of intravenous administration of HUCBC at three different doses on spatial memory and brain morphological changes after HI in newborn Wistar rats. In addition, we tested whether the transplanted HUCBC migrate to the injured brain after transplantation. Seven-day-old animals underwent right carotid artery occlusion and were exposed to 8% O2 inhalation for 2 h. After 24 h, randomly selected animals were assigned to four different experimental groups: HI rats administered with vehicle (HI+vehicle), HI rats treated with 1×106 (HI+low-dose), 1×107 (HI+medium-dose), and 1×108 (HI+high-dose) HUCBC into the jugular vein. A control group (sham-operated) was also included in this study. After 8 weeks of transplantation, spatial memory performance was assessed using the Morris water maze (MWM), and subsequently, the animals were euthanized for brain morphological analysis using stereological methods. In addition, we performed immunofluorescence and polymerase chain reaction (PCR) analyses to identify HUCBC in the rat brain 7 days after transplantation. The MWM test showed a significant spatial memory recovery at the highest HUCBC dose compared with HI+vehicle rats (P<0.05). Furthermore, the brain atrophy was also significantly lower in the HI+medium- and high-dose groups compared with the HI+vehicle animals (P<0.01; 0.001, respectively). In addition, HUCBC were demonstrated to be localized in host brains by immunohistochemistry and PCR analyses 7 days after intravenous administration. These results revealed that HUCBC transplantation has the dose-dependent potential to promote robust tissue repair and stable cognitive improvement after HI brain injury. [Copyright &y& Elsevier]

Abstract: The deregulation of cholinergic system and associated neuronal damage is thought to be a major contributor to the pathophysiologic sequelae of hypobaric hypoxia-induced memory impairment. Uniquely, the muscarinic receptors also play a role in zinc uptake. Despite the potential role of muscarinic receptors in the development of post hypoxia cognitive deficits, no studies to date have evaluated the mechanistic relationship between memory dysfunction and zinc homeostasis in brain. In the present study, we evaluated the effect of Ca2EDTA, a specific zinc chelator in the spatial working and associative memory deficits following hypobaric hypoxia. Our results demonstrate that accumulation of intracellular free chelatable zinc in the hippocampal CA3 pyramidal neurons is accompanied with neuronal loss and memory impairment in hypobaric hypoxic condition. Chelation of this free zinc with Ca2EDTA (1.25 mM/kg) ameliorated the hippocampus-dependent spatial as well as associative memory dysfunction and neuronal damage observed on exposure to hypobaric hypoxia. The zinc chelator significantly alleviated the downregulation in expression of choline acetyltransferase, muscarinic receptor 1 and 4, and acetylcholinesterase activity due to hypobaric hypoxia. Our data suggest that the free chelatable zinc released during hypobaric hypoxia might play a critical role in the neuronal damage and the alteration in cholinergic function associated with hypobaric hypoxia-induced memory impairment. We speculate that zinc chelation might be a potential therapy for hypobaric hypoxia-induced cognitive impairment. [Copyright &y& Elsevier]

Abstract: Neuroinflammation is a key mechanism contributing to long-term neuropathology observed after neonatal hypoxia-ischemia (HI). Minocycline, a second-generation tetracycline, is a potent inhibitor of neuroinflammatory mediators and is successful for at least short-term amelioration of neuronal injury after neonatal HI. However the long-term efficacy of minocycline to prevent injury to a specific neuronal network, such as the serotonergic (5-hydroxytryptamine, 5-HT) system, is not known. In a post-natal day 3 (P3) rat model of preterm HI we found significant reductions in 5-HT levels, 5-HT transporter expression and numbers of 5-HT-positive dorsal raphé neurons 6 weeks after insult compared to control animals. Numbers of activated microglia were significantly elevated in the thalamus and dorsal raphé although the greatest numbers were observed in the thalamus. Brain levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were also significantly elevated on P45 in the thalamus and frontal cortex. Post-insult administration of minocycline for 1 week (P3–P9) attenuated the P3 HI-induced increases in numbers of activated microglia and levels of TNF-α and IL-1β on P45 with concurrent changes in serotonergic outcomes. The parallel prevention of P3 HI-induced serotonergic changes suggests that inhibition of neuroinflammation within the first week after P3 HI injury was sufficient to prevent long-term neuroinflammation as well as serotonergic system damage still evident at 6 weeks. Thus early, post-insult administration of minocycline may target secondary neuroinflammation and represent a long-term therapy to preserve the integrity of the central serotonergic network in the preterm neonate. [Copyright &y& Elsevier]

Abstract: Hypoxic respiratory and cardiovascular responses in mammals are mediated by peripheral chemoreceptor afferents which are relayed centrally via the solitary tract nucleus (NTS) in dorsomedial medulla to other cardiorespiratory-related brainstem regions such as ventrolateral medulla (VLM). Here, we test the hypothesis that peripheral chemoafferents could also be relayed directly to the Kölliker-Fuse/parabrachial complex in dorsolateral pons, an area traditionally thought to subserve pneumotaxic and cardiovascular regulation. Experiments were performed on adult Sprague–Dawley rats. Brainstem neurons with axons projecting to the dorsolateral pons were retrogradely labeled by microinjection with choleras toxin subunit B (CTB). Neurons involved in peripheral chemoreflex were identified by hypoxia-induced c-Fos expression. We found that double-labeled neurons (i.e. immunopositive to both CTB and c-Fos) were localized mostly in the commissural and medial subnuclei of NTS and to a lesser extent in the ventrolateral NTS subnucleus, VLM and ventrolateral pontine A5 region. Extracellular recordings from the commissural and medial NTS subnuclei revealed that some hypoxia-excited NTS neurons could be antidromically activated by electrical stimulations at the dorsolateral pons. These findings demonstrate that hypoxia-activated afferent inputs are relayed to the Kölliker-Fuse/parabrachial complex directly via the commissural and medial NTS and indirectly via the ventrolateral NTS subnucleus, VLM and A5 region. These pontine-projecting peripheral chemoafferent inputs may play an important role in the modulation of cardiorespiratory regulation by dorsolateral pons. [ABSTRACT FROM AUTHOR]

Abstract: The clinical course of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS) is closely related to hypoxia. The normal response to hypoxia involves two pathways in particular: the hypoxia inducible factor 1α (HIF-1α) pathway (which notably controls the synthesis of vascular endothelial growth factor (VEGF)) and the nuclear factor kappa B (NF-κb) pathway (responsible for the production of inflammatory mediators, including prostaglandin E2 (PGE2)). Defects in VEGF gene expression are known to cause motor neuron degeneration in animal models. Circulating monocytes are precursors of the microglia, which are involved in the pathogenesis of ALS. To establish whether the HIF-1 and/or NF-κB pathways are deregulated during hypoxia in early-stage, sporadic ALS, we analyzed the response to acute (1 h) and prolonged (24 h) hypoxia in monocytes from ALS and healthy controls. We measured protein expression and mRNA transcription for VEGF, HIF-1, HIF-2, prolyl hydroxylases 1 and 2 (PHD-1 and -2, part of the HIF proteasome-dependent degradation pathway) and their modulation by PGE2. Our results showed that (i) the HIF-1 (but not HIF-2) and VEGF production induced by acute and prolonged hypoxia was selectively and markedly altered in ALS patients and (ii) this defect was not compensated for by PGE2 addition. Moreover, altered HIF-1α activation was associated with low levels of proteolysis by PHD-2 in cells from sporadic ALS patients (relative to controls). For the first time, we have demonstrated clinical and functional abnormalities in the HIF-1 pathway during hypoxia in monocytes from sporadic ALS patients. [ABSTRACT FROM AUTHOR]

Abstract: The brain''s response to ethanol intake has been extensively investigated using electrophysiological recordings, brain lesion techniques, and c-Fos immunoreactivity. However, few studies have investigated this phenomenon using functional magnetic resonance imaging (fMRI). In the present study, we used fMRI to investigate the blood oxygenation level-dependent (BOLD) signal response to an intragastric (IG) load of ethanol in conscious, ethanol-naive rats. An intragastrically infused 10% ethanol solution induced a significant decrease in the intensity of the BOLD signal in several regions of the brain, including the bilateral amygdala (AMG), nucleus accumbens (NAc), hippocampus, ventral pallidum, insular cortex, and cingulate cortex, and an increase in the BOLD signal in the ventral tegmental area (VTA) and hypothalamic regions. Treatment with bicuculline, which is an antagonist of the gamma-aminobutyric acid A (GABAA) receptor, increased the BOLD signal intensity in the regions that had shown decreases in the BOLD signal after the IG infusion of 10% ethanol solution, but it did not affect the BOLD signal increase in the hypothalamus. Treatment with SCH39166, which is an antagonist of D1-like receptors, eliminated the increase in the BOLD signal intensity in the hypothalamic areas but did not affect the BOLD signal decrease following the 10% ethanol infusion. These results indicate that an IG load of ethanol caused both a GABAA receptor–mediated BOLD decrease in the limbic system and the cortex and a D1-like receptor-mediated BOLD increase in the hypothalamic regions in ethanol-naive rats. [Copyright &y& Elsevier]

Abstract: Spinal nerves and their associated dorsal root ganglion (DRG) cells can be subject to mechanical deformation and hypoxia associated with pathology such as disc herniation, spinal stenosis and spine trauma. There is very limited information on the response of adult DRG neurons to such stressors. In this study we used an in vitro approach to examine the response of adult DRG cells to (a) mechanical, hypoxic, and combined injuries; and (b) to compare the effects on injury on nociceptive and non-nociceptive neurons, as well as on non-neuronal cells. Mechanical injury (20% tensile strain) led to significant neuronal cell death (assessed by ethidium homodimer-1 labelling), which was proportional to strain duration (5 min, 1 h, 6 h or 18 h). Hypoxia (2% O 2 for 24 h) also promoted death of DRG neurons, and was further enhanced when mechanical strain and hypoxia were combined. Both mechanical strain and hypoxia significantly decreased the maximum neurite length. Conversely, death of non-neuronal cells was only increased by hypoxia and not by mechanical strain. Total cell death in response to mechanical injury or hypoxia was similar in both non-nociceptive (neurofilament, NF-200 immunoreactive) and nociceptive (calcitonin gene-related peptide, CGRP immunoreactive) neurons, but apoptosis (assessed by activated caspase-3 immunostaining) was significantly higher in CGRP than NF-200 neurons. Surprisingly, cell death of non-peptidergic nociceptors (identified by Griffonia simplicifolia IB4 lectin binding) was already high in control cultures, and was not increased further by either mechanical stretch or hypoxia. These results provide detailed information on the response of adult DRG subpopulations to hypoxia and mechanical strain, and describe in vitro models that could be useful for screening potential neuroprotective agents. [ABSTRACT FROM AUTHOR]

Abstract: A large part of the literature of functional near-infrared spectroscopy (fNIRS) deals with overt verbal fluency. It has been claimed that fNIRS has a low susceptibility to movement related artefacts as, for example, associated with overt speech. However, so far, no study has investigated this assumption in an experimental design. Therefore, we examined a group of 16 healthy subjects during performance of two verbal fluency tasks (experiment 1: phonological fluency; experiment 2: semantical fluency, paced answers, pronouncing vs. writing). We measured changes of oxygenated (O2Hb) and deoxygenated haemoglobin (HHb) over fronto-temporal (brain) areas via fNIRS, while temporalis muscle activity was simultaneously assessed by means of electromyography (EMG). Statistical analyses indicated comparable word production, higher increases of O2Hb and higher decreases of HHb over fronto-temporal areas during word fluency in contrast to the control task weekday reciting. This fNIRS pattern indicates fluency related activation and was found for pronouncing and for writing in both experiments. Regarding the EMG data, fluency related activity was only found for pronouncing, not for writing. Thus, muscle activity cannot account for fluency related fNIRS activity during writing. Additionally, correlation analyses showed no systematic associations of fNIRS and EMG signals. In conclusion, we found arguments that fNIRS actually allows for the measurement of brain activity over fronto-temporal areas during verbal fluency. Nonetheless, further studies should evaluate more direct associations between fNIRS and EMG signals by specific experimental manipulations and data analysing approaches that allow dealing fNIRS and EMG raw data simultaneously. [ABSTRACT FROM AUTHOR]

Abstract: Methyl-CpG-binding protein 2 (MeCP2) deficiency causes Rett syndrome (RTT), a neurodevelopmental disorder characterized by severe cognitive impairment, synaptic dysfunction, and hyperexcitability. Previously we reported that the hippocampus of MeCP2-deficient mice (Mecp2−/y ), a mouse model for RTT, is more susceptible to hypoxia. To identify the underlying mechanisms we now focused on the anoxic responses of wildtype (WT) and Mecp2−/y CA1 neurons in acute hippocampal slices. Intracellular recordings revealed that Mecp2−/y neurons show only reduced or no hyperpolarizations early during cyanide-induced anoxia, suggesting potassium channel (K+ channel) dysfunction. Blocking adenosine-5′-triphosphate-sensitive K+ channels (KATP-) and big-conductance Ca2+-activated K+ channels (BK-channels) did not affect the early anoxic hyperpolarization in either genotype. However, blocking Ca2+ release from the endoplasmic reticulum almost abolished the anoxic hyperpolarizations in Mecp2−/y neurons. Single-channel recordings confirmed that neither KATP- nor BK-channels are the sole mediators of the early anoxic hyperpolarization. Instead, anoxia Ca2+-dependently activated various small/intermediate-conductance K+ channels in WT neurons, which was less evident in Mecp2−/y neurons. Yet, pharmacologically increasing the Ca2+ sensitivity of small/intermediate-conductance KCa channels fully restored the anoxic hyperpolarization in Mecp2−/y neurons. Furthermore, Ca2+ imaging unveiled lower intracellular Ca2+ levels in resting Mecp2−/y neurons and reduced anoxic Ca2+ transients with diminished Ca2+ release from intracellular stores. In conclusion, the enhanced hypoxia susceptibility of Mecp2−/y hippocampus is primarily associated with disturbed Ca2+ homeostasis and diminished Ca2+ rises during anoxia. This secondarily attenuates the activation of KCa channels and thereby increases the hypoxia susceptibility of Mecp2−/y neuronal networks. Since cytosolic Ca2+ levels also determine neuronal excitability and synaptic plasticity, Ca2+ homeostasis may constitute a promising target for pharmacotherapy in RTT. [Copyright &y& Elsevier]

Abstract: The current etiopathogenesis of spinal cord injury comprises a growing number of nontraumatic causes, including ischemia generating hypoxic-dysmetabolic conditions. To mimic the metabolic disruption accompanying nontraumatic acute spinal cord injury and to characterize the type and dynamics of cell death in relation to locomotor network function, we used, as a model, the rat neonatal spinal cord preparation in vitro transiently (1 h) exposed to a “pathological medium” (PM), i.e. hypoxic/aglycemic solution containing toxic radicals. PM induced, in the ventrolateral spinal region, pyknosis already detectable after 2 h and stabilized 24 h later (affecting 55% of white matter cells). Glial cells were much more vulnerable than neurons. The amplitude of fictive locomotor patterns recorded from lumbar ventral roots was decreased and periodicity delayed by PM, in keeping with substantial preservation of neuronal networks. Repeated application of PM intensified such a functional impairment. White matter astrocytes and oligodendrocytes displayed nucleolytic pyknosis mainly dependent on caspase-mediated death processes as shown by active caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) positivity. Expression of cleaved poly(ADP-ribose) polymerase-1 (PARP-1) (the active caspase-3 executor) also grew with similar time course. The caspase-3 inhibitor II counteracted, in a dose-dependent fashion, white matter pyknosis. Our results suggest the important involvement of apoptotic pathways in early glial cell death during the first 24 h after a hypoxic-dysmetabolic insult, associated with impaired locomotor output. Residual locomotor network activity together with distinctive apoptotic damage to white matter cells suggests that early protection against glial destruction may help to prevent subsequent damage extension responsible for paraplegia. [Copyright &y& Elsevier]

Abstract: Cerebral hypoxia is one of the main causes of cerebral injury. This study was conducted to investigate the potential protective effect of H2S in in vitro hypoxic models by subjecting SH-SY5Y cells to either oxygen–glucose deprivation or Na2S2O4 (an oxygen scavenger) treatment. We found that treatment with NaHS (an H2S donor, 10–100 μM) 15 min prior to hypoxia increased cell viability in a concentration-dependent manner. Time-course study showed that NaHS was able to exert its protective effect even when added 8 h before or less than 4 h after hypoxia induction. Interestingly, endogenous H2S level was markedly reduced by hypoxia induction. Over-expression of cystathionine-β-synthase prevented hypoxia induced cell apoptosis. Blockade of ATP-sensitive K+ (KATP) channels with glibenclamide and HMR-1098, protein kinase C (PKC) with its three specific inhibitors (chelerythrine, bisindolylmaleide I and calphostin C), extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059 and heat shock protein 90 (Hsp90) with geldanamycin and radicicol significantly attenuated the protective effects of NaHS. Western blots showed that NaHS significantly stimulated ERK1/2 activation and Hsp90 expression. In conclusion, H2S exerts a protective effect against cerebral hypoxia induced neuronal cell death via KATP/PKC/ERK1/2/Hsp90 pathway. Our findings emphasize the important neuroprotective role of H2S in the brain during cerebral hypoxia. [Copyright &y& Elsevier]

Abstract: Hypoxia–ischemia is a significant cause of brain damage in the human newborn and can result in long-term neurodevelopmental disability. The loss of oxygen and glucose supply to the developing brain leads to excitotoxic neuronal cell damage and death; such over-excitation of nerve cells can also manifest as seizures. The newborn brain is highly susceptible to seizures although it is unclear what role they have in hypoxic-ischemic (H/I) injury. The aim of this study was to determine an association between seizures and severity of brain injury in a piglet model of perinatal H/I and, whether injury severity was related to type of seizure, i.e. sub-clinical (electrographic seizures only) or clinical (electrographic seizures+physical signs). Hypoxia (4% O2) was induced in anaesthetised newborn piglets for 30 min with a final 10 min period of hypotension; animals were recovered and survived to 72 h. Animals were monitored daily for seizures both visually and with electroencephalogram (EEG) recordings. Brain injury was assessed with magnetic resonance imaging (MRI), 1H-MR spectroscopy (1H-MRS), EEG and by histology (haematoxylin and eosin). EEG seizures were observed in 75% of all H/I animals, 46% displayed clinical seizures and 29% sub-clinical seizures. Seizure animals showed significantly lower background amplitude EEG across all post-insult days. Presence of seizures was associated with lower cortical apparent diffusion coefficient (ADC) scores and changes in 1H-MRS metabolite ratios at both 24 and 72 h post-insult. On post-mortem examination animals with seizures showed the greatest degree of neuropathological injury compared to animals without seizures. Furthermore, clinical seizure animals had significantly greater histological injury compared with sub-clinical seizure animals; this difference was not apparent on MRI or 1H-MRS measures. In conclusion we report that both sub-clinical and clinical seizures are associated with increased severity of H/I injury in a term model of neonatal H/I. [Copyright &y& Elsevier]

Abstract: Ischemia/hypoxia is known to induce the neural stem cells proliferation and neural differentiation in rodent and human brain; however its mechanisms remain largely unknown. In this study we investigated the effect of hypoxia on neural stem cells (NSCs) proliferation with the expression of cyclin D1 and the phosphorylation of mitogen-activated protein kinases (MAPK) signaling molecules. NSCs were cultured from cortex of fetal Sprague–Dawley rats on embryonic day 5.5. The hypoxia was made using a microaerophilic incubation system. The NSCs proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, diameter measurement of neurospheres, bromodeoxyuridine (BrdU) incorporation assay and cell cycle analysis. The cell death of NSCs was evaluated by terminal dUTP nick-end labeling (TUNEL) assay. The expression of cyclin D1, phosphorylated extracellular signal regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK) and p38 were analyzed by immunoblotting assay. The results showed that hypoxia increased NSCs proliferation in cell amount, diameter of neurospheres, BrdU incorporation and cell division, and the highest proliferation of the NSCs was observed with 12 h hypoxic treatment; hypoxia did not decrease cell death of NSCs; after hypoxic treatment, the expression of cyclin D1 increased, meanwhile P-JNK2 level increased, P-p38 decreased, and no significant change in P-ERK2 level compared to normoxic cultures. JNK inhibitor SP600125 attenuated the increase of cyclin D1 induced by hypoxia. These findings propose that hypoxia increases cyclin D1 expression through activation of JNK in NSCs of rat in vitro, suggesting a novel possible mechanism for hypoxia-induced proliferation of NSCs. [Copyright &y& Elsevier]

Abstract: Following CNS injury there is a period of vulnerability when cells will not easily tolerate a secondary insult. However recent studies have shown that following traumatic brain injury (TBI), as well as hypoxic-ischemic injuries, the CNS may experience a period of protection termed “preconditioning.” While there is literature characterizing the properties of vulnerability and preconditioning in the adult rodent, there is an absence of comparable literature in the developing rat. To determine if there is a window of vulnerability in the developing rat, post-natal day 19 animals were subjected to a severe lateral fluid percussion injury followed by pilocarpine (Pc)-induced status epilepticus at 1, 6 or 24 h post TBI. During the first 24 h after TBI, the dorsal hippocampus exhibited less status epilepticus-induced cell death than that normally seen following Pc administration alone. Instead of producing a state of hippocampal vulnerability to activation, TBI produced a state of neuroprotection. However, in a second group of animals evaluated 20 weeks post injury, double-injured animals were statistically indistinguishable in terms of seizure threshold, mossy fiber sprouting and cell survival when compared to those treated with Pc alone. TBI, therefore, produced a temporary state of neuroprotection from seizure-induced cell death in the developing rat; however, this ultimately conferred no long-term protection from altered hippocampal circuit rearrangements, enhanced excitability or later convulsive seizures. [Copyright &y& Elsevier]

Abstract: Previously, we have shown that chronic intermittent hypoxia (CIH) impairs baroreflex control of heart rate and augments aortic baroreceptor afferent function. In the present study, we examined whether CIH induces structural changes of aortic afferent axons and terminals. Young-adult Fischer 344 (F344, 4 months old) rats were exposed to room air (RA) or CIH for 35–45 days. After 14–24 days of exposure, they received tracer DiI injection into the left nodose ganglion to anterogradely label vagal afferent nerves. After surgery, animals were returned to their cages to continue RA or CIH exposure. Twenty-one days after DiI injection, the animals were sacrificed and the aortic arch was examined using confocal microscopy. In both RA and CIH rats, we found that DiI-labeled vagal afferent axons entered the wall of the aortic arch, then fanned out and branched into large receptive fields with numerous terminals (flower-sprays, end-nets and free endings). Vagal afferent axons projected much more to the anterior wall than to the posterior wall. In general, the flower-sprays, end-nets and free endings were widely and similarly distributed in the aortic arch of both groups. However, several salient differences between RA and CIH rats were found. Compared to RA control, CIH rats appeared to have larger vagal afferent receptive fields. The CIH rats had many abnormal flower-sprays, end-nets, and free endings which were intermingled and diffused into “bush-like” structures. However, the total number of flower-sprays was comparable (P>0.05). Since there was a large variance of the size of flower-sprays, we only sampled the 10 largest flower-sprays from each animal. CIH substantially increased the size of large flower-sprays (P<0.01). Numerous free endings with enlarged varicosities were identified, resembling axonal sprouting structures. Taken together, our data indicate that CIH induces significant remodeling of afferent terminal structures in the aortic arch of F344 rats. We suggest that such an enlargement of vagal afferent terminals may contribute to altered aortic baroreceptor function following CIH. [Copyright &y& Elsevier]

Abstract: Deep diving mammals have developed strategies to cope with limited oxygen availability when submerged. These adaptations are associated with an increased neuronal hypoxia tolerance. Brain neurons of the hooded seal Cystophora cristata remain much longer active in hypoxic conditions than those of mice. To understand the cellular basis of neuronal hypoxia tolerance, we studied neuroglobin and cytochrome c in C. cristata brain. Neuroglobin, a respiratory protein typically found in vertebrate neurons, displays three unique amino acid substitutions in hooded seal. However, these substitutions unlikely contribute to a modulation of O2 affinity. Moreover, there is no significant difference in total neuroglobin protein levels in mouse, rat and seal brains. However, in terrestrial mammals neuroglobin resided exclusively in neurons, whereas in seals neuroglobin is mainly located in astrocytes. This unusual localization of neuroglobin is accompanied by a shift in the distribution of cytochrome c. In seals, this marker for oxidative metabolism is mainly localized in astrocytes, whereas in terrestrial mammals it is essentially found in neurons. Our results indicate that in seals aerobic ATP production depends significantly on astrocytes, while neurons rely less on aerobic energy metabolism. This adaptation may imbue seal neurons with an increased tolerance to hypoxia and potentially also to reactive oxygen species, and may explain in part the ability of deep diving mammals to sustain neuronal activity during prolonged dives. [Copyright &y& Elsevier]

Abstract: Neural progenitor cells (NPCs) have the potential to survive brain ischemia and participate in neurogenesis after stroke. However, it is not clear how survival responses are initiated in NPCs. Using embryonic mouse NPCs and the in vitro oxygen and glucose deprivation (OGD) model, we found that angiopoietin-1 (Ang1) could prevent NPCs from OGD-induced apoptosis, as evidenced by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling and annexin V labeling. Ang1 significantly elevated tunica intima endothelial kinase 2 (Tie2) autophosphorylation level, suggesting the existence of functional Tie2 receptors on NPCs. NPCs under OGD conditions exhibited reduction of Akt phosphorylation, decrease of the Bcl-2/Bax ratio, activation of caspase-3, cleavage of PARP, and downregulation of β-catenin and nestin. Ang1 reversed the above changes concomitantly with significant rising of survival rates of NPCs under OGD, but all these effects of Ang1 could be blocked by either soluble extracellular domain of Tie2 Fc fusion protein (sTie2Fc) or the phosphoinositide 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one (LY294002). Our findings suggest the existence of an Ang1–Tie2–PI3K signaling axis that is essential in initiation of survival responses in NPCs against cerebral ischemia and hypoxia. [Copyright &y& Elsevier]

Abstract: Cerebral ischemia is a major cause of death and disability and may be a complication of neurosurgery. Certain anesthetics may improve recovery after ischemia and hypoxia by altering electrophysiological changes during the insult. Intracellular recordings were made from CA1 pyramidal cells in hippocampal slices from adult rats. Desflurane or propofol was applied 10 min before and during 10 min of hypoxia (95% nitrogen, 5% carbon dioxide). None of the untreated CA1 pyramidal neurons, 46% of the 6% desflurane- and 38% of the 12% desflurane-treated neurons recovered their resting and action potentials 1 h after hypoxia (P<0.05). Desflurane (6% or 12%) enhanced the hypoxic hyperpolarization (4.9 or 4.7 vs. 2.6 mV), increased the time until the rapid depolarization (441 or 390 vs. 217 s) and reduced the level of depolarization at 10 min of hypoxia (−13.5 or −13.0 vs. −0.6 mV); these changes may be part of the mechanism of its protective effect. Either chelerythrine (5 μM), a protein kinase C inhibitor, or glybenclamide (5 μM), a KATP channel blocker, prevented the protective effect and the electrophysiological changes with 6% desflurane. Propofol (33 or 120 μM) did not improve recovery (0 or 0% vs. 0%) 1 h after 10 min of hypoxia; it did not significantly enhance the hypoxic hyperpolarization (3.6 or 3.1 vs. 2.6 mV) or increase the latency of the rapid depolarization (282 or 257 vs. 217 s). The average depolarization at 10 m of hypoxia with 33 μM propofol (−4.1 mV) was slightly but significantly different from that in untreated hypoxic tissue (−0.6 mV). Desflurane but not propofol improved recovery of the resting and action potentials in hippocampal slices after hypoxia, this improvement correlated with enhanced hyperpolarization and attenuated depolarization of the membrane potential during hypoxia. Our results demonstrate differential effects of anesthetics on electrophysiological changes during hypoxia. [Copyright &y& Elsevier]

Abstract: Exposure of neurons to a non-lethal hypoxic stress greatly reduces cell death during subsequent severe ischemia (hypoxic preconditioning, HPC). In organotypic cultures of rat hippocampus, we demonstrate that HPC requires inositol triphosphate (IP3) receptor-dependent Ca2+ release from the endoplasmic reticulum (ER) triggered by increased cytosolic NAD(P)H. Ca2+ chelation with intracellular BAPTA, ER Ca2+ store depletion with thapsigargin, IP3 receptor block with xestospongin, and RNA interference against subtype 1 of the IP3 receptor all blunted the moderate increases in [Ca2+]i (50–100 nM) required for tolerance induction. Increases in [Ca2+]i during HPC and neuroprotection following HPC were not prevented with NMDA receptor block or by removing Ca2+ from the bathing medium. Increased NAD(P)H fluorescence in CA1 neurons during hypoxia and demonstration that NADH manipulation increases [Ca2+]i in an IP3R-dependent manner revealed a primary role of cellular redox state in liberation of Ca2+ from the ER. Blockade of IP3Rs and intracellular Ca2+ chelation prevented phosphorylation of known HPC signaling targets, including MAPK p42/44 (ERK), protein kinase B (Akt) and CREB. We conclude that the endoplasmic reticulum, acting via redox/NADH-dependent intracellular Ca2+ store release, is an important mediator of the neuroprotective response to hypoxic stress. [Copyright &y& Elsevier]

Abstract: We have reported that hypoxia affects the hypothalamic–pituitary–adrenal (HPA) axis and behavior by driving the expression of central corticotropin-releasing hormone (CRH) and its receptors in adult mammals, and this effect is modulated by other factors. Here, we address whether or not intermittent hypoxia (IH) or restraint (R) or a combination of both (IH+R) during gestation would result in differential alteration of the HPA axis and behavior of the adult male offspring. Gravid rats were exposed to IH in a hypobaric chamber (10.8% O2, altitude of 5 km), R, or both, daily for 4 h for 21 days. Control parameters were set at sea level (20.9% O2). All the stressors significantly and differentially increased CRH and corticotropin-releasing factor receptor type 1 (CRHR1) expression but decreased corticotropin-releasing factor receptor type 2 (CRHR2) in the paraventricular nucleus of the hypothalamus (PVN), enhanced CRHR1 mRNA and CRHR2 mRNA expression in the anterior pituitary, and increased plasma adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels and adrenal weight in adult male offspring aged 120 days. Furthermore, norepinephrine (NE) and dopamine (DA) levels significantly increased in the locus coeruleus (LC), while the percentage of entries into the open arms of the elevated-plus maze test (EPM) markedly declined. In all the above effects, the combination-induced effect was stronger than each stressor alone. Confocal imaging showed a rich colocalization of CRHR1 with CRH or urocortin I (Ucn I), and CRHR2 with CRH or urocortin III (Ucn III) in the PVN, and CRHR1 with CRH in the LC in EPM-tested groups. In conclusion, IH or R alone or both in combination during gestation sensitize the HPA axis and induce anxiety-like behavior of the adult male offspring, and the combined effects are significantly great than IH or R alone. The CRH-NE neural circuit between the PVN and LC through CRH receptor driving might partly be involved in the effects. The differential colocalization of CRH with CRHR1 might be the neural basis of these effects. [Copyright &y& Elsevier]

Abstract: Intermittent hypoxia (IH) is a major pathological factor in the development of neural deficits associated with sleep-disordered breathing. Here we demonstrate that IH lasting 2 or 30 days, but not sustained hypoxia (SH) of the same duration, was accompanied by several posttranslational modifications of the large subunit of RNA polymerase II, Rpb1, including hydroxylation of proline 1465, phosphorylation of serine 5 residues within the C-terminal domain, and nondegradative ubiquitylation. These modifications were found to occur in two regions of the brain, hippocampal region CA1 and the prefrontal cortex, but not in neocortex, brainstem and CA3 region of hippocampus. We also found that mice exposed to 14 or 30 days of IH, but not SH, demonstrated cognitive deficits in behavioral assays. Furthermore, by using the pheochromocytoma-derived PC12 cell line, we showed that, under in vitro IH conditions, induction of Rpb1 hydroxylation, phosphorylation, and ubiquitylation required that the von Hippel-Lindau protein be present. We hypothesize that the observed modifications of Rpb1 participate in regulating the expression of genes involved in mediating cognitive deficits evoked by chronic IH. [Copyright &y& Elsevier]

Abstract: Recently we discovered that hypoxia causes marked impairment of reproductive neuroendocrine function in Atlantic croaker, a marine teleost, which is due to a decline in hypothalamic serotonergic activity. As a first step in understanding the molecular responses of the hypothalamic serotonergic system to hypoxia, we cloned and characterized the genes for the enzymes regulating the rate-limiting step in serotonin biosynthesis, tryptophan hydroxylase (TPH-1 and TPH-2) in the croaker brain. The full-length croaker TPH-1 and TPH-2 cDNAs contain open reading frames encoding proteins with 479 and 487 amino acids, respectively, which are highly homologous to the TPH-1 (76–93%) and TPH-2 (64–92%) proteins of other vertebrates. Croaker TPH-1 and TPH-2 mRNA expression was detected throughout the brain but was greatest in the hypothalamic region. Both Northern blot analysis and real-time PCR showed that TPH-1 (transcript size ∼2.1 kb) and TPH-2 (∼1.9 kb) mRNA levels were significantly decreased in the hypothalami of croaker exposed for 2 weeks to hypoxic conditions compared with those in fish exposed to normoxic conditions. Immunohistochemistry of hypothalamic neurons with TPH antibodies showed reduced expression of TPHs in hypoxia-exposed fish compared with normoxic fish. Western blot analysis confirmed that hypoxia caused a marked decline in hypothalamic TPH protein levels, which was associated with decreases in hypothalamic TPH enzyme activity and 5-hydroxytryptophan levels. These results suggest that TPH is a major site of hypoxia-induced down-regulation of serotonergic function in croaker brains. Moreover, they provide the first evidence that hypoxia decreases the expression of TPH transcripts in vertebrate brains. [Copyright &y& Elsevier]

Abstract: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in brain development and the etiology of adult cerebral injuries. In this study, we determined the MMP-2 and 9 responses following hypoxic ischemia (HI) injury in the developing brain. First, we characterized the developmental changes of MMP activity in the rat brain from embryonic day 18 (E18) to postnatal day 120 (P120). MMP-2 activity was high from E18 to P3 and decreased with age (P≤0.001), while MMP-9 activity was not detectable. MMP-2 immunoreactivity was closely associated with differentiating cortical plate and subplate neurons. Next, we characterized the proteolytic changes after unilateral HI brain injury in 3- (P3) and 21- (P21) day-old rats. Zymography revealed that in the P21 rat brain, MMP-9 activity (150 and 92 kDa forms) was increased at 6 h and remained elevated 24 h post-injury in the ipsilateral injured hemisphere (P≤0.001), whereas there was a gradual increase in MMP-2 (65 kDa) activity, reaching a peak at 5 days (P≤0.001). Similarly, quantitative real time polymerase chain reaction (qRT-PCR) indicated significant elevations in MMP-9 and MMP-2 mRNA expression in the injured cortex (P≤0.05) and hippocampus (P≤0.05) at 1 and 5 days post-injury, respectively in the P21 rat brain. In the P3 rat brain, zymography results revealed that both pro (92 kDa) and cleaved (87 kDa) MMP-9 activities were upregulated in the ipsilateral injured hemisphere from 6 h to 1 day after injury (P≤0.001). In contrast, cleaved MMP-2 (60 kDa) was only moderately upregulated at 6 h (P≤0.01), while pro MMP-2 (65 kDa) levels were unaffected. MMP-9 mRNA expression was also increased at 6 h (P≤0.05) following injury at P3, whereas MMP-2 expression remained unchanged compared with the uninjured contralateral hemisphere. Immunohistochemistry indicated that MMP-9 protein expression was localized predominantly to neurons and peri-vascular astrocytes in the affected regions at early time points, whereas MMP-2 was present on reactive astrocytes surrounding the infarct at later time points. Together, these results indicate that MMP-2 may be primarily associated with the development and differentiation of cortical plate neurons and wound recovery processes. Conversely, MMP-9 appeared to be associated with more acute processes during the period of lesion development. [Copyright &y& Elsevier]

Abstract: A critical issue in animal models of perinatal brain injury is to adapt the pertinent pathophysiological scenarios to their corresponding developmental window in order to induce neuropathological and behavioral characteristics reminiscent to perinatal cerebral palsy (CP). A major problem in most of these animal models designed up to now is that they do not present motor deficits characteristic of CP. Using a unique rat paradigm of prenatal inflammation combined to an early postnatal hypoxia–ischemia pertinent to the context of very early premature human newborns, we were interested in finding out if such experimental conditions might reproduce both histological damages and behavioral deficits previously described in the human context. We showed that exposure to lipopolysaccharide (LPS) or hypoxia–ischemia (H/I) induced behavioral alterations in animals subjected to forced motor activity. When both LPS and H/I aggressions were combined, the motor deficits reached their highest intensity and affected both spontaneous and forced motor activities. LPS+H/I-exposed animals also showed extensive bilateral cortical and subcortical lesions of the motor networks affecting the frontal cortices and underlying white matters fascicles, lenticular nuclei and the substantia nigra. These neuropathological lesions and their associated motor behavioral deficits are reminiscent of those observed in very preterm human neonates affected by subsequent CP and validate the value of the present animal model to test new therapeutic strategies which might open horizons for perinatal neuroprotection. [Copyright &y& Elsevier]

Abstract: The stimulation of extrasynaptic N-methyl-d-aspartate (NMDA) receptors triggers cell death pathways and has been suggested to play a key role in cell degeneration and neuron loss associated with glutamate-induced excitotoxicity. In contrast, synaptic NMDA receptors promote neuronal survival. One mechanism through which extrasynaptic NMDA receptors damage neurons may involve Clca1, which encodes a putative calcium-activated chloride channel. Here we show that Clca1 expression is induced in cultured rat hippocampal neurons exposed to oxygen/glucose-free media; this induction is mediated by a signaling pathway activated by extrasynaptic NMDA receptors. Clca1 mRNA levels also increased in the gerbil hippocampus following a transient forebrain ischemia caused by bilateral carotid occlusion. Microelectrode array recordings revealed that oxygen-glucose deprivation enhances hippocampal network firing rates, which induces c-fos transcription through a signaling pathway that, in contrast to Clca1, is activated by synaptic but not extrasynaptic NMDA receptors. Thus, conditions of low oxygen/glucose lead to the activation of both extrasynaptic and synaptic NMDA receptors that regulate distinct target genes. Clca1 may be part of the genomic death program triggered by extrasynaptic NMDA receptors; it could be a marker for ischemic brain damage and a possible target for therapeutic interventions. [Copyright &y& Elsevier]

Abstract: The rapid and irreversible brain injury produced by anoxia when stroke occurs is well known. Cumulative evidence suggests that the activation of neuronal ATP-sensitive potassium (KATP) channels may have inherent protective effects during cerebral hypoxia, yet little information regarding the therapeutic effects of KATP channel openers is available. We hypothesized that pretreatment with a KATP channel opener might protect against brain injury induced by cerebral hypoxia. In this study, adult Wistar rats were treated with iptakalim, a new KATP channel opener, which is selective for SUR2 type KATP channels, by intragastric administration at doses of 2, 4, or 8 mg/kg/day for 7 days before being exposed to simulated high altitude equivalent to 8000 m in a decompression chamber for 8 h leading to hypoxic brain injury. By light and electron microscopic images, we observed that hypobaric hypoxia-induced brain injury could be prevented by pretreatment with iptakalim. It was also observed that the permeability of the blood–brain barrier, water content, Na+ and Ca2+ concentration, and activities of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase in rat cerebral cortex were increased and the gene expression of the occludin or aquaporin-4 was down- or upregulated respectively, which could also be prevented by the pretreatment with iptakalim at doses of 2, 4, or 8 mg/kg in a dose-dependent manner. Furthermore, we found that in an oxygen-and-glucose-deprived model in ECV304 cells and rat cortical astrocytes, pretreatment with iptakalim significantly increased survived cell rates and decreased lactate dehydrogenate release, which were significantly antagonized by glibenclamide, a KATP channel blocker. We conclude that iptakalim is a promising drug that may protect against brain injury induced by acute hypobaric hypoxia through multiple pathways associated with SUR2-type KATP channels, suggesting a new therapeutic strategy for stroke treatment. [Copyright &y& Elsevier]

Abstract: Caffeine is a widely used psychostimulant freely crossing the placental barrier. At the doses usually absorbed, it acts as an antagonist of both A1 and A2A adenosine receptors. Pregnant women are generally not advised to limit their caffeine consumption and thus expose their progeny to the drug during the whole of gestation and lactation. The possibility that such caffeine exposure may have long-term consequences on brain development has led to several behavioral investigations on animal models. Despite the crucial role played by adenosine receptor systems in neonatal breathing control, few studies in vitro have been concerned with the consequences of maternal caffeine absorption on breathing, and none in the unrestrained intact animal. The present investigation analyzed the influence of caffeine exposure via placental and milk transfer on resting ventilation and on the response to moderate alveolar hypoxia of 0 to 2-day-old newborn rat (P0–P2) together with the possible underlying mechanisms. Dams absorbed caffeine (46±3 mg/kg/day) via drinking fluid (0.2 g/L) throughout gestation, in conditions mimicking moderate human consumption. Caffeine exposure did not significantly affect basal respiratory parameters. In contrast, it attenuated both the early increase and the secondary decrease in ventilation triggered by moderate alveolar hypoxia (11% O2 inhaled). The abolition of Fos protein expression evoked by hypoxia suggested that caffeine exposure may decrease the activity of O2-sensing peripheral chemoreceptor pathway. From real-time PCR data, those functional alterations were associated to increases in A2A adenosine receptor and α2 GABAA receptor subunit mRNAs in the medulla. This indicates that, even at moderate doses, maternal caffeine consumption may induce a series of subtle developmental alterations that may affect modulation of breathing control in the neonate in pathological situations such hypoxia. [Copyright &y& Elsevier]

Abstract: Transforming growth factor-β1 (TGF-β1) is widely recognized as a prototype of multifunctional growth factors and master switches in the regulation of key events of development, disease and repair. It is localized in neurons, astrocytes and brain macrophages in altered conditions but its localization in the amoeboid microglial cells (AMC), a nascent brain macrophage in the developing brain has remained unexplored. Here we report expression of TGF-β1 and its receptors namely, transforming growth factor-β receptor I (TβRI) and transforming growth factor-β receptor II (TβRII) in AMC and BV-2 cells induced by hypoxia. Firstly, increase in TGF-β1 mRNA expression and TGF-β1 release was observed in the corpus callosum in postnatal rats subjected to a single hypoxic exposure. RT-PCR and Western blot analysis revealed a concomitant upregulation of TβRI and TβRII mRNA and protein. Secondly, immunofluorescence labeling showed that the preponderant AMC in the corpus callosum were immunoreactive for TGF-β1 and its receptors. In rats subjected to hypoxia, immunoexpression of TGF-β1 and both receptors was markedly enhanced. In longer surviving rats, the AMC transformed into ramified microglia but retained in them the immunoreactivity. In BV-2 cells exposed to hypoxia, TGF-β1 mRNA expression and release of TGF-β1 into the medium were significantly increased. It is noteworthy that expression of TβRI and TβRII mRNA and protein in hypoxic BV-2 cells was reduced indicating a differential response of AMC and BV-2 cells to hypoxia. Notwithstanding, it is unequivocal that AMC in the developing brain express and release TGF-β1 into the ambient environment. We suggest that this may be a mechanism to help autoregulate microglial activation in adverse conditions via its receptors. [Copyright &y& Elsevier]

Abstract: Attempts have been made to elevate excitatory amino acid transporter 2 (EAAT2) expression in an effort to compensate for loss of function and expression associated with disease or pathology. Increased EAAT2 expression has been noted following treatment with β-lactam antibiotics, and during ischemic preconditioning (IPC). However, both of these conditions induce multiple changes in addition to alterations in EAAT2 expression that could potentially contribute to neuroprotection. Therefore, the aim of this study was to selectively overexpress EAAT2 in astrocytes and characterize the cell type specific contribution of this transporter to neuroprotection. To accomplish this we used a recombinant adeno-associated virus vector, AAV1–glial fibrillary acidic protein (GFAP)–EAAT2, designed to selectively drive the overexpression of EAAT2 within astrocytes. Both viral-mediated gene delivery and β-lactam antibiotic (penicillin-G) treatment of rat hippocampal slice cultures resulted in a significant increase in both the expression of EAAT2, and dihydrokainate (DHK) sensitive glutamate uptake. Penicillin-G provided significant neuroprotection in rat hippocampal slice cultures under conditions of both moderate and severe oxygen glucose deprivation (OGD). In contrast, viral-mediated overexpression of EAAT2 in astrocytes provided enhanced neuroprotection only following a moderate OGD insult. These results indicate that functional EAAT2 can be selectively overexpressed in astrocytes, leading to enhanced neuroprotection. However, this cell type specific increase in EAAT2 expression offers only limited protection compared to treatment with penicillin-G. [Copyright &y& Elsevier]

Abstract: The present study used an in vitro brainstem preparation from pre-metamorphic tadpoles and adult bullfrogs (Lithobates catesbeiana) to understand the neural mechanisms associated with central O2 chemosensitivity and its maturation. In this species, brainstem hypoxia increases fictive lung ventilation in tadpoles but decreases in adults. Previous studies have shown that α1-adrenoceptor inactivation prevents these responses, suggesting that noradrenergic neurons are involved. We first tested the hypothesis that the pons (which includes noradrenergic neurons from the locus coeruleus; LC) plays a role in the lung burst frequency response to central hypoxia by comparing the effects of brainstem transection at the LC level between pre-metamorphic tadpoles and adults. Data show that brainstem transection prevents the lung burst frequency response in both stage groups. During development, the progressive decrease in the Na+/K+/Cl− co-transporter NKCC1 contributes to the maturation of neural networks. Because NKCC1 becomes activated during hypoxia, we then tested the hypothesis that NKCC1 contributes to maturation of the central O2 chemoreflex. Double labeling experiments showed that the proportion of tyrosine hydroxylase positive neurons expressing NKCC1 in the LC decreases during development. Inactivation of NKCC1 with bumetanide bath application reversed the lung burst response to hypoxia in tadpoles. Bumetanide inhibited the response in adults. These data indicate that a structure within the pons (potentially the LC) is necessary to the central hypoxic chemoreflex and demonstrate that NKCC1 plays a role in central O2 chemosensitivity and its maturation in this species. [Copyright &y& Elsevier]

Abstract: Chronic intermittent hypoxia (CIH) attenuates baroreflex control of heart rate (HR). In this study, we assessed whether CIH exposure reduced nucleus ambiguus (NA) control of HR and induced neural degeneration in the NA. Fischer 344 (age: 3–4 months) rats were exposed to either room air (RA: normoxia) or intermittent hypoxia for 35–50 days. At the end of these exposures, animals were anesthetized with pentobarbital. HR responses to arterial blood pressure (AP) changes induced by phenylephrine (PE) and sodium nitroprusside (SNP) were measured. In another set of rats, HR and AP responses to l-glutamate (l-Glu) microinjections (10 mM, 20 nl) into the left NA and electrical stimulation of the left cervical vagus nerve at 1–30 Hz (0.5 mA, 1 ms) for 20 s were measured. Brainstem slices at the level of −800, −400, 0, +400, +800 μm relative to the obex were processed in additional rats using Nissl staining. The NA was identified by retrogradely labeling vagal motoneurons using the tracer tetramethylrhodamine dextran (TMR-D) which was injected into the ipsilateral nodose ganglion. We found that CIH significantly 1) reduced the baroreflex control of HR (slope RA: −1.2±0.2 bpm/mmHg; CIH −0.5±0.1 bpm/mmHg; P<0.05); 2) attenuated the HR responses to l-Glu injections into the NA [HR: −280±15 (RA) vs. −235±16 (CIH) beats/min; P<0.05]; 3) augmented the HR responses to electrical stimulation of the vagus (P<0.05); 4) induced a significant cellular loss in the NA region (P<0.05). Thus, CIH induces a cell loss in the NA region which may contribute to attenuation of baroreflex sensitivity and NA control of HR following CIH. [Copyright &y& Elsevier]

Abstract: Chronic hypoxia causes neural dysfunction. Oxygen (O2) supplements have been commonly used to increase the O2 supply, yet the therapeutic benefit of this treatment remains controversial due to a lack of cellular and molecular evidence. In this study, we examined the effects of short-burst O2 supplementation on neural behavior and presynaptic protein expression profiles in a simple chronic hypoxia model of snail Lymnaea stagnalis. We reported that hypoxia delayed the animal response to light stimuli, suppressed locomotory activity, induced expression of stress-response proteins, hypoxia inducible factor-1α (HIF-1α) and heat shock protein 70 (HSP70), repressed syntaxin-1 (a membrane-bound presynaptic protein) and elevated vesicle-associated membrane protein-1 (VAMP-1) (a vesicle-bound presynaptic protein) level. O2 supplements relieved suppression of neural behaviors, and corrected hypoxia-induced protein alterations in a dose-dependent manner. The effectiveness of supplemental O2 was further evaluated by determining time courses for recovery of neural behaviors and expression of stress response proteins and presynaptic proteins after relief from hypoxia conditions. Our findings suggest that O2 supplement improves hypoxia-induced adverse alterations of presynaptic protein expression and neurobehaviors, however, the optimal level of O2 required for improvement is protein specific and system specific. [Copyright &y& Elsevier]

Abstract: The mechanisms of tolerance to subsequent episodes of ischemia induced by cortical spreading depression (CSD) are not clear. The effects of CSD on the expression of inducible nitric oxide synthase (iNOS), hypoxia inducible factor-1α (HIF-1α), and lactate dehydrogenase-A (LDH-A) were evaluated in the present experiment. Unilateral CSD was induced in Sprague-Dawley rats by application of KCl on the right cortex and the mRNA levels of iNOS, HIF-1α, and LDH-A were evaluated at 15 min, 2 h, 4 h, 6 h or 24 h after CSD. RT-PCR analysis showed: 1) an increase of iNOS mRNA at 15 min, 2 h, 4 h; 2) an increase of HIF-1α mRNA at 6 h; 3) an increase of LDH-A mRNA at 4 h. In situ hybridization with specific digoxigenin-labeled oligonucleotides revealed that the mRNA levels were increased at 15 min–2 h for iNOS, 2–4 h for LDH-A and 6 h for HIF-1 after CSD. Immunohistochemistry analysis revealed that levels of iNOS and HIF-1α were increased, respectively, at 2 h and 6 h after CSD. These data suggest that CSD promotes the expression of iNOS, HIF-1α, and LDH-A in nervous cells giving a neuroprotective effect. [Copyright &y& Elsevier]

Abstract: Slack (Slo 2.2), a member of the Slo potassium channel family, is activated by both voltage and cytosolic factors, such as Na+ ([Na+]i) and Cl− ([Cl−]i). Since the Slo family is known to play a role in hypoxia, and since hypoxia/ischemia is associated with an increase in H+ and CO2 intracellularly, we hypothesized that the Slack channel may be affected by changes in intracellular concentrations of CO2 and H+. To examine this, we expressed the Slack channel in Xenopus oocytes and the Slo 2.2 protein was allowed to be inserted into the plasma membrane. Inside-out patch recordings were performed to examine the response of Slack to different CO2 concentrations (0.038%, 5%, 12%) and to different pH levels (6.3, 6.8, 7.3, 7.8, 8.3). In the presence of low [Na+]i (5 mM), the Slack channel open probability decreased when exposed to decreased pH or increased CO2 in a dose-dependent fashion (from 0.28±0.03, n=3, at pH 7.3 to 0.006±0.005, n=3, P=0.0004, at pH 6.8; and from 0.65±0.17, n=3, at 0.038% CO2 to 0.22±0.07, n=3, P=0.04 at 12% CO2). In the presence of high [Na+]i (45 mM), Slack open probability increased (from 0.03±0.01 at 5 mM [Na+]i, n=3, to 0.11±0.01, n=3, P=0.01) even in the presence of decreased pH (6.3). Since Slack activity increases significantly when exposed to increased [Na+]i, even in presence of increased H+, we propose that Slack may play an important role in pathological conditions during which there is an increase in the intracellular concentrations of both acid and Na+, such as in ischemia/hypoxia. [Copyright &y& Elsevier]

Abstract: Rett syndrome (RTT) is associated with mutations in the transcriptional repressor gene MeCP2. Although the clinical and neuropathological signs of RTT suggest disrupted synaptic function, the specific role of methyl-CpG binding protein 2 (MeCP2) in postmitotic neurons remains relatively unknown. We examined whether MeCP2 deficiency in central neurons contributes to the neuropathogenesis in RTT. Primary cerebellar granule neuronal cultures from wild-type (WT) and MeCP2−/− mice were exposed to N-methyl-d-aspartate (NMDA) and AMPA-induced excitotoxicity and hypoxic–ischemic insult. The magnitude of cell death in MeCP2−/− cells after excitotoxicity and hypoxia was greater than in the WT littermate control cultures and occurred after shorter exposures that usually, in the WT, would not cause cell death. Pretreatment with the growth factor fibroblast growth factor 1 (FGF-1) under conditions at which WT cells showed complete neuroprotection, only partially protected MeCP2−/− cells. To elucidate specifically the effects of MeCP2 knockout (KO) on cell death, we examined two death cascade pathways. MeCP2−/− neurons exposed to 6 h of hypoxia exhibited enhanced activation of the proapoptotic caspase-3 and increased mitochondrial release of apoptosis inducing factor (AIF) compared with WT neurons, which did not show significant changes. However, pretreatment with the caspase inhibitor ZVAD-FMK had little or no effect on AIF release and its subcellular translocation to the nucleus, suggesting caspase-independent AIF release and their independent contribution to hypoxia-induced cell death. Reintroduction of intact MeCP2 gene in MeCP2−/− cells or MeCP2 gene silencing by MeCP2siRNA in WT cells further confirmed the differential sensitivity of the WT and MeCP2−/− cells and suggest a direct role of MeCP2 in cell death. These results clearly demonstrate increased cell death occurred in neurons lacking MeCP2 expression via both caspase- and AIF-dependent apoptotic mechanisms. Our findings suggest a novel, yet unknown, role for MeCP2 in central neurons in the control of neuronal response to cell death. [Copyright &y& Elsevier]