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Objective: This study aims to investigate the biological significance of heat shock protein (HSP70) in adaptation to high altitude hypoxia. Methods: Ninety male SD rats were randomly divided into 10 groups: Acute plateau hypoxia group (group 5) and chronic high altitude hypoxia group (group 3), and control group (group 2).Changes in HSP gene and HSP protein expression in brain tissues of SD rats at different altitudes were determined by Western blot and conventional RT-PCR, while an optical and transmission electron microscope was used to observe the cell structure changes of animal brain tissues. Results: HSP70 expression rapidly increased at high altitudes in SD rats under high-altitude hypoxia environments, and HSP70 increased with altitude. Morphological and structural damage in SD rats in each group increased with altitude. Conclusion: The rapid synthesis of HSP70 in heat shock response is beneficial for maintaining the normal physiological function of cells during hypoxia stress, and the amount of HSP70 production is positively correlated with hypoxia tolerance ability. [ABSTRACT FROM AUTHOR]

Neonatal hypoxia-ischemia (HI) is associated to cognitive and motor impairments and until the moment there is no proven treatment. The underlying neuroprotective mechanisms of stem cells are partially understood and include decrease in excitotoxicity, apoptosis and inflammation suppression. This study was conducted in order to test the effects of intracardiac transplantation of human dental pulp stem cells (hDPSCs) for treating HI damage. Seven-day-old Wistar rats were divided into four groups: sham-saline, sham-hDPSCs, HI-saline, and HI-hDPSCs. Motor and cognitive tasks were performed from postnatal day 30. HI-induced cognitive deficits in the novel-object recognition test and in spatial reference memory impairment which were prevented by hDPSCs. No motor impairments were observed in HI animals. Immunofluorescence analysis showed human-positive nuclei in hDPSC-treated animals closely associated with anti-GFAP staining in the lesion scar tissue, suggesting that these cells were able to migrate to the injury site and could be providing support to CNS cells. Our study evidence novel evidence that hDPSC can contribute to the recovery following hypoxia-ischemia and highlight the need of further investigation in order to better understand the exact mechanisms underlying its neuroprotective effects. [ABSTRACT FROM AUTHOR]

Highlights • dMCAoH was developed as a new model of HIE with a low intra-experimental variability. • Post-ischemic treatment with DEX was neuroprotective in rat pups subjected to dMCAoH. • DEX reduced injuries in pups subjected to CCAoH showing mild/moderate infarcts. Abstract Dexpramipexole (DEX) is a drug with a good safety profile in humans, known for its ability to increase mitochondrial ATP production and prompt neuroprotection in adult rodents subjected to cerebral ischemia. In the present study we evaluated the effect of DEX in rat pups subjected to common carotid artery occlusion plus hypoxia (CCAoH, the classic Rice-Vannucci model). Because of the wide range of infarct size distribution in the CCAoH model, a priori subanalysis based on the effect of DEX on mild/moderate or severe brain injuries was conducted. The subanalysis showed that the drug (3 mg/kg bid i.p, after the hypoxic insult) decreased the infarction size in pups with mild/moderate injuries. Next, we developed a distal middle cerebral artery occlusion plus hypoxia (dMCAoH) model, characterized by an intra-experimental infarct size variability lower than that of the CCAoH model. Post-ischemic treatment with DEX (3 mg/kg bid i.p, after the hypoxic insult) reduced brain infarcts in pups exposed to dMCAoH. For the first time, we show that DEX reduces brain injury in different models of neonatal HIE. In light of the favorable safety profile of DEX in humans, the drug might have a realistic translational potential to treatment of perinatal cerebrovascular disorders. [ABSTRACT FROM AUTHOR]

Most small rodents reduce energy demand in hypoxia via behavioural strategies. For example, animals may reduce their activity, and/or move to colder environments or alter huddling strategies to take advantage of anapyretical energy savings. Naked mole rats (NMRs) are among the most hypoxia tolerant mammals and are highly social; social interactions also have a significant impact on behaviour. Therefore, this species offers a fascinating model in which to study trade-offs between social interactions and energy conservation in hypoxia. We hypothesized that the need to conserve energy in hypoxia supersedes the impetus of sociality in this species and predicted that, in hypoxia, behaviour would not differ between individuals or groups of NMRs. To test this hypothesis, we placed awake, freely behaving NMRs, alone or in groups of 2 or 4, into a temperature-controlled apparatus and measured behavioural activity during 1 h each of normoxia (21% O 2 ), acute hypoxia (7% O 2 ), and normoxic recovery. We found that in normoxia, groups of 4 NMRs were significantly more active in all temperatures than were groups of 1–2 NMRs. When exposed to hypoxia, individual NMRs were ~50% less active and their speed was reduced relative to normoxic levels. Conversely, groups of 2 or 4 NMRs exhibited minor or insignificant decreases in time spent active and speed in hypoxia and huddling behaviour was not altered. Our findings suggest that social interactions influence behavioural strategies employed by NMRs in hypoxia. [ABSTRACT FROM AUTHOR]

The ion channel arrest hypothesis has been the foundation of three decades of research into the underlying mechanisms of hypoxia/anoxia tolerance in several key species, including: painted turtles, goldfish, crucian carp, naked mole rats, and arctic and ground squirrels. The hypothesis originally stated that hypoxia/anoxia tolerant species ought to have fewer ion channels per area membrane and/or mechanisms to regulate the conductance of ion channels. Today we can add to this and include mechanisms to remove channels from membranes and the expression of low conductance isoforms. Furthermore, possible oxygen sensing mechanisms in brain include a link to mitochondrial function, changes in the concentration of intracellular Ca 2+ and reactive oxygen species, and activation of protein kinase C and a phosphatase. Importantly ion channel arrest leads to a decrease in metabolic rate that is fundamental to survival without oxygen and in brain is reflected in decreased action potential frequency or spike arrest. This results not only from a decrease in excitatory glutamatergic receptor currents but also by an increase in inhibitory GABAergic receptor currents. The surprising finding that ionic conductance through some ion channels increases is novel and contrary to the ion channel arrest hypothesis. The major insight that this offers is that key regulatory events are occurring at the level of the synapse and we therefore propose the “synaptic arrest hypothesis”. [ABSTRACT FROM AUTHOR]

Stroke is a leading cause of death and a major contributor to neurological disability in adults. Tissue plasminogen activator is the only approved treatment. However, due to its narrow therapeutic window, <5% of patients receive treatment. Recently, hypoxic postconditioning (HPC) was shown to reduce stroke induced-injury in mice, but the mechanisms and functional outcomes are still unknown. In the current study, male Sprague Dawley rats were subjected to endothelin-1 induced stroke. HPC (8% O 2 , 1 h/d for 5d) or normoxia treatments were started 24 h after stroke. Behavioural tests were performed at various time-points (pre- and post-surgery, 1 and 6 days post stroke) and brains were collected 6 days after stroke for histological and immunoblotting analysis. HPC improved deficits in neurological score, motor and sensory function after stroke. Furthermore, HPC reduced infarct volume and neuronal loss in the cortex, while it increased the number of astrocytes and of Fluoro-Jade-positive cells in the injured hemisphere. We observed a mild increase in HIF-1 and its target gene, glucose transporter-1. Our data suggest that HPC-induced neuroprotection was mediated by enhanced astrocyte function, which may have contributed to functional recovery after stroke. [ABSTRACT FROM AUTHOR]

Birth asphyxia resulting in brain hypoxia-ischemia (H-I) can cause neonatal death or lead to persistent brain damage. Recent investigations have shown that group II metabotropic glutamate receptor (mGluR2/3) activation can provide neuroprotection against H-I but the mechanism of this effect is not clear. The aim of this study was to investigate whether mGluR2/3 agonists applied a short time after H-I reduce brain damage in an experimental model of birth asphyxia, and whether a decrease in oxidative stress plays a role in neuroprotection. Neonatal H-I in 7-day-old rats was used as an experimental model of birth asphyxia. Rats were injected intra peritoneally with mGluR2 (LY 379268) or mGluR3 (NAAG) agonists 1 h or 6 h after H-I (5 mg/kg). The weight deficit of the ischemic brain hemisphere, radical oxygen species (ROS) content levels, antioxidant enzymes activity and the concentrations of reduced glutathione (GSH) were measured. Both agonists reduced weight loss in the ischemic hemisphere and mitigated neuronal degeneration in the CA1 hippocampal region and cerebral cortex. Both agonists reduced the elevated levels of ROS in the ipsilateral hemisphere observed after H-I and prevented an increase in antioxidant enzymes activity in the injured hemisphere restoring them to control levels. A decrease in GSH level was also restored after agonists application. The results show that the activation of mGluR2 and mGluR3 a short time after H-I triggers neuroprotective mechanisms that act through the inhibition of oxidative stress and ROS production. The prevention of ROS production by the inhibition of glutamate release and decrease in its extracellular concentration is likely the main mechanism involved in the observed neuroprotection. [ABSTRACT FROM AUTHOR]

Sirtuin 3 (SIRT3) mediates cellular resistance toward various forms of stress. SIRT3 expression in the developing brain, especially its localization in various glial cell types, has not been fully explored. This study aimed to determine SIRT3 expression in the brain of neonatal rats subjected to hypoxia. By immunohistochemistry, immunofluorescence and Western blotting, we show here that SIRT3 expression in the periventricular white matter was up-regulated in hypoxia group compared with the control group at the corresponding time points. Intense SIRT3 expression was detected in microglia at early time points after hypoxia whose cell number was increased with reduced ramifications in hypoxia group compared with the control group. Furthermore, SIRT3 immunoreactivity was obviously enhanced at 24 h, 3 and 7d, but was declined at 14d after hypoxia so that SIRT3 expression between the two groups was comparable. SIRT3 immunofluorescence was also localized in astrocytes labeled with GFAP which was augmented at different time points in hypoxia group. GPAP positive astrocytes exhibited long extending processes being most pronounced at 3d. SIRT3 was moderately expressed at 24 h, 3 and 7d, but was markedly increased at 14d after hypoxia. Moderate SIRT3 expression was also localized in oligodendrocytes labeled with CNPase in the control group. The incidence of CNPase positive oligodendrocytes showing colocalization of SIRT3 increased significantly at 24 h, 3 and 7d after hypoxia. In conclusion, SIRT3 expression was differentially up-regulated in all three major glial cell types following hypoxia. It is suggested that increased SIRT3 expression in the respective glial cell types following hypoxia is involved in different signaling pathways that protect against hypoxic stress in the developing brain. [ABSTRACT FROM AUTHOR]

Introduction The serum proteome following traumatic brain injury (TBI) could provide information for outcome prediction and injury monitoring. The aim with this affinity proteomic study was to identify serum proteins over time and between normoxic and hypoxic conditions in focal TBI. Material and methods Sprague Dawley rats (n = 73) received a 3 mm deep controlled cortical impact (“severe injury”). Following injury, the rats inhaled either a normoxic (22% O 2 ) or hypoxic (11% O 2 ) air mixture for 30 min before resuscitation. The rats were sacrificed at day 1, 3, 7, 14 and 28 after trauma. A total of 204 antibodies targeting 143 unique proteins of interest in TBI research, were selected. The sample proteome was analyzed in a suspension bead array set-up. Comparative statistics and factor analysis were used to detect differences as well as variance in the data. Results We found that complement factor 9 (C9), complement factor B (CFB) and aldolase c (ALDOC) were detected at higher levels the first days after trauma. In contrast, hypoxia inducing factor (HIF)1α, amyloid precursor protein (APP) and WBSCR17 increased over the subsequent weeks. S100A9 levels were higher in hypoxic-compared to normoxic rats, together with a majority of the analyzed proteins, albeit few reached statistical significance. The principal component analysis revealed a variance in the data, highlighting clusters of proteins. Conclusions Protein profiling of serum following TBI using an antibody based microarray revealed temporal changes of several proteins over an extended period of up to four weeks. Further studies are warranted to confirm our findings. [ABSTRACT FROM AUTHOR]

We performed ultrastructural study of cerebral cortex mitochondria in rats with different tolerance to oxygen deficiency (low resistant and highly resistant specimens). Low resistant rats were characterized by the prevalence of mitochondria with lightened matrix due to the nondense packing of cristae. By contrast, mitochondria of highly resistant animals had the dense packing of cristae. The structure of mitochondria underwent adaptive changes at 14-10% O in the inspired air. Under these conditions, structural characteristics of the cerebral cortex in hypoxia-sensitive rats resembled those in resistant animals. The decrease in O concentration to 8% was accompanied by ultrastructural signs of mitochondrial damage, which correlated with de-energization of the cell and dysfunction of adaptive signaling systems. Ultrastructural features of cerebral cortex mitochondria in animals with low and high tolerance to acute oxygen deficiency confirm the hypothesis that they are associated with two different 'functionaland-metabolic portraits'. [ABSTRACT FROM AUTHOR]

Hypoxia–ischemia (HI)-induced perinatal encephalopathy frequently causes chronic neurological morbidities and acute mortality. Bone mesenchymal stem cell (BMSC) transplantation could potentially promote functional and anatomical recovery of ischemic tissue. In vitro hypoxic preconditioning is an effective strategy to improve the survival of BMSCs in ischemic tissue. In this study, cobalt chloride (CoCl 2 ) preconditioned medium from BMSC cultures was injected into the left lateral ventricle of HI rats using a micro-osmotic pump at a flow rate 1.0 μl/h for 7 days. The protein levels of HIF-1α and its target genes, vascular endothelial growth factor and erythropoietin, markedly increased after CoCl 2 preconditioning in BMSCs. In 7-week-old rats that received CoCl 2 preconditioned BMSC medium, results of the Morris water maze test indicated ameliorated spatial working memory function following hypoxia-ischemia damage. Neuronal loss, cellular disorganization, and shrinkage in brain tissue were also ameliorated. Extracellular field excitatory postsynaptic potentials (fEPSPs) in the brain slices of 8-week-old rats were recorded; administration of CoCl 2 preconditioned BMSC culture medium induced a progressive increment of baseline and amplitude of the fEPSPs. Immunohistochemical quantification showed that GluR2 protein expression increased. In conclusion, CoCl 2 activates HIF-1α signals in BMSCs. CoCl 2 preconditioned BMSC culture medium likely effects neuroprotection by inducing long-term potentiation (LTP), which could be associated with GluR2 expression. The paracrine effects of hypoxia preconditioning on BMSCs could have applications in novel cell-based therapeutic strategies for hypoxic and ischemic brain injury. [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]

Background Anoxia leads to a robust generation of reactive oxygen species/nitrogen species which can result in mitochondrial dysfunction and associated cell death in the cerebral cortex of neonates. Aim The present study investigated the pharmacological role of tempol in the treatment of rat neonatal cortical mitochondrial dysfunction induced insult progression (day-1 to day-7) and associated neurobehavioral alterations post-anoxia. Methods Rat pups of 30 h age or postnatal day 2 (PND2) were randomly divided into 5 groups ( n = 5 per group): (1) Control; (2) Anoxia; (3) Anoxia + Tempol 75 mg/kg; (4) Anoxia + Tempol 150 mg/kg; and (5) Anoxia + Tempol 300 mg/kg, and subjected to two episode of anoxia (10 min each) at 24 h of time interval in an enclosed chamber supplied with 100% N 2 . Results Tempol significantly decreased nitric oxide ( NO) formation and simultaneously improved superoxide dismutase (SOD) and catalase (CAT) activities. Further, we observed a significantly ( P < 0.05) improvement in mitochondrial respiration, complex enzyme activities, mitochondrial membrane potential (MMP) along with attenuation of transition pore opening (MPT) after treatment with tempol. Furthermore, tempol decreased expression of mitochondrial Bax, cytochrome-C, caspase-9 and caspase-3 while the increase in expression of cytoplasmic Bax, mitochondrial Bcl-2 on day-7 in cortical region indicating regulation of intrinsic pathway of apoptosis. Further, it improved anoxia-induced neurobehavioral outcome (hanging and reflex latencies). Conclusion Biochemical, molecular and behavioral studies suggest the role of tempol in preserving mitochondrial function and associated neurobehavioral outcomes after neonatal anoxia. [ABSTRACT FROM AUTHOR]

Anoxia is one of the most prevalent causes of neonatal morbidity and mortality, especially in preterm neonates, constituting an important public health problem due to permanent neurological sequelae observed in patients. Oxygen deprivation triggers a series of simultaneous cascades, culminating in cell death mainly located in more vulnerable metabolic brain regions, such as the hippocampus. In the process of cell death by oxygen deprivation, cytosolic calcium plays crucial roles. Intracellular inositol 1,4,5-trisphosphate receptors (IP3Rs) are important regulators of cytosolic calcium levels, although the role of these receptors in neonatal anoxia is completely unknown. This study focused on the functional role of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in rat hippocampus after neonatal anoxia. Quantitative real-time PCR revealed a decrease of IP3R1 gene expression 24 hours after neonatal anoxia. We detected that IP3R1 accumulates specially in CA1, and this spatial pattern did not change after neonatal anoxia. Interestingly, we observed that anoxia triggers translocation of IP3R1 to nucleus in hippocampal cells. We were able to observe that anoxia changes distribution of IP3R1 immunofluorescence signals, as revealed by cluster size analysis. We next examined the role of IP3R1 in the neuronal cell loss triggered by neonatal anoxia. Intrahippocampal injection of non-specific IP3R1 blocker 2-APB clearly reduced the number of Fluoro-Jade C and Tunel positive cells, revealing that activation of IP3R1 increases cell death after neonatal anoxia. Finally, we aimed to disclose mechanistics of IP3R1 in cell death. We were able to determine that blockade of IP3R1 did not reduced the distribution and pixel density of activated caspase 3-positive cells, indicating that the participation of IP3R1 in neuronal cell loss is not related to classical caspase-mediated apoptosis. In summary, this study may contribute to new perspectives in the investigation of neurodegenerative mechanisms triggered by oxygen deprivation. [ABSTRACT FROM AUTHOR]

Objective: To investigate the permeability of β-NGF through blood-brain-barrier (BBB) in neonatal and adult rats, and the spatial distribution of β-NGF in different brain regions in hypoxic-ischemic (HI) and normal neonatal rats. Methods: To investigate the overall permeability of β-NGF through BBB, β-NGF labeled with I was injected into adult rats, neonatal rats and HI neonatal rats via tail vein. The radioactivity of brain tissue and blood was examined and analyzed 30 min after injection. Also, brain regions including the basal forebrain, frontal cortex, hippocampus, hypothalamus, cerebellum, bulbus olfactorius and hypophysis, of all the rats were dissected and radioactivity was examined to investigate the spatial specificity of NGF permeation through BBB. Results: Statistically significant results were observed in I-β-NGF contents in brain tissues of adult rats group, neonatal rats group and HI neonatal rats group (P < 0.05). Compared to the HI neonatal rats' brain with the highest I-β-NGF contents, normal neonatal rats ranks the second while the adult rats were the lowest. While for the spatial specificity examination part, I-β-NGF in both HI group and control group were widely distributed in basal forebrain, frontal cortex, hippocampus, cerebellum and bulbus olfactorius. But the radioactivity in frontal cortex, hippocampus and cerebellum of HI groups are statistically higher than control groups (P < 0.05). Conclusion: β-NGF can more easily penetrate the BBB of newborn rats than adult rats via peripheral venous administration and this effect can be enhanced by HI insult. Also, this HI-induced permeation of β-NGF through BBB is more obvious in frontal cortex, hippocampus and cerebellum. [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]

In hypoxic-ischemic injury of the brain of neonates, the level of caspase-3 was found to be aberrantly activated. Its overexpression leads to the alteration of cytoskeleton protein fodrin and loss of DNA repair enzyme which ultimately results in neurological impairment and disability. Concerning this, the present study was intended to develop novel nicotine hydrazide analogues as caspase inhibitors via efficient synthetic route. These compounds were subsequently tested for inhibitory activity against caspase-3 and -7 where they exhibit highly potent activity against caspase-3 revealing compound 5k as most potent inhibitor (IC 50 = 19.4 ± 2.5 μM). In Western blot analysis, 5k considerably inhibits the overexpression of caspase-3. The aryl nicotinate of compound 5k , as indicated by molecular docking was found to engage His121 and critical enzyme thiols, i.e., Cys163 of caspase-3 for its potent activity. Moreover, histopathological examination of brain tissues and hippocampus neurons showed that compound 5k considerably improves the brain injury and exert neuroprotective effects in hypoxic-ischemic (HI). In brain homogenate, 5k significantly improves the activity of MDA, SOD, GSH-Px, CAT and T-AOC to exert its beneficial effect against oxidative stress induced by HI injury. [ABSTRACT FROM AUTHOR]

The present study was designed to set up a reliable model of severe hypoxia-ischemia brain damage (HIBD) in neonatal rats and several methods were used to identify whether the model was successful. A total of 40 healthy 7-day-old Sprague-Dawley rats were randomly divided into 2 groups: The sham-surgery group (n=18) and the HIBD model group (n=22). The HIBD model was produced according to the traditional Rice method. The rats were anesthetized with ethyl ether. The left common carotid artery (CCA) was exposed, ligated and cut. Following this, the rats were exposed to hypoxia in a normobaric chamber filled with 8% oxygen and 92% nitrogen for 2 h. In the sham-surgery group, the left CCA was exposed but was not ligated, cut or exposed to hypoxia. The neurobehavioral changes of the rats were observed in the 24 h after HIBD. The brains were collected after 72 h to observe the pathological morphological changes of the brain tissue. The behavioral ability and neurobehavioral changes were studied in each group. The water maze test was used for evaluating the learning-memory ability when the rats were 28 days old. Compared with the sham-surgery group, all the HIBD model rats had a lag of motor development. The rats had evident changes in anatomy and Nissl staining, and cognitive impairment was shown through the result of the water maze. Therefore, the model of HIBD in neonatal rats is feasible and provides a reliable model for subsequent studies. [ABSTRACT FROM AUTHOR]

Acetylation of nucleosome histones results in relaxation of DNA and its availability for the transcriptional regulators, and is generally associated with the enhancement of gene expression. Although it is well known that activation of a variety of pro-adaptive genes represents a key event in the development of brain hypoxic/ischemic tolerance, the role of epigenetic mechanisms, in particular histone acetylation, in this process is still unexplored. The aim of the present study was to investigate changes in acetylation of histones in vulnerable brain neurons using original well-standardized model of hypobaric hypoxia and preconditioning-induced tolerance of the brain. Using quantitative immunohistochemistry and Western blot, effects of severe injurious hypobaric hypoxia (SH, 180 mm Hg, 3 h) and neuroprotective preconditioning mode (three episodes of 360 mm Hg for 2 h spaced at 24 h) on the levels of the acetylated proteins and acetylated H3 Lys24 (H3K24ac) in the neocortex and hippocampus of rats were studied. SH caused global repression of the acetylation processes in the neocortex (layers II–III, V) and hippocampus (CA1, CA3) by 3–24 h, and this effect was prevented by the preconditioning. Moreover, hypoxic preconditioning remarkably increased the acetylation of H3K24 in response to SH in the brain areas examined. The preconditioning hypoxia without subsequent SH also stimulated acetylation processes in the neocortex and hippocampus. The moderately enhanced expression of the acetylated proteins in the preconditioned rats was maintained for 24 h, whereas acetylation of H3K24 was intense but transient, peaked at 3 h. The novel data obtained in the present study indicate that large activation of the acetylation processes, in particular acetylation of histones might be essential for the development of brain hypoxic tolerance. [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]

The expression of neurogenesis marker - NeuroD2 transcription factor - in the hippocampal dentate gyrus was studied in rats exposed to severe destructive hypoxia, a single or three episodes of moderate hypobaric hypoxia, preconditioned severe hypoxia, and severe hypoxia followed by 3 sessions of postconditioning by moderate hypobaric hypoxia. All the studied hypoxic exposure modes led to an increase of NeuroD2 level. Three-fold moderate hypoxia per se and in the preconditioning mode (followed by exposure to severe hypoxia) produced most pronounced up-regulatory effect on NeuroD2 expression. The results indicated that stimulation of neurogenesis processes seemed to be one of the aspects of the neuroprotective effect of three-fold preconditioning moderate hypoxia, but not of hypoxic postconditioning. [ABSTRACT FROM AUTHOR]

Aim: Infants with birth asphyxia frequently require resuscitation. Current guidance is to start newborn resuscitation in 21% oxygen. However, infants with severe hypoxia-ischaemia may require prolonged resuscitation with oxygen. To date, no study has looked at the effect of resuscitation in 100% oxygen following a severe hypoxic-ischaemic insult.Methods: Postnatal day 7 Wistar rats underwent a severe hypoxic-ischaemic insult (modified Vannucci unilateral brain injury model) followed by immediate resuscitation in either 21% or 100% oxygen for 30 min. Seven days following the insult, negative geotaxis testing was performed in survivors, and the brains were harvested. Relative ipsilateral cortical and hippocampal area loss was assessed histologically.Results: Total area loss in the affected hemisphere and area loss within the hippocampus did not significantly differ between the two groups. The same results were seen for short-term neurological assessment. No difference was seen in weight gain between pups resuscitated in 21% and 100% oxygen.Conclusion: Resuscitation in 100% oxygen does not cause a deleterious effect on brain injury following a severe hypoxic-ischaemic insult in a rat model of hypoxia-ischaemia. Further work investigating the effects of resuscitation in 100% oxygen is warranted, especially for newborn infants with severe hypoxic-ischaemic encephalopathy. [ABSTRACT FROM AUTHOR]

Background: It is well documented that the nitric oxide (NO) might be directly involved in brain response to hypobaric hypoxia, and could contribute to memory deficiencies. Recent studies have shown that melatonin could attenuate hypoxia or ischemia-induced nerve injuries by decreasing the production of free radicals. The present study, using immunohistochemical and immunoblot methods, aimed to explore whether melatonin treatment may affect the expression of nitric oxide system and protein nitration, and provide neuroprotection in the rat hippocampus injured by hypobaric hypoxia. Prior to hypoxic treatment, adult rats were pretreated with melatonin (100 mg/kg, i.p.) before they were exposed to the altitude chamber with 48 Torr of the partial oxygen concentration (pO2) for 7 h to mimic the ambience of being at 9000 m in height. They were then sacrificed after 0 h, 1, and 3 days of reoxygenation.Results: The results obtained from the immunohistochemical and immunoblotting analyses showed that the expressions of neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nitrotyrosine (Ntyr) and Caspase 3 in the hypoxic hippocampus were increased from 0 h to 3 days of reoxygenation. Interestingly, the hypoxia-induced increase of nNOS, eNOS, iNOS, Ntyr and Caspase 3 protein expression was significantly depressed in the hypoxic rats treated with melatonin.Conclusions: Activation of the nitric oxide system and protein nitration constitutes a hippocampal response to hypobaric hypoxia and administration of melatonin could provide new therapeutic avenues to prevent and/or treat the symptoms produced by hypobaric hypoxia. [ABSTRACT FROM AUTHOR]

Oxymatrine (OMT), an active constituent of Chinese herb Sophora flavescens Ait, has been proved to possess anti-tumor, anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Previous study has demonstrated that OMT had protective roles on multiple in vitro and in vivo brain injury models including regulation of apoptosis-related proteins caspase-3, Bax and Bcl-2. In this study, we investigated whether this protective effect could apply to neonatal hypoxic-ischemic brain damage. Seven-day-old Sprague–Dawley rats were treated with the left carotid artery ligation followed by exposure to 8% oxygen (balanced with nitrogen) for 2.5 h at 37 °C. In sham group rats, neither ligation nor hypoxia was performed. After two successive days intraperitoneal injection with OMT (30, 60 and 120 mg/kg), Nimodipine (1 mg/kg), and saline, brain infarct volume was estimated, histomorphology changes were performed by hematoxylin–eosin (HE) staining as well as electron microscopy. In addition, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and total antioxidant capacity (T-AOC), as well as production of malondialdehyde (MDA) were assayed in ipsilateral hemisphere homogenates to evaluate the redox status after hypoxic-ischemic. Expression of apoptosis-related proteins Caspase-3, Bax and Bcl-2 in brain were analyzed by western-blot analysis and immunofluorescence. Administration of OMT significantly decreased brain infarct volume and the percentage of injured cells, and ameliorated histopathology and morphological injury as well. Furthermore, OMT obviously increased the activities of SOD, GSH-Px, CAT and T-AOC, and decreased MDA content. Western-blot analysis showed a marked decrease in Caspase-3 expression and increase in the ratio of Bcl-2/Bax after OMT (120 mg/kg) post-treatment as compared with hypoxic-ischemic group. These results suggest that OMT exerts a neuroprotective effect against hypoxic-ischemic brain damage in neonatal rats, which is likely to be mediated through increasing anti-oxidant enzyme activities and inhibiting cell death. [ABSTRACT FROM AUTHOR]

Increased autophagy/mitophagy is thought to contribute to cerebellar dysfunction in Purkinje cell degeneration mice. Intriguingly, cerebellar Purkinje cells are highly vulnerable to hypoxia–ischemia (HI), related at least in part to their high metabolic activity. Whether or not excessive or supraphysiologic autophagy plays a role in Purkinje cell susceptibility to HI is unknown. Accordingly, we evaluated the role of autophagy in the cerebellum after global ischemia produced by asphyxial cardiac arrest in postnatal day (PND) 16–18 rats, using siRNA-targeted inhibition of Atg7, necessary for microtubule-associated protein light chain 3-II (LC3-II) and Atg12–Atg5 complex formation. Two days before a 9 min asphyxial cardiac arrest or sham surgery, Atg7 or control siRNA was injected intracisternally to target the cerebellum. Treatment with Atg7 siRNA: 1) reduced Atg7 protein expression in the cerebellum by 56%; 2) prevented the typical ischemia-induced formation of LC3-II in the cerebellum 24 h after asphyxial cardiac arrest; 3) improved performance on the beam-balance apparatus on days 1–5; and 4) increased calbindin-labeled Purkinje cell survival assessed on day 14. Improved Purkinje cell survival was more consistent in female vs. male rats, and improved beam-balance performance was only seen in female rats. Similar responses to Atg7 siRNA i.e. reduced autophagy and neurodegeneration vs. control siRNA were seen when exposing sex-segregated green fluorescent protein-LC3 tagged mouse primary cortical neurons to oxygen glucose deprivation in vitro. Thus, inhibition of autophagy after global ischemia in PND 16–18 rats leads to increased survival of Purkinje cells and improved motor performance in a sex-dependent manner. [ABSTRACT FROM AUTHOR]

Ineffective contractions and prolonged labor are common birth complications in primiparous women, and oxytocin is the most common agent given for induction or augmentation of labor. Clinical studies in humans suggest oxytocin might adversely affect the CNS response to hypoxia at birth. In this study, we used a rat model of global anoxia during Cesarean section birth to test if administering oxytocin to pregnant dams prior to birth affects the acute neonatal CNS response to birth anoxia. Anoxic pups born from dams pre-treated with intravenous injections or infusions of oxytocin before birth showed significantly increased brain lactate, a metabolic indicator of CNS hypoxia, compared to anoxic pups from dams pre-treated with saline. Anoxic pups born from dams given oxytocin before birth also showed decreased brain ATP compared to anoxic pups from saline dams. Direct injection of oxytocin to postnatal day 2 rat pups followed by exposure to anoxia also resulted in increased brain lactate and decreased brain ATP, compared to anoxia exposure alone. Oxytocin pre-treatment of the dam decreased brain malondialdehyde, a marker of lipid peroxidation, as well as protein kinase C activity, both in anoxic pups and controls, suggesting oxytocin may reduce aspects of oxidative stress. Finally, when dams were pretreated with indomethacin, a cyclooxygenase (COX) inhibitor, maternal oxytocin no longer potentiated effects of anoxia on neonatal brain lactate, suggesting this effect of oxytocin may be mediated via prostaglandin production or other COX-derived products. The results indicate that maternal oxytocin administration may have multiple acute effects on CNS metabolic responses to anoxia at birth. [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]

Periventricular leukomalacia (PVL) is a common ischemic brain injury in premature infants for which there is no effective treatment. The objective of this study was to determine whether transplanted mouse oligodendrocyte progenitor cells (OPCs) have neuroprotective effects in a rat model of PVL. Hypoxia-ischemia (HI) was induced in 3-day-old rat pups by left carotid artery ligation, followed by exposure to 6% oxygen for 2.5 h. Animals were assigned to OPC transplantation or sham control groups and injected with OPCs or PBS, respectively, and sacrificed up to 6 weeks later for immunohistochemical analysis to investigate the survival and differentiation of transplanted OPCs. Apoptosis was evaluated by double immunolabeling of brain sections for caspase-3 and neuronal nuclei (NeuN), while proliferation was assessed using a combination of anti-Nestin and -bromodeoxyuridine antibodies. The expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 was examined 7 days after OPC transplantation. The Morris water maze was used to test spatial learning and memory. The results showed that transplanted OPCs survived and formed a myelin sheath, and stimulated BDNF and Bcl-2 expression and the proliferation of neural stem cells (NSC), while inhibiting HI-induced neuronal apoptosis relative to control animals. Moreover, deficits in spatial learning and memory resulting from HI were improved by OPC transplantation. These results demonstrate an important neuroprotective role for OPCs that can potentially be exploited in cell-based therapeutic approaches to minimize HI-induced brain injury. [ABSTRACT FROM AUTHOR]

Ischemic brain injury is a dynamic process involving oxidative stress, inflammation, cell death and the activation of endogenous adaptive and regenerative mechanisms depending on the activation of transcription factors such as hypoxia-inducible factor 1-alpha. Accordingly, we have previously described a new focal hypoxia model by direct intracerebral cobalt chloride injection. In turn, oleanolic acid, a plant-derived triterpenoid, has been extensively used in Asian countries for its anti-inflammatory and anti-tumor properties. A variety of novel pharmacological effects have been attributed to this triterpenoid, including beneficial effects on neurodegenerative disorders – including experimental autoimmune encephalomyelitis – due to its immunomodulatory activities at systemic level, as well as within the central nervous system. In this context, we hypothesize that this triterpenoid may be capable of exerting neuroprotective effects in ischemic brain, suppressing glial activities that contribute to neurotoxicity while promoting those that support neuronal survival. In order to test this hypothesis, we used the intraperitoneal administration of oleanoic acid in adult rats for seven days previous to focal cortical hypoxia induced by cobalt chloride brain injection. We analyzed the neuroprotective effect of oleanoic acid from a morphological point of view, focusing on neuronal survival and glial reaction. [ABSTRACT FROM AUTHOR]

Noxious stimuli applied at doses close to but below the threshold of cell injury induce adaptive responses that provide a defense against additional stress. Epileptic preconditioning protects neurons against status epilepticus and ischemia; however, it is not known if the converse is true. During hypoxia/ischemia (H/I), lactate released from astrocytes is taken up by neurons and is stored for energy, a process mediated by monocarboxylate transporter 4 (MCT4) in astroglia. The present study investigated whether H/I preconditioning can provide protection to neurons against epilepsy through upregulation of MCT4 expression in astrocytes in vitro and in vivo. An oxygen/glucose deprivation protocol was used in primary astrocyte cultures, while rats were subjected to an intermittent hypoxia preconditioning (IHP) paradigm followed by lithium-pilocarpine-induced epilepsy as well as lactate transportation inhibitor injection, with a subsequent evaluation of protein expression as well as behavior. H/I induced an upregulation of MCT4 expression, while an IHP time course of 5 days provided the greatest protection against epileptic seizures, which was most apparent by 3 days after IHP. However, lactate transport function disturbances can block the protective effect induced by IHP. These findings provide a potential basis for the clinical treatment of epilepsy. [ABSTRACT FROM AUTHOR]

Parameters of respiration, transmembrane potential, and Ca-binding capacity of mitochondria isolated from Langendorff-perfused hearts of rats adapted to normobaric hypoxia were analyzed. Ischemia and reperfusion modeling in intact and adapted animals reduced Ca-binding capacity of mitochondria, which indicated increased sensitivity of mitochondrial permeability transition pores (MPTP) to calcium ions. These changes were accompanied by a decrease in transmembrane potential, ADP/O coeffi cient (ratio of added ADP to oxygen consumption in State 3), and inhibition of State 3 respiration. At the same time, adaptation attenuated the negative effect of ischemia and reperfusion on the functional state of mitochondria. [ABSTRACT FROM AUTHOR]

Abstract: Perinatal nicotine exposure downregulated angiotensin II type 2 receptor (AT2R) in the developing brain and increased brain vulnerability to hypoxic–ischemic injury in male neonatal rats. We tested the hypothesis that site-specific CpG methylation at AT2R gene promoter contributes to the increased vulnerability of brain injury in the neonate. Nicotine was administered to pregnant rats from day 4 of gestation to day 10 after birth. Brain hypoxic–ischemic injury was induced in day 10 male pups. CpG methylation at AT2R promoter was determined in the brain by quantitative methylation-specific PCR. Nicotine exposure significantly increased the methylation of a single CpG−52 locus near the TATA-box at AT2R promoter. Electrophoretic mobility shift assay indicated that the methylation of CpG−52 significantly decreased the binding affinity of TATA-binding protein (TBP). Chromatin immunoprecipitation assay further demonstrated an increase in the binding of a methyl-binding protein and a decrease in TBP binding to AT2R promoter in vivo in neonatal brains of nicotine-treated animals. This resulted in AT2R gene repression in the brain. Intracerebroventricular administration of a demethylating agent 5-aza-2′-deoxycytidine abrogated the enhanced methylation of CpG−52, rescued the TBP binding, and restored AT2R gene expression. Of importance, 5-aza-2′-deoxycytidine reversed the nicotine-increased vulnerability of brain hypoxic–ischemic injury in the neonate. The finding provides mechanistic evidence of increased promoter methylation and resultant AT2R gene repression in the developing brain linking perinatal stress and a pathophysiological consequence of heightened vulnerability of brain hypoxic–ischemic encephalopathy in the neonate. [Copyright &y& Elsevier]

Highlights: [•] Neonatal rats reared in hyperoxia have a sustained HVR younger than expected. [•] We used systemic PPADS to isolate contributions of CNS hypoxia. [•] Control rats exhibit hypoxic ventilatory depression at P4-5 but not at P13-15. [•] Hyperoxia-reared rats exhibited no hypoxic ventilatory depression at either age. [•] Developmental hyperoxia elicits plasticity in the CNS response to hypoxia. [Copyright &y& Elsevier]

We report here the first studies of the effects of acute hypobaric hypoxia (AHH)during the period of early organogenesis on three sequential generations: the generation including pregnant females exposed to AHH (F0) and the two subsequent generations – F1 and F2. All three generations showed significant abnormalities in behavioral measures of motor and orientational-investigative activity, reflecting the state of anxiety, while generations F1 and F2 also showed changes in the ability of offspring to learn with positive reinforcement. These changes persisted to the pubertal period of life. In addition, females of generations F0 and F1 showed influences of AHH on maternal behavior, these influences being in different directions. The possible mechanisms of the effects of gestational stress during early organogenesis on subsequent generations are discussed. [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: The hippocampus is comprised of two distinct subfields that show different responses to hypoxic-ischemic brain injury: the CA1 region is particularly susceptible whereas the dentate gyrus (DG) is quite resistant. Our aim was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of A2A adenosine receptor antagonism to recovery of synaptic activity after OGD. Extracellular recordings of field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG in brain slices prepared from male Wistar rats were used. A 9-min OGD is needed in the DG to always induce the appearance of anoxic depolarization (AD) and the irreversible block of synaptic activity, as recorded up to 24 h from the end of the insult, whereas only 7-min OGD is required in the CA1 region. Selective antagonism of A2A adenosine receptors by ZM241385 significantly prevents or delays the appearance of AD and protects from the irreversible block of neurotransmission induced by 9-min OGD in the DG. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone of DG in slices prepared from 5-bromo-2′-deoxyuridine (BrdU) treated rats was investigated. Slices were further incubated with an immature neuronal marker, doublecortin (DCX). The number of BrdU+ cells was significantly decreased 6 h after 9-min OGD and this effect was antagonized by ZM241385. After 24 h from the end of 9-min OGD, the number of BrdU+ cells returned to that found before OGD and increased arborization of tertiary dendrites of DCX+ cells was observed. The adenosine A2A antagonist ZM241385 protects from synaptic failure and from decreased proliferation of immature neuronal cells at a precocious time after OGD. [Copyright &y& Elsevier]

Background: Intra-uterine growth restriction (IUGR) has been associated with increased predisposition to age-related complications. We tested the hypothesis that rat offspring models of IUGR would exhibit exacerbated, age-related retinal dysfunction. Methods: Female Sprague-Dawley rats (maintained at 11.5% O2 from gestational day 15 to 21 to induce IUGR) and control offspring (maintained at 21% O2 throughout pregnancy) had retinal function assessed at 2 months (young) and 14 months of age (aged) with electroretinogram (ERG) recordings. Retinal anatomy was assessed by immunofluorescence. Results: Deficits in rod-driven retina function were observed in aged IUGR offspring, as evidenced by reduced amplitudes of dark-adapted mixed a-wave Vmax (by 49.3%, P<0.01), b-wave Vmax (by 42.1%, P<0.001) and dark-adapted peak oscillatory potentials (by 42.3%, P<0.01). In contrast to the rod-driven defects specific to aged IUGR offspring, light adapted ERG recordings revealed cone defects in young animals, that were stationary until old age. At 2 months, IUGR offspring had amplitude reductions for both b-wave (Vmax by 46%, P<0.01) and peak oscillatory potential (Vmax by 38%, P<0.05). Finally, defects in cone-driven responses were further confirmed by reduced maximal photopic flicker amplitudes at 2 (by 42%, P<0.001) and 14 months (by 34%, P = 0.06) and critical flicker fusion frequencies at 14 months (Control: 42±1 Hz, IUGR: 35±2 Hz, P<0.05). These functional changes were not paralleled by anatomical losses in IUGR offspring retinas. Conclusions: These data support that the developing retina is sensitive to stressors, and that pathways governing cone- and rod-driven function differ in their susceptibilities. In the case of prenatal hypoxia, cone- and rod-driven dysfunction manifest at young and old ages, respectively. We must, therefore, take into account the specific impact that fetal programming might exert on age-related retinal dystrophies when considering related diagnoses and therapeutic applications. [ABSTRACT FROM AUTHOR]

It has been reported that both activation of N-methyl-D-aspartate receptors and acid-sensing ion channels during cerebral ischemic insult contributed to brain injury. But which of these two molecular targets plays a more pivotal role in hypoxia-induced brain injury during ischemia is not known. In this study, the neuroprotective effects of an acid-sensing cation channel blocker and an N-methyl-D-aspartate receptor blocker were evaluated in a rat model of cardiac arrest-induced cerebral hypoxia. We found that intracisternal injection of amiloride, an acid-sensing ion channel blocker, dose-dependently reduced cerebral hypoxia-induced neurodegeneration, seizures, and audiogenic myoclonic jerks. In contrast, intracisternal injection of memantine, a selective uncompetitive N-methyl-D-aspartate receptor blocker, had no significant effect on cerebral hypoxia-induced neurodegeneration, seizure and audiogenic myoclonic jerks. Intracisternal injection of zoniporide, a specific sodium-hydrogen exchanger inhibitor, before cardiac arrest-induced cerebral hypoxia, also did not reduce cerebral hypoxia-induced neurodegeneration, seizures and myoclonic jerks. These results suggest that acid-sensing ion channels play a more pivotal role than N-methyl-D-aspartate receptors in mediating cerebral hypoxia-induced brain injury during ischemic insult. [ABSTRACT FROM AUTHOR]

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: 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: Hypoxia is an important pathogenic factor for the induction of vascular leakage and brain edema formation. Recent studies suggest a role for TNF-α in the induction of brain edema. Ghrelin attenuates the synthesis of TNF-α following subarachnoid hemorrhage and traumatic brain injury (TBI). Therefore, we examined the effects of ghrelin on the brain edema, serum TNF-α levels and body weight in a systemic hypoxia model. Adult male Wistar rats were divided into acute and chronic controls, acute or chronic hypoxia and ghrelin-treated (80μg/kg/ip/daily) acute or chronic hypoxia groups. Systemic hypoxia was induced in rats by a normobaric hypoxic chamber (O2 11%) for two days (acute) or ten days (chronic). Effect of ghrelin on brain edema and serum TNF-α levels was assessed by dry–wet and ELISA method, respectively. The results showed that acute (P <0.001) and chronic (P <0.05) hypoxia caused an increase of brain water content. Administration of ghrelin only in the acute hypoxia group significantly (P <0.001) reduced brain water content. Acute hypoxia caused an increase of serum TNF-α level (P <0.001) and ghrelin significantly (P <0.001) reduced it. TNF-α level in chronic hypoxia did not change significantly. Both acute and chronic hypoxia decreased body weight significantly (P <0.001) and administration of ghrelin only could prevent further weight loss in chronic hypoxia group (P <0.001). Our findings show that administration of ghrelin may be useful in reducing brain edema induced by acute systemic hypoxia and at least part of the anti-edematous effects of ghrelin is due to decrease of serum TNF-α levels. [Copyright &y& Elsevier]

Abstract: Tetramethylpyrazine (TMP), one of the active ingredients of the Chinese herb Lingusticum Wallichii Frantchat (Chuan Xiong), plays an important role in neuroprotection. However, the protective effect of TMP on the medulla oblongata, the most important region of the brain for cardiovascular and respiratory control, during chronic hypoxia remains unclear. In this study, we examined the neuroprotective effect of TMP on the medulla oblongata after chronic hypoxic injury in rats. Male Sprague–Dawley rats were randomly divided into four groups: control group, TMP group, chronic hypoxia group, and chronic hypoxia+TMP group. Rats were exposed to hypoxia (10% (v/v) O2) or normoxia for 6h daily for 14days. TMP (80mg/kg) or vehicle (saline) was injected intraperitoneally 30min before experimentation. Loss of neurons in the pre-Bötzinger complex, the nucleus ambiguus, the nucleus tractus solitarius, the hypoglossal nucleus and the facial nucleus were evaluated by Nissl staining. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured, and apoptosis was monitored using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. The level of Bcl-2 mRNA and Bax mRNA was quantitatively measured by RT-PCR analysis. TMP protected Nissl bodies of neurons from injury in all nuclei observed, and reduced the loss of neurons in the nucleus ambiguus, the nucleus tractus solitarius, and the hypoglossal nucleus in rats subjected to chronic hypoxia. TMP upregulated SOD activity and inhibited the increase in MDA content in the medulla oblongata of hypoxic rats. In addition, TMP decreased the rate of apoptosis index (the percentage of apoptotic cells against the total number of cells) in all medullary structures examined, excepting the nucleus ambiguus and inhibited the decrease in Bcl-2 mRNA levels in the medulla oblongata following hypoxia. Our findings indicate that TMP may protect the medullary structures that are involved in cardiovascular and respiratory control from injury induced by chronic hypoxia in rats via its anti-oxidant and anti-apoptotic effects. [Copyright &y& Elsevier]

As a consequence of obstetric complications, neonatal hypoxia has been discussed as an environmental factor in the pathophysiology of schizophrenia. However, the biological consequences of hypoxia are unclear. The neurodevelopmental hypothesis of schizophrenia suggests that the onset of abnormal brain development and neuropathology occurs perinatally, whereas symptoms of the disease appear in early adulthood. In our animal model of chronic neonatal hypoxia, we have detected behavioral alterations resembling those known from schizophrenia. Disturbances in cell proliferation possibly contribute to the pathophysiology of this disease. In the present study, we used postnatal rats to investigate cell proliferation in several brain areas following neonatal hypoxia. Rats were repeatedly exposed to hypoxia (89 % N, 11 % O) from postnatal day (PD) 4-8. We then evaluated cell proliferation on PD 13 and 39, respectively. These investigations were performed in the anterior cingulate cortex (ACC), caudate-putamen (CPU), dentate gyrus, and subventricular zone. Rats exposed to hypoxia exhibited increased cell proliferation in the ACC at PD 13, normalizing at PD 39. In other brain regions, no alterations have been detected. Additionally, hypoxia-treated rats showed decreased CPU volume at PD 13. The results of the present study on the one hand support the assumption of chronic hypoxia influencing transient cell proliferation in the ACC, and on the other hand reveal normalization during ageing. [ABSTRACT FROM AUTHOR]

Abstract: We have previously shown that when siRNA against Int6 (siRNA-Int6) was used, hypoxia-inducible factor 2α (HIF2α) activity was stabilized even under normoxic conditions, and the expression of several angiogenic factors was increased. In neuronal tissues, the mechanism underlying angiogenesis remains largely unknown. In the current study, we investigate the role of the tumor suppressor Int6/eIF3e in the regulation of the expression of angiogenic factors in neuronal cells. In addition, we test whether siRNA-Int6 reduces cold-induced brain damage in rats. We used human neuroblastoma SHSY5Y cells transfected with either siRNA-Int6, or a negative control siRNA. Real-time PCR and supersensitive multiplex assay were used to detect gene and protein expression of several angiogenic factors after transfection. For the animal studies, Wistar rats were subjected to brain damage by cold injury, and 50μg siRNA-Int6, 100μg siRNA-Int6, or negative control was administrated. At day 7 post-treatment, brain sections were stained and image analysis system was used to determine the damaged area. Our experiments using SHSY5Y cells revealed a significant effect of siRNA-Int6 on the expression of HIF2α but not HIF1α, both at 8 and 24h after transfection. The siRNA-Int6 led to significant up-regulation of angiogenic factors, including vascular endothelial growth factor and platelet-derived growth factor-B, both at the mRNA and protein levels. Furthermore, our animal studies revealed significantly reduced area of cold-induced damage in rats receiving siRNA-Int6, compared to negative controls. Our findings indicate that Int6 act as a hypoxia-independent master switch of angiogenesis in neuronal cells, and that inhibition of Int6 by siRNA may be an effective therapeutic strategy in treating ischemic diseases such as brain ischemia and injury. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to investigate the antioxidant and possible neuroprotective and antioxidant effects of five alkyl hydroxytyrosyl (HT) ethers (ethyl, butyl, hexyl, octyl and dodecyl) in rat brain slices. None of the compounds modified lipid peroxidation or glutathione concentrations (GSH) in oxygenated samples. The effects of oxidative stress were investigated with ferrous salts to induce lipid peroxidation and diethylmaleate (DEM) to reduce GSH. All compounds inhibited lipid peroxidation with an inhibitory concentration 50% (IC50) one tenth that of HT. These compounds, especially the butyl derivative, prevented GSH depletion after incubation with DEM. We also explored the neuroprotective effect of these compounds in an experimental model of hypoxia-reoxygenation in rat brain slices. All compounds showed neuroprotective and antioxidant effects. Our results established a relationship between these effects and the length of the carbon chain (maximum effect in the range of C4–C8). [Copyright &y& Elsevier]

The Kölliker-Fuse nucleus (KFN) in dorsolateral pons has been implicated in many physiological functions via its extensive efferent connections. Here, we combine iontophoretic anterograde tracing with posthypoxia c-Fos immunohistology to map KFN axonal terminations among hypoxia-activated/nonactivated brainstem and spinal structures in rats. Using a set of stringent inclusion/exclusion criteria to align visualized axons across multiple coronal brain sections, we were able to unequivocally trace axonal trajectories over a long rostrocaudal distance perpendicular to the coronal plane. Structures that were both richly innervated by KFN axonal projections and immunopositive to c-Fos included KFN (contralateral side), ventrolateral pontine area, areas ventral to rostral compact/subcompact ambiguus nucleus, caudal (lateral) ambiguus nucleus, nucleus retroambiguus, and commissural-medial subdivisions of solitary tract nucleus. The intertrigeminal nucleus, facial and hypoglossal nuclei, retrotrapezoid nucleus, parafacial region and spinal cord segment 5 were also richly innervated by KFN axonal projections but were only weakly (or not) immunopositive to c-Fos. The most striking finding was that some descending axons from KFN sent out branches to innervate multiple (up to seven) pontomedullary target structures including facial nucleus, trigeminal sensory nucleus, and various parts of ambiguus nucleus and its surrounding areas. The extensive axonal fan-out from single KFN neurons to multiple brainstem and spinal cord structures ('one-to-many relationship') provides anatomical evidence that KFN may coordinate diverse physiological functions including hypoxic and hypercapnic respiratory responses, respiratory pattern generation and motor output, diving reflex, modulation of upper airways patency, coughing and vomiting abdominal expiratory reflex, as well as cardiovascular regulation and cardiorespiratory coupling. [ABSTRACT FROM AUTHOR]

Studies suggest that neuroprotective effects of normobaric oxygen (NBO) therapy in acute stroke are partly mediated by hemodynamic alterations. We investigated cerebral hemodynamic effects of repeated NBO exposures. Serial magnetic resonance imaging (MRI) was performed in Wistar rats subjected to focal ischemic stroke. Normobaric oxygen-induced functional cerebral blood volume (fCBV) responses were analyzed. All rats had diffusion-weighted MRI (DWI) lesions within larger perfusion deficits, with DWI lesion expansion after 3 hours. Functional cerebral blood volume responses to NBO were spatially and temporally heterogeneous. Contralateral healthy tissue responded consistently with vasoconstriction that increased with time. No significant responses were evident in the acute DWI lesion. In hypoperfused regions surrounding the acute DWI lesion, tissue that remained viable until the end of the experiment showed relative preservation of mean fCBV at early time points, with some rats showing increased fCBV (vasodilation); however, these regions later exhibited significantly decreased fCBV (vasoconstriction). Tissue that became DWI abnormal by study-end initially showed marginal fCBV changes that later became moderate fCBV reductions. Our results suggest that a reverse-steal hemodynamic effect may occur in peripheral ischemic zones during NBO treatment of focal stroke. In addition, CBV responses to NBO challenge may have potential as an imaging marker to distinguish ischemic core from salvageable tissues. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies showed that inflammation and apoptosis were involved in the pathogenesis of hypoxic–ischemic brain injury. The immature brain is particularly vulnerable to damage. Intracerebral injection of glial cell line-derived neurotrophic factor (GDNF) has been shown to reduce the injury induced by hypoxia–ischemia (HI). In this study, the neuroprotective effect of intracerebral ultrasound-induced dissolution of lipid-coated GDNF microbubbles was investigated in a neonatal rat model of hypoxic–ischemic brain injury. Hypoxic–ischemic injury was induced in 7-day-old rats in the present study. The rats with hypoxia–ischemia received intracerebral injections of GDNF-containing microbubbles (0.5mg/kg). They then received low frequency ultrasound stimulation (20kHz, 2h intervals for a total of 24h and each time lasted for 1min) to induce release of GDNF into the right striatum. We found that low frequency ultrasound stimulation can induce lipid-coated GDNF microbubbles to release GDNF. Ultrasound-induced dissolution of lipid-coated GDNF microbubbles treatment reduced infarction volume and improved neurological outcomes in neonatal rats. In the meanwhile, the microbubbles attenuated the production of inducible nitric oxide synthase, nitric oxide and tumor necrosis factor-alpha, as well as the activation of caspase-3 in insulted side of brain in neonatal rats. These data demonstrated that ultrasound-induced dissolution of lipid-coated GDNF microbubbles treatment can provide a neuroprotective effect against hypoxia–ischemia in neonatal rats. [Copyright &y& Elsevier]

Doxycycline may potentially be a neuroprotective treatment for neonatal hypoxic-ischemic brain injury through its anti-inflammatory effects. The aim of this study was to examine any long-term neuroprotection by doxycycline treatment on cerebral gray and white matter. Hypoxic-ischemic brain injury was induced in 7-day-old rats. Pups were treated with either doxycycline (HI+doxy) or saline (HI+vehicle) by intraperitoneal injection at 1 h after hypoxia-ischemia (HI). At 6 h after HI, MnCl2 was injected intraperitoneally for later manganese-enhanced magnetic resonance imaging (MRI). MRI was performed with diffusion-weighted imaging on day 1 and T1-weighted imaging and diffusion tensor imaging at 7, 21 and 42 days after HI. Animals were killed after MRI on day 42 and histological examinations of the brains were performed. There was a tendency towards lower lesion volumes on diffusion maps among HI+doxy than HI+vehicle rats at 1 day after HI. Volumetric MRI showed increasing differences between groups with time after HI, with less cyst formation and less cerebral tissue loss among HI+doxy than HI+vehicle pups. HI+doxy pups had less manganese enhancement on day 7 after HI, indicating reduced inflammation. HI+doxy pups had higher fractional anisotropy on diffusion tensor imaging in major white matter tracts in the injured hemisphere than HI+vehicle pups, indicating less injury to white matter and better myelination. Histological examinations supported the MRI results. Lesion size on early MRI was highly correlated with final injury measures. In conclusion, a single dose of doxycycline reduced long-term cerebral tissue loss and white matter injury after neonatal HI, with an increasing effect of treatment with time after injury. [ABSTRACT FROM AUTHOR]