Combined treatment with naloxone and the alpha2 adrenoceptor antagonist atipamezole reversed brain hypoxia induced by a fentanyl-xylazine mixture in a rat model.

Xylazine, a veterinary tranquillizer known by drug users as "Tranq", is being increasingly detected in people who overdose on opioid drugs, indicating enhanced health risk of fentanyl-xylazine mixtures. We recently found that xylazine potentiates fentanyl- and heroin-induced brain hypoxia and eliminates the rebound-like post-hypoxic oxygen increases. Here, we used oxygen sensors coupled with high-speed amperometry in rats of both sexes to explore the treatment potential of naloxone plus atipamezole, a selective α2-adrenoceptor antagonist, in reversing brain (nucleus accumbens) and periphery (subcutaneous space) hypoxia induced by a fentanyl-xylazine mixture. Pretreatment with naloxone (0.2 mg/kg, IV) fully blocked brain and peripheral hypoxia induced by fentanyl (20 µg/kg, IV), but only partially decreased hypoxia induced by a fentanyl-xylazine mixture. Pretreatment with atipamezole (0.25 mg/kg, IV) fully blocked the hypoxic effects of xylazine (1.0 mg/kg, IV), but not fentanyl. Pretreatment with atipamezole + naloxone was more potent than naloxone alone in blocking the hypoxic effects of the fentanyl-xylazine mixture. Both naloxone and naloxone + atipamezole, delivered at the peak of brain hypoxia (3 min post fentanyl-xylazine exposure), reversed the rapid initial brain hypoxia, but only naloxone + atipamezole decreased the prolonged weaker hypoxia. There were no sex differences in the effects of the different drugs and their combinations on brain and peripheral oxygen responses. Results indicate that combined treatment with naloxone and atipamezole is more effective than naloxone alone in reversing the hypoxic effects of fentanyl-xylazine mixtures. Naloxone + atipamezole treatment should be considered in preventing overdoses induced by fentanyl-xylazine mixtures in humans.

Isoflurane lowers the cerebral metabolic rate of oxygen and prevents hypoxia during cortical spreading depolarization in vitro: An integrative experimental and modeling study.

Cortical spreading depolarization (SD) imposes a massive increase in energy demand and therefore evolves as a target for treatment following acute brain injuries. Anesthetics are empirically used to reduce energy metabolism in critical brain conditions, yet their effect on metabolism during SD remains largely unknown. We investigated oxidative metabolism during SD in brain slices from Wistar rats. Extracellular potassium ([K+]o), local field potential and partial tissue oxygen pressure (ptiO2) were measured simultaneously. The cerebral metabolic rate of oxygen (CMRO2) was calculated using a reaction-diffusion model. By that, we tested the effect of clinically relevant concentrations of isoflurane on CMRO2 during SD and modeled tissue oxygenation for different capillary pO2 values. During SD, CMRO2 increased 2.7-fold, resulting in transient hypoxia in the slice core. Isoflurane decreased CMRO2, reduced peak [K+]o, and prolonged [K+]o clearance, which indicates reduced synaptic transmission and sodium-potassium ATPase inhibition. Modeling tissue oxygenation during SD illustrates the need for increased capillary pO2 levels to prevent hypoxia. In the absence thereof, isoflurane could improve tissue oxygenation by lowering CMRO2. Therefore, isoflurane is a promising candidate for pre-clinical studies on neuronal survival in conditions involving SD.

Silibinin exerts neuroprotective effects against cerebral hypoxia/reoxygenation injury by activating the GAS6/Axl pathway.

Ischemic stroke is regarded one of the most common causes of brain vulnerability. Silibinin (SIL), extracted from the seeds of Silybinisus laborinum L., has been found to exhibit obvious therapeutic effects on neurodegenerative diseases. GAS6 has been proven to have significant neuroprotective effects; however, the role of SIL and GAS6 in ischemic stroke remains unclear. This study aimed to investigate the protective effects of SIL against cerebral ischemia-reperfusion injury in neuroblastoma N2a cells, as well as the mechanisms involved. Firstly, the toxicity of SIL was evaluated, and safe concentrations were chosen for subsequent experiments. Then, SIL exerts significant neuroprotection against hypoxia/reoxygenation (HR) injury in N2a cells, as manifested by increased cell viability, decreased apoptotic rate, LDH, and ROS generation. Additionally, SIL was found to inhibit HR-induced apoptosis, mitochondria dysfunction, and oxidative stress. However, silencing of GAS6 inhibited the neuroprotective effects of SIL. To sum up, these results suggest that SIL may be a promising therapeutic agent for the treatment of ischemic stroke.

Blockade of adenosine A2A receptor alleviates cognitive dysfunction after chronic exposure to intermittent hypoxia in mice.

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is widely known for its multiple systems damage, especially neurocognitive deficits in children. Since their discovery, adenosine A2A receptors (A2ARs) have been considered as key elements in signaling pathways mediating neurodegenerative diseases such as Huntington's and Alzheimer's, as well as cognitive function regulation. Herein, we investigated A2AR role in cognitive impairment induced by chronic intermittent hypoxia (CIH). Mice were exposed to CIH 7 h every day for 4 weeks, and intraperitoneally injected with A2AR agonist CGS21680 or A2AR antagonist SCH58261 half an hour before IH exposure daily. The 8-arm radial arm maze was utilized to assess spatial memory after CIH exposures.To validate findings using pharmacology, the impact of intermittent hypoxia was investigated in A2AR knockout mice. CIH-induced memory dysfunction was manifested by increased error rates in the radial arm maze test. The behavioral changes were associated with hippocampal pathology, neuronal apoptosis, and synaptic plasticity impairment. The stimulation of adenosine A2AR exacerbated memory impairment with more serious neuropathological damage, attenuated long-term potentiation (LTP), syntaxin down-regulation, and increased BDNF protein. Moreover, apoptosis-promoting protein cleaved caspase-3 was upregulated while anti-apoptotic protein Bcl-2 was downregulated. Consistent with these findings, A2AR inhibition with SCH58261 and A2AR deletion exhibited the opposite result. Overall, these findings suggest that A2AR plays a critical role in CIH-induced impairment of learning and memory by accelerating hippocampal neuronal apoptosis and reducing synaptic plasticity. Blockade of adenosine A2A receptor alleviates cognitive dysfunction after chronic exposure to intermittent hypoxia in mice.

Posttreatment Strategy Against Hypoxia and Ischemia Based on Selective Targeting of Nonnuclear Estrogen Receptors with PaPE-1.

Newly synthesized Pathway Preferential Estrogen-1 (PaPE-1) selectively activates membrane estrogen receptors (mERs), namely, mERα and mERß, and has been shown to evoke neuroprotection; however, its effectiveness in protecting brain tissue against hypoxia and ischemia has not been verified in a posttreatment paradigm. This is the first study showing that a 6-h delayed posttreatment with PaPE-1 inhibited hypoxia/ischemia-induced neuronal death, as indicated by neutral red uptake in mouse primary cell cultures in vitro. The effect was accompanied by substantial decreases in neurotoxicity and neurodegeneration in terms of LDH release and Fluoro-Jade C staining of damaged cells, respectively. The mechanisms of the neuroprotective action of PaPE-1 also involved apoptosis inhibition demonstrated by normalization of both mitochondrial membrane potential and expression levels of apoptosis-related genes and proteins such as Fas, Fasl, Bcl2, FAS, FASL, BCL2, BAX, and GSK3ß. Furthermore, PaPE-1-evoked neuroprotection was mediated through a reduction in ROS formation and restoration of cellular metabolic activity that had become dysregulated due to hypoxia and ischemia. These data provide evidence that targeting membrane non-GPER estrogen receptors with PaPE-1 is an effective therapy that protects brain neurons from hypoxic/ischemic damage, even when applied with a 6-h delay from injury onset.

Brain erythropoietin fine-tunes a counterbalance between neurodifferentiation and microglia in the adult hippocampus.

In adult cornu ammonis hippocampi, erythropoietin (EPO) expression drives the differentiation of new neurons, independent of DNA synthesis, and increases dendritic spine density. This substantial brain hardware upgrade is part of a regulatory circle: during motor-cognitive challenge, neurons experience "functional" hypoxia, triggering neuronal EPO production, which in turn promotes improved performance. Here, we show an unexpected involvement of resident microglia. During EPO upregulation and stimulated neurodifferentiation, either by functional or inspiratory hypoxia, microglia numbers decrease. Treating mice with recombinant human (rh)EPO or exposure to hypoxia recapitulates these changes and reveals the involvement of neuronally expressed IL-34 and microglial CSF1R. Surprisingly, EPO affects microglia in phases, initially by inducing apoptosis, later by reducing proliferation, and overall dampens microglia activity and metabolism, as verified by selective genetic targeting of either the microglial or pyramidal neuronal EPO receptor. We suggest that during accelerating neuronal differentiation, EPO acts as regulator of the CSF1R-dependent microglia.

Galectin-3 protects against ischemic stroke by promoting neuro-angiogenesis via apoptosis inhibition and Akt/Caspase regulation.

Post-stroke neurological deficits and mortality are often associated with vascular disruption and neuronal apoptosis. Galectin-3 (Gal3) is a potent pro-survival and angiogenic factor. However, little is known about its protective role in the cerebral ischemia/reperfusion (I/R) injury. We have previously shown significant up-regulation of Gal3 in the post-stroke rat brain, and that blocking of Gal3 with neutralizing antibody decreases the cerebral blood vessel density. Our current study demonstrates that intracerebral local delivery of the Gal3 into rat brain at the time of reperfusion exerts neuroprotection. Ischemic lesion volume and neuronal cell death were significantly reduced as compared with the vehicle-treated MCAO rat brains. Gal3 increased vessel density and neuronal survival after I/R in rat brains. Importantly, Gal3-treated groups showed significant improvement in motor and sensory functional recovery. Gal3 increased neuronal cell viability under in vitro oxygen-glucose deprivation conditions in association with increased phosphorylated-Akt, decreased phosphorylated-ERK1/2, and reduced caspase-3 activity. Gene expression analysis showed down regulation of pro-apoptotic and inflammatory genes including Fas-ligand, and upregulation of pro-survival and pro-angiogenic genes including Bcl-2, PECAM, and occludin. These results indicate a key role for Gal3 in neuro-vascular protection and functional recovery following ischemic stroke through modulation of angiogenic and apoptotic pathways.

MicroRNA-9 mediated the protective effect of ferulic acid on hypoxic-ischemic brain damage in neonatal rats.

Neonatal hypoxic-ischemic brain damage (HIBD) is prone to cognitive and memory impairments, and there is no effective clinical treatment until now. Ferulic acid (FA) is found within members of the genus Angelica, reportedly shows protective effects on neuronal damage. However, the protective effects of FA on HIBD remains unclear. In this study, using the Morris water maze task, we herein found that the impairment of spatial memory formation in adult rats exposed to HIBD was significantly reversed by FA treatment and the administration of LNA-miR-9. The expression of miRNA-9 was detected by RT-PCR analyses, and the results shown that miRNA-9 was significantly increased in the hippocampus of neonatal rats following HIBD and in the PC12 cells following hypoxic-ischemic injury, while FA and LNA-miR-9 both inhibited the expression of miRNA-9, suggesting that the therapeutic effect of FA was mainly attributed to the inhibition of miRNA-9 expression. Indeed, the silencing of miR-9 by LNA-miR-9 or FA similarly attenuated neuronal damage and cerebral atrophy in the rat hippocampus after HIBD, which was consistent with the restored expression levels of brain-derived neurotrophic factor (BDNF). Therefore, our findings indicate that FA treatment may protect against neuronal death through the inhibition of miRNA-9 induction in the rat hippocampus following hypoxic-ischemic damage.

Resveratrol attenuates hypoxia-induced neuronal cell death, inflammation and mitochondrial oxidative stress by modulation of TRPM2 channel.

Hypoxia (HYPX) induced-overload Ca2+ entry results in increase of mitochondrial oxidative stress, inflammation and apoptosis in several neurons. Ca2+ permeable TRPM2 channel was gated by ADP-ribose (ADPR) and reactive oxygen species (ROS), although its activity was modulated in HYPX-exposed neurons by resveratrol (RSV). The aim of this study was to evaluate if a therapy of RSV can modulate the effect of HYPX in the TRPM2 expressing SH-SY5Y neuronal and HEK293 (no expression of TRPM2) cell lines. The SH-SY5Y and HEK293 cells were divided into four groups as control, RSV (50 µM and 24 hours), and HYPX and RSV + HYPX. For induction of HYPX in the cells, CoCl2 (200 µM and 24 hours) incubation was used. HYPX-induced intracellular Ca2+ responses to TRPM2 activation were increased in the SH-SY5Y cells but not in the HEK293 cells from coming H2O2 and ADPR. RSV treatment improved intracellular Ca2+ responses, mitochondrial function, suppressed the generation of cytokine (IL-1ß and TNF-α), cytosolic and mitochondrial ROS in the SH-SY5Y cells. Intracellular free Zn2+, apoptosis, cell death, PARP-1, TRPM2 expression, caspase -3 and -9 levels are increased through activating TRPM2 in the SH-SY5Y cells exposed to the HYPX. However, the values were decreased in the cells by RSV and TRPM2 blockers (ACA and 2-APB). In SH-SY5Y neuronal cells exposed to HYPX conditions, the neuroprotective effects of RSV were shown to be exerted via modulation of oxidative stress, inflammation, apoptosis and death through modulation of TRPM2 channel. RSV could be used as an effective agent in the treatment of neurodegeneration exposure to HYPX.

Cerebral oxygen metabolism in adults with sickle cell disease.

In sickle cell disease (SCD), oxygen delivery is impaired due to anemia, especially during times of increased metabolic demand, and cerebral blood flow (CBF) must increase to meet changing physiologic needs. But hyperemia limits cerebrovascular reserve (CVR) and ischemic risk prevails despite elevated CBF. The cerebral metabolic rate of oxygen (CMRO2 ) directly reflects oxygen supply and consumption and may therefore be more insightful than flow-based CVR measures for ischemic risk in SCD. We hypothesized that adults with SCD have impaired CMRO2 at rest and that a vasodilatory challenge with acetazolamide would improve CMRO2 . CMRO2 was calculated from CBF and oxygen extraction fraction (OEF), measured with arterial spin labeling and T2 -prepared tissue relaxation with inversion recovery (T2 -TRIR) MRI. We studied 36 adults with SCD without a clinical history of overt stroke, and nine healthy controls. As expected, CBF was higher in patients with SCD versus controls (mean ± SD: 74 ± 16 versus 46 ± 5 mL/100 g/min, P < .001), resulting in similar oxygen delivery (SCD: 377 ± 67 versus controls: 368 ± 42 µmol O2 /100g/min, P = .69). OEF was lower in patients versus controls (27 ± 4 versus 35 ± 4%, P < .001), resulting in lower CMRO2 in patients versus controls (102 ± 24 versus 127 ± 20 µmol O2 /100g/min, P = .002). After acetazolamide, CMRO2 declined further in patients (P < .01) and did not decline significantly in controls (P = .78), indicating that forcing higher CBF worsened oxygen utilization in SCD patients. This lower CMRO2 could reflect variation between healthy and unhealthy vascular beds in terms of dilatory capacity and resistance whereby dysfunctional vessels become more oxygen-deprived, hence increasing the risk of localized ischemia.

The Role of Peripheral Opioid Receptors in Triggering Heroin-induced Brain Hypoxia.

While it is known that opioid receptors (ORs) are densely expressed in both the brain and periphery, it is widely accepted that hypoxic effects of opioids result solely from their direct action in the CNS. To examine the role of peripheral ORs in triggering brain hypoxia, we used oxygen sensors in freely moving rats to examine how naloxone-HCl and naloxone-methiodide, the latter which is commonly believed to be peripherally restricted, affect brain oxygen responses induced by intravenous heroin at low, human-relevant doses. Similar to naloxone-HCl, naloxone-methiodide at a relatively low dose (2 mg/kg) fully blocked heroin-induced decreases in brain oxygen levels. As measured by mass spectrometry, naloxone-methiodide was found to be ~40-fold less permeable than naloxone-HCl across the blood-brain barrier, thus acting as a selective blocker of peripheral ORs. Despite this selectivity, a low but detectable amount of naloxone was found in brain tissue after naloxone-methiodide administration, potentially influencing our results. Therefore, we examined the effects of naloxone-methiodide at a very low dose (0.2 mg/kg; at which naloxone was undetectable in brain tissue) and found that this drug still powerfully attenuates heroin-induced brain oxygen responses. These data demonstrate the role of peripheral ORs in triggering heroin-induced respiratory depression and subsequent brain hypoxia.

C1 Esterase Inhibitor Reduces BBB Leakage and Apoptosis in the Hypoxic Developing Mouse Brain.

Inflammatory pathways involved in blood-brain barrier (BBB) vulnerability and hypoxic brain oedema in models of perinatal brain injury seem to provide putative therapeutic targets. To investigate impacts of C1-esterase inhibitor (C1-INH; 7.5-30 IU/kg, i.p.) on functional BBB properties in the hypoxic developing mouse brain (P7; 8% O2 for 6 h), expression of pro-apoptotic genes (BNIP3, DUSP1), inflammatory markers (IL-1ß, TNF-alpha, IL-6, MMP), and tight junction proteins (ZO-1, occludin, claudin-1, -5), and S100b protein concentrations were analysed after a regeneration period of 24 h. Apoptotic cell death was quantified by CC3 immunohistochemistry and TUNEL staining. In addition to increased apoptosis in the parietal cortex, hippocampus, and subventricular zone, hypoxia significantly enhanced the brain-to-plasma albumin ratio, the cerebral S100b protein levels, BNIP3 and DUSP1 mRNA concentrations as well as mRNA expression of pro-inflammatory cytokines (IL-1ß, TNF-alpha). In response to C1-INH, albumin ratio and S100b concentrations were similar to those of controls. However, the mRNA expression of BNIP3 and DUSP1 and pro-inflammatory cytokines as well as the degree of apoptosis were significantly decreased compared to non-treated controls. In addition, occludin mRNA levels were elevated in response to C1-INH (p < 0.01). Here, we demonstrate for the first time that C1-INH significantly decreased hypoxia-induced BBB leakage and apoptosis in the developing mouse brain, indicating its significance as a promising target for neuroprotective therapy.

Total flavones of Rhododendron simsii Planch flower protect rat hippocampal neuron from hypoxia-reoxygenation injury via activation of BKCa channel.

OBJECTIVES: To study the effects of total flavones of Rhododendra simsii Planch flower (TFR) on hypoxia/reoxygenation (H/R) injury in rat hippocampal neurons and its underlying mechanism. METHOD: Model of H/R was established in newborn rat primary cultured hippocampal neuron. Lactate dehydrogenase (LDH) and neuron-specific enolase (NSE) activity as well as malondialdehyde (MDA) content in cultured supernatants of the neurons were examined. Methyl thiazolyl tetrazolium assay and Hoechst33258 staining were, respectively, used to detect cell viability and apoptosis of neurons. Protein expression and current of BKCa channel were assessed by using Western blotting and whole-cell patch-clamp methods, respectively. KEY FINDINGS: In the ranges of 3.7-300 mg/l, TFR significantly inhibited H/R-induced decrease of neuronal viability and increases of LDH, NSE and MDA in the supernatants as well as apoptosis; TFR 33.3, 100 and 300 mg/l markedly increased current of BKCa channel rather than the BKCa channel protein expression in the neurons. CONCLUSIONS: Total flavones of R. simsii Planch flower had a protective effect against H/R injury in rat hippocampal neuron, and activation of BKCa channel may contribute to the neuroprotection.

Neurotrophic Factors in the Treatment of Acute Brain Hypoxia Secondary to Cardiac Arrest: a Case Report.

Finding neuroprotective agents to counteract the deleterious effects of hypoxia on neuronal cells successfully is one of the most critical targets of clinical research since preclinical studies have identified potential neuroprotective strategies. In clinical practice, amantadine and piracetam are used with reasonable success. We present the cases of three patients with acute brain hypoxia secondary to cardiac arrest, to whom Cerebrolysin was added to the standard neuroprotective treatment regimen, leading to a notable improvement in functional outcome.

Trans-Himalayan Phytococktail Confers Protection Against Hypobaric Hypoxia-Induced Hippocampal Neurodegeneration and Memory Impairment in Male Sprague Dawley Rats.

Background: Exposure to hypobaric hypoxia (HH) has been reported to cause neurodegeneration and memory impairment. Hippophae rhamnoides, Prunus armeniaca, and Rhodiola imbricata, the indigenous plants of Indian Trans-Himalaya are widely used in traditional Tibetan and Amchi system of medicine. These are rich sources of diverse bioactive metabolites having prophylactic and therapeutic uses against a wide array of neurodegenerative diseases. The objective of this study was to elucidate the prophylactic and neuroprotective efficacy of formulated phytococktail (PC) against simulated HH-induced neurodegeneration in male Sprague Dawley (SD) rats. Materials and Methods: A PC containing H. rhamnoides fruit pulp, P. armeniaca fruit pulp, and R. imbricata dry root extract (100:50:1) was formulated. The neuroprotective efficacy of PC was evaluated in male SD rats following exposure to 7 day HH at simulated altitude (25,000 ft, 282 mm Hg). Rats were divided into four groups viz., normoxia group (NOR), normoxic group treated with PC (NORPC), 7 day hypoxic group treated with vehicle (7DH), and 7 day hypoxic group treated with PC (7DHPC). Memory impairment and neuromorphological alterations were measured. Targeted protein expression was analyzed by immunoblotting study. Results: PC supplementation significantly reduced the oxidative stress markers during exposure to HH. Spatial memory impairment by HH was significantly ameliorated by PC. HH-induced augmented pyknosis, decreased dendritic arborization, and increased Hoechst-positive neurons in hippocampal CA3 region were significantly ameliorated by PC. Immunoblotting study showed upregulation of BDNF and TrkB expression by PC. PC also prevented the hippocampal neurodegeneration by activating the PI3K/AKT signaling pathway, which leads to GSK-3ß inactivation by its phosphorylation and alleviation of hippocampal Caspase3 expression leading to inhibition of apoptotic neuronal cell death. Conclusion: The present study advocates the potential role of PC as an effective neuroprotective supplement in preventing HH-induced neurodegeneration. Activation of the PI3K/Akt pathway through BDNF/TrkB interaction following PC supplementation after exposure to HH inhibits hippocampal neuronal apoptosis and memory impairment.

Increasing Evidence for the Use of Sodium Oxybate in Multi-Drug-Resistant Lance-Adams Syndrome.

Background: Treatment of posthypoxic myoclonus (PHM) can be a challenge in patients not responsive to first-line medications. PMH is a rare condition that has a dramatic impact on patients' quality of life. Refractory cases are not uncommon. Case report: We report a patient with PHM non-responsive to conventional treatments who showed a dramatic improvement with sodium oxybate (SBX). Cases of PHM treated with SBX reported in the literature were reviewed. Discussion: Resting and stimulus-induced myoclonus respond robustly to SBX, with significant improvement in patients' quality of life. SBX may be considered in patients with PHM resistant to first-line medications.

Association of antiepileptic drugs with resolution of epileptiform activity after cardiac arrest.

INTRODUCTION: We tested the impact of antiepileptic drug (AED) administration on post-cardiac arrest epileptiform electroencephalographic (EEG) activity. METHODS: We studied an observational cohort of comatose subjects treated at a single academic medical center after cardiac arrest from September 2010 to January 2018. We aggregated the observed EEG patterns into 5 categories: suppressed; discontinuous background with superimposed epileptiform activity; discontinuous background without epileptiform features; continuous background with epileptiform activity; and continuous background without epileptiform activity. We calculated overall probabilities of transitions between EEG states in a multistate model, then used Aalen's additive regression to test if AEDs or hypothermia are associated with a change in these probabilities. RESULTS: Overall, 828 subjects had EEG-monitoring for 42,840 h with a median of 40 [IQR 23-64] h per subject. Among patients with epileptiform findings on initial monitoring, 50% transitioned at least once to a non-epileptiform, non-suppressed state. By contrast, 19% with non-epileptiform initial activity transitioned to an epileptiform state at least once. Overall, 568 (78%) patients received at least one AED. Among patients with continuous EEG background activity, valproate, levetiracetam and lower body temperature were each associated with an increased probability of transition from epileptiform states to non-epileptiform states, where patients with discontinuous EEG background activity no agent linked to an increased probability of transitioning from epileptiform states. CONCLUSION: After cardiac arrest, the impact of AEDs may depend on the presence of continuous cortical background activity. These data serve to inform experimental work to better define the opportunities to improve neurologic care post-cardiac arrest.

Minocycline mitigates the effect of neonatal hypoxic insult on human brain organoids.

Neonatal hypoxic injury (NHI) is a devastating cause of disease that affects >60% of babies born with a very low birth weight, resulting in significant morbidity and mortality, including life-long neurological consequences such as seizures, cerebral palsy, and intellectual disability. Hypoxic injury results in increased neuronal death, which disrupts normal brain development. Although animal model systems have been useful to study the effects of NHI, they do not fully represent the uniqueness and complexities of the human brain. To better understand the effects of hypoxia on human brain development, we have generated a brain organoid protocol and evaluated these cells over the course of 6 months. As anticipated, the expression of a forebrain marker, FOXG1, increased and then remained expressed over time, while there was a transition in the expression of the deep-layer (TBR1) and upper-layer (SATB2) cortical markers. In addition, ventral genes (Eng1 and Nkx2.1) as well as markers of specialized nonneuronal cells (Olig2 and GFAP) also increased at later time points. We next tested the development of our in vitro cerebral organoid model at different oxygen concentrations and found that hypoxia repressed gene markers for forebrain, oligodendrocytes, glial cells, and cortical layers, as well as genes important for the migration of cortical neurons. In contrast, ventral markers were either unaffected or even increased in expression with hypoxic insult. Interestingly, the negative effect of hypoxia on the dorsal brain genes as well as oligodendrocytes, and neuronal progenitors could be mitigated by the use of minocycline, an FDA-approved small molecule. Taken together, we have generated a unique and relevant in vitro human brain model system to study diseases such as NHI as well as their potential treatments. Using this system, we have shown the efficacy of minocycline for human NHI.

OCT4B-190 protects against ischemic stroke by modulating GSK-3ß/HDAC6.

OCT4 is a key regulator in maintaining the pluripotency and self-renewal of embryonic stem cells (ESCs). Human OCT4 gene has three mRNA isoforms, termed OCT4A, OCT4B and OCT4B1. The 190-amino-acid protein isoform (OCT4B-190) is one of the major products of OCT4B mRNA, the biological function of which is still not well defined. Recent evidence suggests that OCT4B-190 may function in the cellular stress response. The glycogen synthase kinase-3ß (GSK-3ß) and histone deacetylase 6 (HDAC6) are also key stress modulators that play critical roles in the ischemic cascades of stroke. Hence, we here further investigated the effects of OCT4B-190 in the experimental stroke, and explored the underlying roles of GSK-3ß and HDAC6. We found that OCT4B-190 overexpression enhanced neuronal viability at 24 h after oxygen-glucose deprivation (OGD) treatment. Moreover, in male C57BL/6 mice subjected to transit middle cerebral artery occlusion (MCAO), OCT4B-190 overexpression reduced infarct volume and improved neurological function after stroke. Notably, we found spatio-temporal alterations of GSK-3ß and HDAC6 in the ischemic cortex and striatum, which were affected by adenovirus-mediated OCT4B-190 overexpression. OCT4B-190 demonstrated similar impacts on neuronal cultures in vitro, downregulating OGD-induced GSK-3ß activity and HDAC6 expression. In addition, we found that GSK-3ß and HDAC6 were co-expressed in the cytoplasm of neurons, and OCT4B-190 had an effect on interactions between GSK-3ß and HDAC6 in neuronal cultures subjected to OGD treatment. These findings suggest that OCT4B-190 exerts neuroprotection in the experimental stroke potentially by regulating actions of GSK-3ß and HDAC6 simultaneously, which may be an attractive therapeutic strategy for ischemic stroke.

Calcium channel blockade with nimodipine reverses MRI evidence of cerebral oedema following acute hypoxia.

Acute cerebral hypoxia causes rapid calcium shifts leading to neuronal damage and death. Calcium channel antagonists improve outcomes in some clinical conditions, but mechanisms remain unclear. In 18 healthy participants we: (i) quantified with multiparametric MRI the effect of hypoxia on the thalamus, a region particularly sensitive to hypoxia, and on the whole brain in general; (ii) investigated how calcium channel antagonism with the drug nimodipine affects the brain response to hypoxia. Hypoxia resulted in a significant decrease in apparent diffusion coefficient (ADC), a measure particularly sensitive to cell swelling, in a widespread network of regions across the brain, and the thalamus in particular. In hypoxia, nimodipine significantly increased ADC in the same brain regions, normalizing ADC towards normoxia baseline. There was positive correlation between blood nimodipine levels and ADC change. In the thalamus, there was a significant decrease in the amplitude of low frequency fluctuations (ALFF) in resting state functional MRI and an apparent increase of grey matter volume in hypoxia, with the ALFF partially normalized towards normoxia baseline with nimodipine. This study provides further evidence that the brain response to acute hypoxia is mediated by calcium, and importantly that manipulation of intracellular calcium flux following hypoxia may reduce cerebral cytotoxic oedema.