Neuroprotective Effect of Clemastine Improved Oligodendrocyte Proliferation through the MAPK/ERK Pathway in a Neonatal Hypoxia Ischemia Rat Model.

Neonatal hypoxic-ischemic encephalopathy is the most common cause of long-term disability in term neonates, and white matter injury is the primary cause of cerebral palsy. Therapies that focus on the neuroprotection of myelination and oligodendrocyte proliferation could potentially ameliorate long-lasting neurological impairments after hypoxic-ischemic encephalopathy. Clemastine, a histamine H1 antagonist, has been shown to exert neuroprotective effects in multiple sclerosis and spinal cord injury by promoting oligodendrogenesis and re-myelination. In this study, we demonstrated the neuroprotective effects of clemastine in our rat model of neonatal hypoxic-ischemic brain injury. Animals received a single intraperitoneal injection of either vehicle or clemastine (10 mg/kg) for 6 consecutive days. Our results showed a significant reduction in white matter loss after treatment, with a clear effect of clemastine on oligodendrocytes, showing a significant increase in the number of Olig2+ cells. We characterized the MAPK/ERK pathway as a potential mechanistic pathway underlying the neuroprotective effects of clemastine. Altogether, our results demonstrate that clemastine is a potential compound for the treatment of hypoxic-ischemic encephalopathy, with a clear neuroprotective effect on white matter injury by promoting oligodendrogenesis.

Amorfrutin B Compromises Hypoxia/Ischemia-induced Activation of Human Microglia in a PPARγ-dependent Manner: Effects on Inflammation, Proliferation Potential, and Mitochondrial Status.

Amorfrutin B is a selective PPARγ modulator that we demonstrated to be a promising neuroprotective compound in cellular models of stroke and perinatal asphyxia. Although neuronal mechanisms of amorfrutin B-evoked neuroprotection have been identified, none of them reflects the actions of the compound on microglia, which play a pivotal role in brain response to hypoxia/ischemia. Here, we provide evidence for amorfrutin B-induced effects on human microglia subjected to hypoxia/ischemia; the compound counteracts inflammation, and influences mitochondrial status and proliferation potential in a PPARγ-dependent manner. Post-treatment with amorfrutin B decreased the IBA1 fluorescence intensity, reduced caspase-1 activity, and downregulated IL1B/IL-1ß and TNFA but not IL10/IL-10 expression, which was upregulated. Amorfrutin B also stimulated PPARγ signaling, as evidenced by increased mRNA and/or protein levels of PPARγ and PGC1α. In addition, amorfrutin B reversed the hypoxia/ischemia-evoked effects on mitochondria-related parameters, such as mitochondrial membrane potential, BCL2/BCL2 expression and metabolic activity, which were correlated with diminished proliferation potential of microglia. Interestingly, the inhibitory effect of amorfrutin B on the proliferation potential and mitochondrial function of microglia is opposite to the stimulatory effect of amorfrutin B on mouse neuronal survival, as evidenced by increased neuronal viability and reduced neurodegeneration. In summary, this study showed for the first time that amorfrutin B compromises hypoxia/ischemia-induced activation of human microglia in a PPARγ-dependent manner, which involves inhibiting inflammation, normalizing mitochondrial status, and controlling proliferation potential. These data extend the protective potential of amorfrutin B in the pharmacotherapy of hypoxic/ischemic brain injury, targeting not only neurons but also activated microglia.

Hydrogen alleviates hypoxic-ischaemic brain damage in neonatal rats by inhibiting injury of brain pericytes.

Hypoxia-ischaemia (HI) can induce the death of cerebrovascular constituent cells through oxidative stress. Hydrogen is a powerful antioxidant which can activate the antioxidant system. A hypoxia-ischaemia brain damage (HIBD) model was established in 7-day-old SD rats. Rats were treated with different doses of hydrogen-rich water (HRW), and brain pericyte oxidative stress damage, cerebrovascular function and brain tissue damage were assessed. Meanwhile, in vitro-cultured pericytes were subjected to oxygen-glucose deprivation and treated with different concentrations of HRW. Oxidative injury was measured and the molecular mechanism of how HRW alleviated oxidative injury of pericytes was also examined. The results showed that HRW significantly attenuated HI-induced oxidative stress in the brain pericytes of neonatal rats, partly through the Nrf2-HO-1 pathway, further improving cerebrovascular function and reducing brain injury and dysfunction. Furthermore, HRW is superior to a single-cell death inhibitor for apoptosis, ferroptosis, parthanatos, necroptosis and autophagy and can better inhibit HI-induced pericyte death. The liver and kidney functions of rats were not affected by present used HRW dose. This study elucidates the role and mechanism of hydrogen in treating HIBD from the perspective of pericytes, providing new theoretical evidence and mechanistic references for the clinical application of hydrogen in neonatal HIE.

Hypoxic-Ischemic Insult Alters Polyamine and Neurotransmitter Abundance in the Specific Neonatal Rat Brain Subregions.

Neonatal hypoxic-ischemic (HI) brain insult is a major cause of neonatal mortality and morbidity. To assess the underlying pathological mechanisms, we mapped the spatiotemporal changes in polyamine, amino acid, and neurotransmitter levels, following HI insult (by the Rice-Vannucci method) in the brains of seven-day-old rat pups. Matrix-assisted laser desorption/ionization mass spectrometry imaging of chemically modified small-molecule metabolites by 4-(anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide revealed critical HI-related metabolomic changes of 22 metabolites in 14 rat brain subregions, much earlier than light microscopy detected signs of neuronal damage. For the first time, we demonstrated excessive polyamine oxidation and accumulation of 3-aminopropanal in HI neonatal brains, which was later accompanied by neuronal apoptosis enhanced by increases in glycine and norepinephrine in critically affected brain regions. Specifically, putrescine, cadaverine, and 3-aminopropanal increased significantly as early as 12 h postinsult, mainly in motor and somatosensory cortex, hippocampus, and midbrain, followed by an increase in norepinephrine 24 h postinsult, which was predominant in the caudate putamen, the region most vulnerable to HI. The decrease of γ-aminobutyric acid (GABA) and the continuous dysregulation of the GABAergic system together with low taurine levels up to 36 h sustained progressive neurodegenerative cellular processes. The molecular alterations presented here at the subregional rat brain level provided unprecedented insight into early metabolomic changes in HI-insulted neonatal brains, which may further aid in the identification of novel therapeutic targets for the treatment of neonatal HI encephalopathy.

Criteria for brain death in Denmark.

In 1990, the Danish brain death legislation was adopted by the Danish Parliament. Each year, around 100 patients in Denmark fulfil criteria for brain death/death by neurological criteria (BD/DNC). In this review of current Danish criteria including the indication for ancillary investigation, which in Denmark is digital subtraction angiography (DSA), we conclude that the time has come to revise the national BD/DNC criteria. We propose that visible anoxic-ischaemic encephalopathy on brain CT after cardiac arrest does not require evaluation by ancillary testing, and that CT-angiography can be used instead of DSA.

Aloesin ameliorates hypoxic-ischemic brain damage in neonatal mice by suppressing TLR4-mediated neuroinflammation.

BACKGROUND: At present, neonatal hypoxic-ischemic encephalopathy (HIE), especially moderate to severe HIE, is a challenging disease for neonatologists to treat, and new alternative/complementary treatments are urgently needed. The neuroinflammatory cascade triggered by hypoxia-ischemia (HI) insult is one of the core pathological mechanisms of HIE. Early inhibition of neuroinflammation provides long-term neuroprotection. Plant-derived monomers have impressive anti-inflammatory effects. Aloesin (ALO) has been shown to have significant anti-inflammatory and antioxidant effects in diseases such as ulcerative colitis, but its role in HIE is unclear. To this end, we conducted a series of experiments to explore the potential mechanism of ALO in preventing and treating brain damage caused by HI insult. MATERIALS AND METHODS: Hypoxic-ischemic brain damage (HIBD) was induced in 7-day-old Institute of Cancer Research (ICR) mice, which were then treated with 20 mg/kg ALO. The neuroprotective effects of ALO on HIBD and the underlying mechanism were evaluated through neurobehavioral testing, infarct size measurement, apoptosis detection, protein and messenger RNA level determination, immunofluorescence, and molecular docking. RESULTS: ALO alleviated the long-term neurobehavioral deficits caused by HI insult; reduced the extent of cerebral infarction; inhibited cell apoptosis; decreased the levels of the inflammatory factors interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α; activated microglia and astrocytes; and downregulated the protein expression of members in the TLR4 signaling pathway. In addition, molecular docking showed that ALO can bind stably to TLR4. CONCLUSION: ALO ameliorated HIBD in neonatal mice by inhibiting the neuroinflammatory response mediated by TLR4 signaling.

CYR61 is Involved in Neonatal Hypoxic-ischemic Brain Damage Via Modulating Astrocyte-mediated Neuroinflammation.

The activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in astrocytes has been found in the hypoxic-ischemic brain damage (HIBD) model. Cysteine rich angiogenic inducer 61 (CYR61) is secreted by reactive astrocytes. However, the effects of CYR61 on HIBD and its related mechanisms remain unclear. This study sought to explore the role of CYR61 in the activation of astrocytes and the NLRP3 inflammasome in neonatal HIBD. HIBD models were established in 7-day Sprague-Dawley rat pups. Neurobehavioral evaluation and 2,3,5-triphenyl-tetrazolium chloride staining were performed. In addition, rat primary astrocytes were used to establish the cell model of HIBD in vitro by oxygen-glucose deprivation/reperfusion (OGD/R). Then, CYR61-overexpression and sh-CYR61 viruses mediated by lentivirus were transduced into ODG/R-treated primary astrocytes. The expressions of related genes were evaluated using real-time quantitative PCR, western blot, immunofluorescence staining, and Enzyme-linked immunosorbent assay. The results showed that hypoxia-ischemia induced short-term neurological deficits, neuronal damage, and cerebral infarction in neonatal rats. In vivo, the expressions of CYR61, NLRP3, and glial fibrillary acidic protein (GFAP) were up-regulated in the HIBD model. In vitro, CYR61 exhibited high expression. CYR61 overexpression increased the expressions of GFAP and C3, whereas decreased S100A10 expression. CYR61 overexpression increased the expression of NLRP3, ASC, caspase-1 p20 and IL-1ß. CYR61 overexpression activated NF-κB by promoting the phosphorylation of IκBα and p65. Thus, CYR61 is involved in neonatal HIBD progress, which may be related to the activation of astrocytes, the NLRP3 inflammasome, and the NF-κB signaling pathway.

Protocol to optimize the Rice-Vannucci rat pup model of perinatal asphyxia to ensure predictable hypoxic-ischemic cerebral lesions.

The Rice-Vannucci model in rodent pups is subject to substantial loss of animals, result inconsistency, and high lab-to-lab variability in extent and composition of induced injury. This protocol allows for highly predictable and reproducible hypoxic-ischemic cerebral injury lesions in post-natal day 10 Wistar rat pups with no mortality. We describe steps for common carotid artery ligation, brief post-operative normothermia, exposure to hypoxia, and post-hypoxic normothermia. Precise timing and temperature control in each step are crucial for a successful procedure. For complete details on the use and execution of this protocol, please refer to Hartman et al.1.

Neuroprotective effects of miRNA-326 knockout in neonatal hypoxic-ischemic brain damage mice via the δ-opioid receptor.

Hypoxic-ischemic brain damage (HIBD) in the perinatal period is an important cause of cerebral damage and long-term neurological sequelae, and can place much pressure on families and society. Our previous study demonstrated that miRNA-326 reduces neuronal apoptosis by up-regulating the δ-opioid receptor (DOR) under oxygen-glucose deprivation in vitro. In the present study, we aimed to explore the neuroprotective effects of the miRNA-326/DOR axis by inhibiting apoptosis in HIBD using neonatal miRNA-326 knockout mice. Neonatal C57BL/6 mice, neonatal miRNA-326 knockout mice, and neonatal miRNA-326 knockout mice intraperitoneally injected with the DOR inhibitor naltrindole were treated with hypoxic-ischemia (HI). Neurological deficit scores, magnetic resonance imaging, terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling, and Caspase-3, Bax, and B-cell lymphoma 2 (Bcl-2) expression were evaluated on day 2 after HI. Neurobehavioral analyses were performed on days 2 and 28 after HI. Additionally, the Morris water maze test was conducted on days 28. Compared with HI-treated neonatal C57BL/6 mice, HI-treated neonatal miRNA-326 knockout mice had higher neurological deficit scores, smaller cerebral infarction areas, and improved motor function, reaction ability, and long-term spatial learning and memory. These effects were likely the result of inhibiting apoptosis; the DOR inhibitor reversed these neuroprotective effects. Our findings indicate that miRNA-326 knockout plays a neuroprotective effect in neonatal HIBD by inhibiting apoptosis via the target gene DOR.

Emodin attenuates hypoxic-ischemic brain damage by inhibiting neuronal apoptosis in neonatal mice.

Neonatal hypoxic-ischemic brain damage (HIBD) can lead to mortality and severe neurological dysfunction. Emodin is a natural anthraquinone derivative that is easy to obtain and has good neuroprotective effects. This study aimed to investigate the neuroprotective effect of emodin on neonatal mouse HIBD. The modified Rice-Vannucci method was used to induce HIBD in mouse pups. Eighty postnatal 7-day (P7) C57BL/6 neonatal mice were randomly divided into the sham group (sham), vehicle group (vehicle), and emodin group (emodin). TTC staining and whole-brain morphology were used to evaluate the infarct volume and morphology of the brain tissue. The condition of the neurons was observed through Nissl staining, HE staining, FJC staining, immunofluorescence and Western blot for NeuN, IBA-1, and GFAP. The physiological status of the mice was evaluated using weight measurements. The neural function of the mice was assessed using the negative geotaxis test, righting reflex test, and grip test. TUNEL staining was used to detect apoptosis in brain cells. Finally, Western blot and immunofluorescence were used to detect the expression levels of apoptosis-related proteins, such as P53, cleaved caspase-3, Bax and Bcl-2, in the brain. Experiments have shown that emodin can reduce the cerebral infarct volume, brain oedema, neuronal apoptosis, and degeneration and improve the reconstruction of brain tissue morphology, neuronal morphology, physiological conditions, and neural function. Additionally, emodin inhibited the expression of proapoptotic proteins such as P53, Bax and cleaved caspase-3 and promoted the expression of the antiapoptotic protein Bcl-2. Emodin attenuates HIBD by inhibiting neuronal apoptosis in neonatal mice.

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OBJECTIVES: To observe the effects of melatonin on autophagy in cortical neurons of neonatal rats with hypoxic-ischemic brain damage (HIBD) and to explore its mechanisms via the PI3K/AKT signaling pathway, aiming to provide a basis for the clinical application of melatonin. METHODS: Seven-day-old Sprague-Dawley neonatal rats were randomly divided into a sham operation group, an HIBD group, and a melatonin group (n=9 each). The neonatal rat HIBD model was established using the classic Rice-Vannucci method. Neuronal morphology in the neonatal rat cerebral cortex was observed with hematoxylin-eosin staining and Nissl staining. Autophagy-related protein levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 were detected by immunofluorescence staining and Western blot analysis. Phosphorylated phosphoinositide 3-kinase (p-PI3K) and phosphorylated protein kinase B (p-AKT) protein expression levels were measured by immunohistochemistry and Western blot. The correlation between autophagy and the PI3K pathway in the melatonin group and the HIBD group was analyzed using Pearson correlation analysis. RESULTS: Twenty-four hours post-modeling, neurons in the sham operation group displayed normal size and orderly arrangement. In contrast, neurons in the HIBD group showed swelling and disorderly arrangement, while those in the melatonin group had relatively normal morphology and more orderly arrangement. Nissl bodies were normal in the sham operation group but distorted in the HIBD group; however, they remained relatively intact in the melatonin group. The average fluorescence intensity of LC3 and Beclin-1 was higher in the HIBD group compared to the sham operation group, but was reduced in the melatonin group compared to the HIBD group (P<0.05). The number of p-PI3K+ and p-AKT+ cells decreased in the HIBD group compared to the sham operation group but increased in the melatonin group compared to the HIBD group (P<0.05). LC3 and Beclin-1 protein expression levels were higher, and p-PI3K and p-AKT levels were lower in the HIBD group compared to the sham operation group (P<0.05); however, in the melatonin group, LC3 and Beclin-1 levels decreased, and p-PI3K and p-AKT increased compared to the HIBD group (P<0.05). The correlation analysis results showed that the difference of the mean fluorescence intensity of LC3 and Beclin-1 protein in the injured cerebral cortex between the melatonin and HIBD groups was negatively correlated with the difference of the number of p-PI3K+ and p-AKT+ cells between the two groups (P<0.05). CONCLUSIONS: Melatonin can inhibit excessive autophagy in cortical neurons of neonatal rats with HIBD, thereby alleviating HIBD. This mechanism is associated with the PI3K/AKT pathway.

Echinatin alleviates inflammation and pyroptosis in hypoxic-ischemic brain damage by inhibiting TLR4/ NF-κB pathway.

Hypoxic ischemic encephalopathy (HIE) is a primary cause of neonatal death and disabilities. The pathogenetic process of HIE is closely associated with neuroinflammation. Therefore, targeting and suppressing inflammatory pathways presents a promising therapeutic strategy for the treatment of HIE. Echinatin is an active component of glycyrrhiza, with anti-inflammatory and anti-oxidative properties. It is commonly combined with other traditional Chinese herbs to exert heat-clearing and detoxifying effects. This study aimed to investigate the anti-inflammatory and neuroprotective effects of Echinatin in neonatal rats with hypoxic-ischemic brain damage, as well as in PC12 cells exposed to oxygen-glucose deprivation (OGD). In vivo, Echinatin effectively reduced cerebral edema and infarct volume, protected brain tissue morphology, improved long-term behavioral functions, and inhibited microglia activation. These effects were accompanied by the downregulation of inflammatory factors and pyroptosis markers. The RNA sequencing analysis revealed an enrichment of inflammatory genes in rats with hypoxic-ischemic brain damage, and Protein-protein interaction (PPI) network analysis identified TLR4, MyD88, and NF-κB as the key regulators. In vitro, Echinatin reduced the levels of TLR4 relevant proteins, inhibited nuclear translocation of NF-κB, reduced the expression of downstreams inflammatory cytokines and pyroptosis proteins, and prevented cell membrane destructions. These findings demonstrated that Echinatin could inhibit the TLR4/NF-κB pathway, thereby alleviating neuroinflammation and pyroptosis. This suggests that Echinatin could be a potential candidate for the treatment of HIE.

CXCL10 is a crucial chemoattractant for efficient intranasal delivery of mesenchymal stem cells to the neonatal hypoxic-ischemic brain.

BACKGROUND: Hypoxic-Ischemic Encephalopathy (HIE) is a leading cause of mortality and morbidity in newborns. Recent research has shown promise in using intranasal mesenchymal stem cell (MSC) therapy if administered within 10 days after Hypoxia-Ischemia (HI) in neonatal mice. MSCs migrate from the nasal cavity to the cerebral lesion in response to chemotactic cues. Which exact chemokines are crucial for MSC guidance to the HI lesion is currently not fully understood. This study investigates the role of CXCL10 in MSC migration towards the HI-injured brain. METHODS: HI was induced in male and female 9-day-old C57BL/6 mice followed by intranasal MSC treatment at day 10 or 17 post-HI. CXCL10 protein levels, PKH26-labeled MSCs and lesion size were assessed by ELISA, immunofluorescent imaging and MAP2 staining respectively. At day 17 post-HI, when CXCL10 levels were reduced, intracranial CXCL10 injection and intranasal PKH26-labeled MSC administration were combined to assess CXCL10-guided MSC migration. MSC treatment efficacy was evaluated after 18 days, measuring lesion size, motor outcome (cylinder rearing task), glial scarring (GFAP staining) and neuronal density (NeuN staining) around the lesion. Expression of the receptor for CXCL10, i.e. CXCR3, on MSCs was confirmed by qPCR and Western Blot. Moreover, CXCL10-guided MSC migration was assessed through an in vitro transwell migration assay. RESULTS: Intranasal MSC treatment at day 17 post-HI did not reduce lesion size in contrast to earlier treatment timepoints. Cerebral CXCL10 levels were significantly decreased at 17 days versus 10 days post-HI and correlated with reduced MSC migration towards the brain. In vitro experiments demonstrated that CXCR3 receptor inhibition prevented CXCL10-guided migration of MSCs. Intracranial CXCL10 injection at day 17 post-HI significantly increased the number of MSCs reaching the lesion which was accompanied by repair of the HI lesion as measured by reduced lesion size and glial scarring, and an increased number of neurons around the lesion. CONCLUSIONS: This study underscores the crucial role of the chemoattractant CXCL10 in guiding MSCs to the HI lesion after intranasal administration. Strategies to enhance CXCR3-mediated migration of MSCs may improve the efficacy of MSC therapy or extend its regenerative therapeutic window.

Protecting effects of 4-octyl itaconate on neonatal hypoxic-ischemic encephalopathy via Nrf2 pathway in astrocytes.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the most common neurological problems occurring in the perinatal period. However, there still is not a promising approach to reduce long-term neurodevelopmental outcomes of HIE. Recently, itaconate has been found to exhibit anti-oxidative and anti-inflammatory effects. However, the therapeutic efficacy of itaconate in HIE remains inconclusive. Therefore, this study attempts to explore the pathophysiological mechanisms of oxidative stress and inflammatory responses in HIE as well as the potential therapeutic role of a derivative of itaconate, 4-octyl itaconate (4OI). METHODS: We used 7-day-old mice to induce hypoxic-ischemic (HI) model by right common carotid artery ligation followed by 1 h of hypoxia. Behavioral experiments including the Y-maze and novel object recognition test were performed on HI mice at P60 to evaluate long-term neurodevelopmental outcomes. We employed an approach combining non-targeted metabolomics with transcriptomics to screen alterations in metabolic profiles and gene expression in the hippocampal tissue of the mice at 8 h after hypoxia. Immunofluorescence staining and RT-PCR were used to evaluate the pathological changes in brain tissue cells and the expression of mRNA and proteins. 4OI was intraperitoneally injected into HI model mice to assess its anti-inflammatory and antioxidant effects. BV2 and C8D1A cells were cultured in vitro to study the effect of 4OI on the expression and nuclear translocation of Nrf2. We also used Nrf2-siRNA to further validate 4OI-induced Nrf2 pathway in astrocytes. RESULTS: We found that in the acute phase of HI, there was an accumulation of pyruvate and lactate in the hippocampal tissue, accompanied by oxidative stress and pro-inflammatory, as well as increased expression of antioxidative stress and anti-inflammatory genes. Treatment of 4OI could inhibit activation and proliferation of microglial cells and astrocytes, reduce neuronal death and relieve cognitive dysfunction in HI mice. Furthermore, 4OI enhanced nuclear factor erythroid-2-related factor (Nfe2l2; Nrf2) expression and nuclear translocation in astrocytes, reduced pro-inflammatory cytokine production, and increased antioxidant enzyme expression. CONCLUSION: Our study demonstrates that 4OI has a potential therapeutic effect on neuronal damage and cognitive deficits in HIE, potentially through the modulation of inflammation and oxidative stress pathways by Nrf2 in astrocytes.

Neuronal autosis is Na+/K+-ATPase alpha 3-dependent and involved in hypoxic-ischemic neuronal death.

Macroautophagy (hereafter called autophagy) is an essential physiological process of degradation of organelles and long-lived proteins. The discovery of autosis, a Na+/K+-ATPase (ATP1)-dependent type of autophagic cell death with specific morphological and biochemical features, has strongly contributed to the acceptance of a pro-death role of autophagy. However, the occurrence and relevance of autosis in neurons has never been clearly investigated, whereas we previously provided evidence that autophagy mechanisms could be involved in neuronal death in different in vitro and in vivo rodent models of hypoxia-ischemia (HI) and that morphological features of autosis were observed in dying neurons following rat perinatal cerebral HI. In the present study, we demonstrated that neuronal autosis could occur in primary cortical neurons using two different stimulations enhancing autophagy flux and neuronal death: a neurotoxic concentration of Tat-BECN1 (an autophagy-inducing peptide) and a hypoxic/excitotoxic stimulus (mimicking neuronal death induced by cerebral HI). Both stimulations induce autophagic neuronal death (dependent on canonical autophagic genes and independent on apoptotic, necroptotic or ferroptotic pathways) with all morphological and biochemical (ATP1a-dependent) features of autosis. However, we demonstrated that autosis is not dependent on the ubiquitous subunit ATP1a1 in neurons, as in dividing cell types, but on the neuronal specific ATP1a3 subunit. We also provided evidence that, in different in vitro and in vivo models where autosis is induced, ATP1a3-BECN1 interaction is increased and prevented by cardiac glycosides treatment. Interestingly, an increase in ATP1a3-BECN1 interaction is also detected in dying neurons in the autoptic brains of human newborns with severe hypoxic-ischemic encephalopathy (HIE). Altogether, these results suggest that ATP1a3-BECN1-dependent autosis could play an important role in neuronal death in HI conditions, paving the way for the development of new neuroprotective strategies in hypoxic-ischemic conditions including in severe case of human HIE.

Sevoflurane postconditioning ameliorates cerebral hypoxia/reoxygenation injury in zebrafish involving the Akt/GSK-3ß pathway activation and the microtubule-associated protein 2 promotion.

Sevoflurane postconditioning has been shown to provide neuroprotection against cerebral hypoxia-ischemia injury, but the mechanisms remain elusive. Microtubule-associated protein 2 (MAP2) is implicated in early neuronal hypoxia-ischemia injury. This study aimed to investigate whether the neuroprotective effects of sevoflurane postconditioning are related to the Akt/GSK-3ß pathway and its downstream target MAP2 in zebrafish hypoxia/reoxygenation (H/R) model. Sevoflurane postconditioning or GSK-3ß inhibitor TDZD-8 were used to treat H/R zebrafish. The cerebral infarction, neuronal apoptosis, and mitochondrial changes were evaluated using TTC staining, TUNEL staining, and transmission electron microscopy, respectively. The distribution of MAP2 in the brain was determined by immunofluorescence imaging. The levels of Akt, p-Akt, GSK-3ß, p-GSK-3ß, and MAP2 proteins were evaluated by Western blotting. The neurobehavioral recovery of zebrafish was assessed based on optokinetic response behavior. Our results indicated that sevoflurane postconditioning and TDZD-8 significantly reduced the cerebral infarction area, suppressed cell apoptosis, and improved mitochondrial integrity in zebrafish subjected to H/R. Furthermore, sevoflurane postconditioning and TDZD-8 elevated the ratios of p-Akt/Akt and p-GSK-3ß/GSK-3ß. However, the neuroprotective effect of sevoflurane postconditioning was effectively abolished upon suppression of MAP2 expression. In conclusion, sevoflurane postconditioning ameliorated cerebral H/R injury and facilitated the restoration of neurobehavioral function through the activation of Akt/GSK-3ß pathway and promotion of MAP2 expression.

Menaquinone-4 alleviates hypoxic-ischemic brain damage in neonatal rats by reducing mitochondrial dysfunction via Sirt1-PGC-1α-TFAM signaling pathway.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a major contributor to neonatal mortality and neurodevelopmental disorders, but currently there is no effective therapy drug for HIE. Mitochondrial dysfunction plays a pivotal role in hypoxic-ischemic brain damage(HIBD). Menaquinone-4 (MK-4), a subtype of vitamin K2 prevalent in the brain, has been shown to enhance mitochondrial function and exhibit protective effects against ischemia-reperfusion injury. However, the impact and underlying molecular mechanism of MK-4 in HIE have not been fully elucidated. METHODS: In this study, we established the neonatal rats HIBD model in vivo and oxygen-glucose deprivation and reperfusion (OGD/R) of primary neurons in vitro to explore the neuroprotective effects of MK-4 on HI damage, and illuminate the potential mechanism. RESULTS: Our findings revealed that MK-4 ameliorated mitochondrial dysfunction, reduced oxidative stress, and prevented HI-induced neuronal apoptosis by activating the Sirt1-PGC-1α-TFAM signaling pathway through Sirt1 mediation. Importantly, these protective effects were partially reversed by EX-527, a Sirt1 inhibitor. CONCLUSION: Our study elucidated the potential therapeutic mechanism of MK-4 in neonatal HIE, suggesting its viability as an agent for enhancing recovery from HI-induced cerebral damage in newborns. Further exploration into MK-4 could lead to novel interventions for HIE therapy.

Intermittent theta-burst stimulation alleviates hypoxia-ischemia-caused myelin damage and neurologic disability.

Neonatal hypoxia-ischemia (HI) results in behavioral deficits, characterized by neuronal injury and retarded myelin formation. To date, limited treatment methods are available to prevent or alleviate neurologic sequelae of HI. Intermittent theta-burst stimulation (iTBS), a non-invasive therapeutic procedure, is considered a promising therapeutic tool for treating some neurocognitive disorders and neuropsychiatric diseases. Hence, this study aims to investigate whether iTBS can prevent the negative behavioral manifestations of HI and explore the mechanisms for associations. We exposed postnatal day 10 Sprague-Dawley male and female rats to 2 h of hypoxia (6% O2) following right common carotid artery ligation, resulting in oligodendrocyte (OL) dysfunction, including reduced proliferation and differentiation of oligodendrocyte precursor cells (OPCs), decreased OL survival, and compromised myelin in the corpus callosum (CC) and hippocampal dentate gyrus (DG). These alterations were concomitant with cognitive dysfunction and depression-like behaviors. Crucially, early iTBS treatment (15 G, 190 s, seven days, initiated one day post-HI) significantly alleviated HI-caused myelin damage and mitigated the neurologic sequelae both in male and female rats. However, the late iTBS treatment (initiated 18 days after HI insult) could not significantly impact these behavioral deficits. In summary, our findings support that early iTBS treatment may be a promising strategy to improve HI-induced neurologic disability. The underlying mechanisms of iTBS treatment are associated with promoting the differentiation of OPCs and alleviating myelin damage.

The Temporal Relationship between Blood-Brain Barrier Integrity and Microglial Response following Neonatal Hypoxia Ischemia.

Blood-brain barrier (BBB) dysfunction and neuroinflammation are key mechanisms of brain injury. We performed a time-course study following neonatal hypoxia-ischemia (HI) to characterize these events. HI brain injury was induced in postnatal day 10 rats by single carotid artery ligation followed by hypoxia (8% oxygen, 90 min). At 6, 12, 24, and 72 h (h) post-HI, brains were collected to assess neuropathology and BBB dysfunction. A significant breakdown of the BBB was observed in the HI injury group compared to the sham group from 6 h in the cortex and hippocampus (p < 0.001), including a significant increase in albumin extravasation (p < 0.0033) and decrease in basal lamina integrity and tight-junction proteins. There was a decrease in resting microglia (p < 0.0001) transitioning to an intermediate state from as early as 6 h post-HI, with the intermediate microglia peaking at 12 h (p < 0.0001), which significantly correlated to the peak of microbleeds. Neonatal HI insult leads to significant brain injury over the first 72 h that is mediated by BBB disruption within 6 h and a transitioning state of the resident microglia. Key BBB events coincide with the appearance of the intermediate microglial state and this relationship warrants further research and may be a key target for therapeutic intervention.

TSG-6-Mediated Extracellular Matrix Modifications Regulate Hypoxic-Ischemic Brain Injury.

Proteoglycans containing link domains modify the extracellular matrix (ECM) to regulate cellular homeostasis and can also sensitize tissues/organs to injury and stress. Hypoxic-ischemic (H-I) injury disrupts cellular homeostasis by activating inflammation and attenuating regeneration and repair pathways. In the brain, the main component of the ECM is the glycosaminoglycan hyaluronic acid (HA), but whether HA modifications of the ECM regulate cellular homeostasis and response to H-I injury is not known. In this report, employing both male and female mice, we demonstrate that link-domain-containing proteoglycan, TNFα-stimulated gene-6 (TSG-6), is active in the brain from birth onward and differentially modifies ECM HA during discrete neurodevelopmental windows. ECM HA modification by TSG-6 enables it to serve as a developmental switch to regulate the activity of the Hippo pathway effector protein, yes-associated protein 1 (YAP1), in the maturing brain and in response to H-I injury. Mice that lack TSG-6 expression display dysregulated expression of YAP1 targets, excitatory amino acid transporter 1 (EAAT1; glutamate-aspartate transporter) and 2 (EAAT2; glutamate transporter-1). Dysregulation of YAP1 activation in TSG-6-/- mice coincides with age- and sex-dependent sensitization of the brain to H-I injury such that 1-week-old neonates display an anti-inflammatory response in contrast to an enhanced proinflammatory injury reaction in 3-month-old adult males but not females. Our findings thus support that a key regulator of age- and sex-dependent H-I injury response in the mouse brain is modulation of the Hippo-YAP1 pathway by TSG-6-dependent ECM modifications.