Mild brain hypothermia (31-34 °C) after neonatal hypoxia-ischemia (HI) improves neurodevelopmental outcomes in human and animal neonates. Using an asphyxia model with neonatal mice treated with mild hypothermia after HI, we investigated whether (1)H nuclear magnetic resonance (NMR) metabolomics of brain extracts could suggest biomarkers and distinguish different treatments and outcome groups.

Central nervous system diseases are associated with hypoxia, which usually cause irreversible nerve damage, but the underlying mechanism is unclear and effective intervention strategies are lacking. This study was designed to explore the mechanism and treatment strategy of hypoxia-induced nerve injury.

Chronic hypoxia leads to irreversible cognitive impairment, primarily due to hippocampal neurodegeneration, for which the underlying mechanism remains poorly understood. We administered hypoxia (13%) to C57BL mice for 1-14 days in this study. Chronic hypoxia for 7 or 14 d, but not 1 or 3 d, resulted in alpha-synuclein hyperphosphorylation at serine129 (α-Syn p-S129) and protein aggregation, hippocampal neurodegeneration, and cognitive deficits, whereas the latter could be prevented by alpha-synuclein knockdown or an administered short peptide competing at α-Syn S129. These results suggest that α-Syn p-S129 mediates hippocampal degeneration and cognitive impairment following chronic hypoxia. Furthermore, we found that chronic hypoxia enhanced ceramide catabolism by inducing hypoxia-inducible factor (HIF)-2α and HIF-2α-dependent transcriptional activation of alkaline ceramidase 2 (Acer2). Thus, the enzymatic activity of protein phosphatase 2A (PP2A), a specific phosphatase for α-syn, is inhibited, leading to the sustained induction of α-Syn p-S129. Finally, we found that intermittent hypoxic preconditioning protected against subsequent chronic hypoxia-induced hippocampal neurodegeneration and cognitive impairment by preventing α-Syn p-S129. These results proved the critical role of α-syn pathology in chronic hypoxia-afforded cognitive impairment and revealed a novel mechanism underlying α-syn hyperphosphorylation during chronic hypoxia. The findings bear implications in developing novel therapeutic interventions for chronic hypoxia-related brain disorders.

Neonatal hypoxic-ischemic encephalopathy is a serious neurological disease, often resulting in long-term neurodevelopmental disorders among surviving children. However, whether these neurodevelopmental issues can be passed to offspring remains unclear. The right common carotid artery of 7-day-old parental-generation rats was subjected to permanent ligation using a vessel electrocoagulator. Neonatal hypoxic-ischemic rat models were established by subjecting the rats to 8% O2-92% N2 for 2 hours. The results showed that 24 hours after hypoxia and ischemia, pathological damage, cerebral atrophy, liquefaction, and impairment were found, and Zea-Longa scores were significantly increased. The parental-generation rats were propagated at 3 months old, and offspring were obtained. No changes in the overall brain structures of these offspring rats were identified by magnetic resonance imaging. However, the escape latency was longer and the number of platform crossings was reduced among these offspring compared with normal rats. These results indicated that the offspring of hypoxic-ischemic encephalopathy model rats displayed cognitive impairments in learning and memory. This study was approved by the Animal Care & Welfare Committee of Kunming Medical University, China in 2018 (approval No. kmmu2019072).

Brain-derived neurotrophic factor (BDNF) regulates many neurological functions and plays a vital role during the recovery from central nervous system injuries. However, the changes in BDNF expression and associated factors following hypoxia-ischemia induced neonatal brain damage, and the significance of these changes are not fully understood. In the present study, a rat model of hypoxic-ischemic brain damage was established through the occlusion of the right common carotid artery, followed by 2 hours in a hypoxic-ischemic environment. Rats with hypoxic-ischemic brain damage presented deficits in both sensory and motor functions, and obvious pathological changes could be detected in brain tissues. The mRNA expression levels of BDNF and its processing enzymes and receptors (Furin, matrix metallopeptidase 9, tissue-type plasminogen activator, tyrosine Kinase receptor B, plasminogen activator inhibitor-1, and Sortilin) were upregulated in the ipsilateral hippocampus and cerebral cortex 6 hours after injury; however, the expression levels of these mRNAs were found to be downregulated in the contralateral hippocampus and cerebral cortex. These findings suggest that BDNF and its processing enzymes and receptors may play important roles in the pathogenesis and recovery from neonatal hypoxic-ischemic brain damage. This study was approved by the Animal Ethics Committee of the University of South Australia (approval No. U12-18) on July 30, 2018.

Neonatal hypoxic-ischemic encephalopathy (HIE) always results in severe neurologic dysfunction, nevertheless effective treatments are limited and the underlying mechanism also remains unclear. In this study, we firstly established the neonatal HIE model in the postnatal day 7 SD rats, Zea-Longa score and TTC staining were employed to assess the neurological behavior and infarct volume of the brain after cerebral hypoxia-ischemia (HI). Afterwards, protein chip was adopted to detect the differential proteins in the right cortex, hippocampus and lung, ultimately, PDGF was noticed. Then, immunohistochemistry, immunofluorescence double staining of NeuN/PDGF, and western blot were used to validate the expression level of PDGF in the cortex and hippocampus at 6 hours (h), 12 h and 24 h after HI. To determine the role of PDGF, the primary cortical neurons were prepared and performed PDGF shRNA administration. The results showed that HIE induced a severe behavioral dysfunction and brain infarction in neonatal rats, and the expression of PDGF in right cortex and hippocampus was remarkably increased after HI. Whereas, suppressing PDGF resulted in a significant loss of neurons and inhibition of neurite growth. Moreover, the protein level of P-PI3K and P-AKT signaling pathways were largely decreased following PDGF-shRNA application in the cortical neurons. In conclusion, PDGF suppression aggravated neuronal dysfunction, and the underlying mechanism is associated with inhibiting the phosphorylation of P-PI3K and P-AKT. Together, PDGF regulating PI3K and AKT may be an important panel in HIE events and therefore may provide possible strategy for the treatment of HIE in future clinic trail.

Defining the relationship between maternal care, sensory development and brain gene expression in neonates is important to understand the impact of environmental challenges during sensitive periods in early life. In this study, we used a selection approach to test the hypothesis that variation in maternal licking and grooming (LG) during the first week of life influences sensory development in Wistar rat pups. We tracked the onset of the auditory brainstem response (ABR), the timing of eye opening (EO), middle ear development with micro-CT X-ray tomography, and used qRT-PCR to monitor changes in gene expression of the hypoxia-sensitive pathway and neurotrophin signaling in pups reared by low-LG or high-LG dams. The results show the first evidence that the transcription of genes involved in the hypoxia-sensitive pathway and neurotrophin signaling is regulated during separate sensitive periods that occur before and after hearing onset, respectively. Although the timing of ABR onset, EO, and the relative mRNA levels of genes involved in the hypoxia-sensitive pathway did not differ between pups from different LG groups, we found statistically significant increases in the relative mRNA levels of four genes involved in neurotrophin signaling in auditory brain regions from pups of different LG backgrounds. These results suggest that sensitivity to hypoxic challenge might be widespread in the auditory system of neonate rats before hearing onset, and that maternal LG may affect the transcription of genes involved in experience-dependent neuroplasticity.

Neonatal hypoxic ischemic encephalopathy (HIE) due to birth asphyxia is common and causes severe neurological deficits, without any effective therapies currently available. Neuronal death is an important driving factors of neurological disorders after HIE, but the regulatory mechanisms are still uncertain. Long non-coding RNA (lncRNA) or ceRNA network act as a significant regulator in neuroregeneration and neuronal apoptosis, thus owning a great potential as therapeutic targets in HIE. Here, we found a new lncRNA, is the most functional in targeting the Igfbp3 gene in HIE, which enriched in the cell growth and cell apoptosis processes. In addition, luciferase reporter assay showed competitive regulatory binding sites to the target gene Igfbp3 between TCONS00044054 (Vi4) and miR-185-5p. The change in blood miR-185-5p and Igfbp3 expression is further confirmed in patients with brain ischemia. Moreover, Vi4 overexpression and miR-185-5p knock-out promote the neuron survival and neurite growth, and suppress the cell apoptosis, then further improve the motor and cognitive deficits in rats with HIE, while Igfbp3 interfering got the opposite results. Together, Vi4-miR-185-5p-Igfbp3 regulatory network plays an important role in neuron survival and cell apoptosis and further promote the neuro-functional recovery from HIE, therefore is a likely a drug target for HIE therapy.

Fructose-1,6-bisphosphate (FBP), an endogenous intracellular metabolite in glycolysis, was found in many preclinical studies to be neuroprotective during hypoxia-ischemia (HI) when administered exogenously. We looked for HI neuroprotection from FBP in a neonatal rat brain slice model, using 14.1 T (1)H/(31)P/(13)C NMR spectroscopy of perchloric acid slice extracts to ask: 1) if FBP preserves high energy phosphates during HI; and 2) if exogenous [1-(13)C]FBP enters cells and is glycolytically metabolized to [3-(13)C]lactate. We also asked: 3) if substantial superoxide production occurs during and after HI, thinking such might be treatable by exogenous FBP's antioxidant effects. Superfused P7 rat cerebrocortical slices (350 mum) were treated with 2 mM FBP before and during 30 min of HI, and then given 4 h of recovery with an FBP-free oxygenated superfusate. Slices were removed before HI, at the end of HI, and at 1 and 4 h after HI. FBP did not improve high energy phosphate levels or change (1)H metabolite profiles. Large increases in [3-(13)C]lactate were seen with (13)C NMR, but the lactate fractional enrichment was always (1.1+/-0.5)%, implying that all of lactate's (13)C was natural abundance (13)C, that none was from metabolism of (13)C-FBP. FBP had no effect on the fluorescence of ethidium produced from superoxide oxidation of hydroethidine. Compared to control slices, ethidium fluorescence was 25% higher during HI and 50% higher at the end of recovery. Exogenous FBP did not provide protection or enter glycolysis. Its use as an antioxidant might be worth studying at higher FBP concentrations.

The etiology of Parkinson's disease (PD) is complex and the mechanism is unclear. It has become a top priority to find common factors that induce and affect PD pathology. We explored the key role of hypoxia in promoting the pathological propagation of α-synuclein (α-syn) and the progression of PD.

Hypoxic stress occurs in various physiological and pathological states, such as aging, disease, or high-altitude exposure, all of which pose a challenge to many organs in the body, necessitating adaptation. However, the exact mechanisms by which hypoxia affects advanced brain function (learning and memory skills in particular) remain unclear. In this study, we investigated the effects of hypoxic stress on hippocampal function. Specifically, we studied the effects of the dysfunction of mitochondrial oxidative phosphorylation using global proteomics. First, we found that hypoxic stress impaired cognitive and motor abilities, whereas it caused no substantial changes in the brain morphology or structure of mice. Second, bioinformatics analysis indicated that hypoxia affected the expression of 516 proteins, of which 71.1% were upregulated and 28.5% were downregulated. We demonstrated that mitochondrial function was altered and manifested as a decrease in NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 expression, accompanied by increased reactive oxygen species generation, resulting in further neuronal injury. These results may provide some new insights into how hypoxic stress alters hippocampal function via the dysfunction of mitochondrial oxidative phosphorylation.

Defining the relationship between vascular development and the expression of hypoxia-inducible factors (Hifs) and vascular endothelial growth factor (Vegf) in the auditory brainstem is important to understand how tissue hypoxia caused by oxygen shortage contributes to sensory deficits in neonates. In this study, we used histology, molecular labeling, confocal microscopy and 3D image processing methods to test the hypothesis that significant maturation of the vascular bed in the medial nucleus of the trapezoid body (MNTB) occurs during the postnatal period that precedes hearing onset. Isolectin-B4 histochemistry experiments suggested that the MNTB vasculature becomes more elaborate between P5 and P10. When combined with a cell proliferation marker and immunohistochemistry, we found that vascular growth coincides with a switch in the localization of proliferating cells to perivascular locations, and an increase in the density of microglia within the MNTB. Furthermore, microglia were identified as perivascular cells with proliferative activity during the period of vascular maturation. Lastly, combined in situ hybridization and immunohistochemistry experiments showed distinct profiles of Hif1a and Vegf mRNA localization in microglia, astrocytes and MNTB principal neurons. These results suggest that different cells of the neuro-glio-vascular unit are likely targets of hypoxic insult in the auditory brainstem of neonate rats.

Neonatal hypoxic-ischemic encephalopathy (HIE), is a major cause of neurologic disorders in terms of neonates, with the unclear underlying mechanisms. In the study, triphenyl tetrazolium chloride (TTC) staining and Zea-longa score were performed to examine the neurologic damage in hypoxia and ischemia (HI) rats. The results showed that HI induced obviously infarct and serious neurologic impairment in neonatal rats. Then, protein chip was applied to detect the differential expression genes in cortex and hippocampus and found the brain-derived neurotrophic factor (BDNF) down-regulated both in cortex and hippocampus. Moreover, low expression of BDNF after HI in right cortex and hippocampus was validate by immunohistochemistry (IHC) and Western Blotting (WB). Afterwards, overexpressing and interfering HSV vector were produced, then verified by immunofluorescent staining and real-time quantitative polymerase chain reaction (qRT-PCR). The results of Tuj1 staining indicated that overexpression of BDNF could promote axonal regeneration and inhibit neuron swelling, whereas BDNF interference take an opposite effect after Oxygen glucose deprivation (OGD) injury. Finally, the interaction network among BDNF and associated proteins as examined by Genemania and confirmed by qRT-PCR. We found that the expression of VDAC1 was decreased and Stx1b was increased when BDNF overexpressing, which indicated that BDNF promoted neurite regrowth after OGD might be related to downregulation of VDAC1 and upregulation of Stx1b. Our results might provide novel strategy for the treatment of neurological defects induced by cerebral ischemia and hypoxia.

Neuronal apoptosis is a major pathological hallmark of the neonatal hypoxic-ischemic brain damage (HIBD); however, the role of miR-7a-2-3p in the regulation of HIBD remains unknown. The purpose of this study was to explore the possible roles of miR-7a-2-3p in brain injury using a hypoxia-ischemia model in rats and oxygen-glucose deprivation (OGD) model in vitro. Firstly, we established the hypoxia-ischemia (HI) model and verified the model using Zea Longa scores and MRI in rats. Next, the changes of miR-7a-2-3p were screened in the ischemic cortex of neonatal rats by qRT-PCR at 12, 48, and 96 h after HIBD. We have found that the expression of miR-7a-2-3p in the HI rats decreased significantly, compared with the sham group (P < 0.01). Then, we established the OGD model in PC12 cells, SH-SY5Y cells and primary cortical neurons in vitro and qRT-PCR was used to confirm the changes of miR-7a-2-3p in these cells after the OGD. In order to determine the function of miR-7a-2-3p, PC12 cells, SH-SY5Y cells and rat primary cortical neurons were randomly divided into normal, OGD, mimic negative control (mimic-NC) and miR-7a-2-3p groups. Then, Tuj1+ (neuronal marker) staining, TUNEL assay (to detect apoptotic cells) and MTT assay (to investigate cell viability) were performed. We have found that the number of PC12 cells, SH-SY5Y cells and cortical neurons in the miR-7a-2-3p groups increased significantly (P < 0.01) in comparison to the OGD groups. The survival of cortical neurons in the miR-7a-2-3p group was improved markedly (P < 0.01), while the apoptosis of neurons in the miR-7a-2-3p group was significantly decreased (P < 0.01), compared with the normal group. Lastly, we investigated the target genes of miR-7a-2-3p by using the prediction databases (miRDB, TargetScan, miRWalk, and miRmap) and verified the target genes with qRT-PCR in the HI rats. Bioinformatics prediction showed that Vimentin (VIM), pleiomorphic adenoma gene 1(PLAG1), dual specificity phosphatase 10 (DUSP10), NAD(P)H dehydrogenase, quinone 1 (NQO1) and tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) might be the targets of miR-7a-2-3p and the qRT-PCR confirmed that VIM increased in the HI rats (P < 0.01). In conclusion, miR-7a-2-3p plays a crucial role in the hypoxic-ischemic injury, and is associated with regulation of VIM.

Interleukin 10 (IL-10) is a synthetic inhibitor of human cytokines with immunomodulatory and anti-inflammatory effects. This study was designed to investigate the expression variation of IL-10 in the multiple sites including cortex, hippocampus, and lung tissues of neonatal hypoxic-ischemic (HI) rats and explore the crucial role of IL-10 in alleviating HI brain damage. In this study, neonatal Sprague-Dawley rats were subjected to the right common carotid artery ligation, followed by 2 h of hypoxia. The expression of IL-10 in the cortex, hippocampus, and lung tissues was measured with immunohistochemistry, real-time quantitative polymerase chain reaction (RT-qPCR), and western blot (WB). Immunofluorescence double staining was performed to observe the localization of IL-10 in neurons and astrocytes. Moreover, not-targeting and targeting IL-10 siRNA lentivirus vectors were injected into the rats of the negative control (NC) and IL-10 group, respectively, and the mRNA levels of B-cell lymphoma 2 (Bcl-2) and endoplasmic reticulum protein 29 (ERp29) were detected by RT-qPCR following IL-10 silence. The results demonstrated that the IL-10 expression was markedly increased after HI and IL-10 were colocalized with neurons and astrocytes which were badly injured by HI insult. In addition, Bcl-2 and ERp29 were remarkably decreased following IL-10 mRNA interference compared with the NC group. Our findings revealed that IL-10 exerted its antiapoptotic and neuroprotective effects by regulating the expression of Bcl-2 and ERp29, indicating that IL-10 may be a promising molecule target for HIE treatment.