Hypoxia-ischemia Research Articles

Enhanced EEG seizure recognition after hypoxia-ischemia in fetal sheep using transformer-based wavelet-scalogram deep learning

This study introduces transformer-based deep learning models as superior alternatives to convolutional neural network (CNN) architectures for seizure detection after hypoxia–ischemia (HI) in electroencephalography (EEG) recordings in the developing brain. We further demonstrate that these models excel in classifying of varied and subtle morphologies of electrographic seizures observed in the EEG of fetal sheep after HI. The study explores model training combinations from four cohorts of Romney/Suffolk fetal sheep (HI-normothermia-term (n = 7), HI-hypothermia-term (n = 14), sham-normothermia-term (n = 5), and HI-normothermia-preterm (n = 14)), totalling 31,015 EEG segments from 17,300 h of recordings. We evaluated multiple Transformer architectures using both 2D wavelet-scalograms (WS) and 1D raw EEG signals through leave-one-out and k-fold cross-validation, comparing models’ performance with or without sham-normothermia-term data and contrasting them with CNN-based results from our previous work. The Data-Efficient image Transformer (DeiT) emerged as the top-performing model, achieving 99.60 % accuracy (AUC = 0.992) when fed with WS, surpassing the 2D WS CNN result (98.94 % accuracy, AUC = 0.967) from our prior study. Notably, the Wav2Vec2 transformer model, fed with raw 1D EEG segments, demonstrated comparable performance with 99.60 % accuracy (AUC = 0.992), outperforming the 1D CNN result (98.43 % accuracy, AUC = 0.961) from the previous study. More importantly, the transformers can adeptly distinguish between seizures in both the preterm and term brain, and under the influence of treatment with 99.92 % accuracy (AUC = 0.997). Our findings highlight the superior ability of transformer models to capture long-range dependencies within EEG data, enhancing seizure detection without the added complexity of generating 2D WS images. Transformer models show promise for generalized automated seizure detection, irrespective of the perinatal brain maturation stage and the influence of hypothermia, offering potential improvements for seizure detection after HI in newborns.

2,3-Diphosphoglyceric Acid Alleviating Hypoxic-Ischemic Brain Damage through p38 MAPK Modulation.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a critical condition characterized by significant brain damage due to insufficient blood flow and oxygen delivery at birth, leading to high rates of neonatal mortality and long-term neurological deficits worldwide. 2,3-Diphosphoglyceric acid (2,3-DPG), a small molecule metabolite prevalent in erythrocytes, plays an important role in regulating oxygen delivery, but its potential neuroprotective role in hypoxic-ischemic brain damage (HIBD) has yet to be fully elucidated. Our research reveals that the administration of 2,3-DPG effectively reduces neuron damage caused by hypoxia-ischemia (HI) both in vitro and in vivo. We observed a notable decrease in HI-induced neuronal cell apoptosis, attributed to the downregulation of Bax and cleaved-caspase 3, alongside an upregulation of Bcl-2 expression. Furthermore, 2,3-DPG significantly alleviates oxidative stress and mitochondrial damage induced by oxygen-glucose deprivation/reperfusion (OGD/R). The administration of 2,3-DPG in rats subjected to HIBD resulted in a marked reduction in brain edema and infarct volume, achieved through the suppression of neuronal apoptosis and neuroinflammation. Using RNA-seq analysis, we validated that 2,3-DPG offers protection against neuronal apoptosis under HI conditions by modulating the p38 MAPK pathway. These insights indicated that 2,3-DPG might act as a promising novel therapeutic candidate for HIE.

MicroRNA-128-3p Affects Neuronal Apoptosis and Neurobehavior in Cerebral Palsy Rats by Targeting E3 Ubiquitin-Linking Enzyme Smurf2 and Regulating YY1 Expression.

This study was dedicated to investigating the effects of microRNA-128-3p (miR-128-3p) on neuronal apoptosis and neurobehavior in cerebral palsy (CP) rats via the Smurf2/YY1 axis.In vivo modeling of hypoxic-ischemic (HI) CP was established in neonatal rats. Neurobehavioral tests (geotaxis reflex, cliff avoidance reaction, and grip test) were measured after HI induction. The HI-induced neurological injury was evaluated by HE staining, Nissl staining, TUNEL staining, immunohistochemical staining, and RT-qPCR. The expression of miR-128-3p, Smurf2, and YY1 was determined by RT-qPCR and western blot techniques. Moreover, primary cortical neurons were used to establish the oxygen and glucose deprivation (OGD) model in vitro, cell viability was detected by CCK-8 assay, neuronal apoptosis was assessed by flow cytometry and western blot, and the underlying mechanism between miR-128-3p, Smurf2 and YY1 was verified by bioinformatics analysis, dual luciferase reporter assay, RIP, Co-IP, ubiquitination assay, western blot, and RT-qPCR.In vivo, miR-128-3p and YY1 expression was elevated, and Smurf2 expression was decreased in brain tissues of hypoxic-ischemic CP rats. Downregulation of miR-128-3p or overexpression of Smurf2 improved neurobehavioral performance, reduced neuronal apoptosis, and elevated Nestin and NGF expression in hypoxic-ischemic CP rats, and downregulation of Smurf2 reversed the effects of downregulation of miR-128-3p on neurobehavioral performance, neuronal apoptosis, and Nestin and NGF expression in hypoxic-ischemic CP rats, while overexpression of YY1 reversed the effects of Smurf2 on neurobehavioral performance, neuronal apoptosis, and Nestin and NGF expression in hypoxic-ischemic CP rats. In vitro, downregulation of miR-128-3p effectively promoted the neuronal survival, reduced the apoptosis rate, and decreased caspase3 protein expression after OGD, and overexpression of YY1 reversed the ameliorative effect of downregulation of miR-128-3p on OGD-induced neuronal injury. miR-128-3p targeted to suppress Smurf2 to lower YY1 ubiquitination degradation and decrease its expression.Inhibition of miR-128-3p improves neuronal apoptosis and neurobehavioral changes in hypoxic-ischemic CP rats by promoting Smurf2 to promote YY1 ubiquitination degradation and reduce YY1 expression.

Neuroserpin normalization by mesenchymal stem cell therapy after encephalopathy of prematurity in neonatal rats.

Hypoxic-ischemia (HI), infection/inflammation and reperfusion injury are pathogenic factors of encephalopathy of prematurity, which involves maturational/neurotrophic disturbances in oligodendrocyte progenitor cells (OPC) and neurons/axons. Mesenchymal stem cells (MSCs) might facilitate neuroserpin production, which is neurotrophic for OPC/neurons. This study investigated MSC effects on developmental disturbances after lipopolysaccharide (LPS)-sensitized HI/reperfusion (LHIR) injury and the relation to neuroserpin expression. Postnatal day 2 (P2) rat pups received intraperitoneal LPS (5 µg/kg) injection followed by HI (unilateral common-carotid-artery ligation and 6.5% oxygen exposure for 90 min) and post-HI reperfusion (release of ligation). MSCs (5 × 104 cells) were injected into the left lateral ventricle at 24 h post-LHIR. Neurological tests and brain tissue examinations were performed between P5 and P56. After LHIR injury, MSC therapy significantly reduced cell death in subplate neurons, attenuated axonal damage, and facilitated synaptophysin synthesis in the cortex. It also alleviated OPC maturation arrest and preserved the complexity of myelinated axons in the white matter, leading to cognitive, motor and behavioral functional improvements. These beneficial effects were linked to restored neuroserpin expression in subplate neurons. MSC therapy ameliorated developmental disturbances after LHIR injury through protection of neuroserpin expression, serving as a promising approach for treating encephalopathy of prematurity. Neuroserpin is secreted by subplate neurons and may regulate the development of neurons and oligodendrocyte-axon contact for myelination in the premature brain. LPS-sensitized hypoxic-ischemia/reperfusion (LHIR) injury caused the developmental disturbances of neurons/axons and oligodendrocytes, and lowered neuroserpin levels in a neonatal rat model simulating encephalopathy of prematurity. Mesenchymal stem cell therapy alleviated the developmental disturbances after LHIR injury through protection of neuroserpin expression in subplate neurons, offering a new perspective on potential treatment for encephalopathy of prematurity.

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.

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.

Transcriptomic Hallmarks of Hypoxic-Ischemic Brain Injury: Insights from an in Vitro Model.

Hypoxic-ischemic injury of neurons is a pathological process observed in several neurological conditions, including ischemic stroke and neonatal hypoxic-ischemic brain injury (HIBI). An optimal treatment strategy for these conditions remains elusive. The present study delved deeper into the molecular alterations occurring during the injury process in order to identify potential therapeutic targets. Oxygen-glucose deprivation/reperfusion (OGD/R) serves as an established in vitro model for the simulation of HIBI. This study utilized RNA sequencing to analyze rat primary hippocampal neurons that were subjected to either 0.5 or 2 h of OGD, followed by 0, 9, or 18 h of reperfusion. Differential expression analysis was conducted to identify genes dysregulated during OGD/R. Time-series analysis was used to identify genes exhibiting similar expression patterns over time. Additionally, functional enrichment analysis was conducted to explore their biological functions, and protein-protein interaction (PPI) network analyses were performed to identify hub genes. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for validation of hub-gene expression. The study included a total of 24 samples. Analysis revealed distinct transcriptomic alterations after OGD/R processes, with significant dysregulation of genes such as Txnip, Btg2, Egr1 and Egr2. In the OGD process, 76 genes, in two identified clusters, showed a consistent increase in expression; functional analysis showed involvement of inflammatory responses and signaling pathways like tumor necrosis factor (TNF), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and interleukin 17 (IL-17). PPI network analysis suggested that Ccl2, Jun, Cxcl1, Ptprc, and Atf3 were potential hub genes. In the reperfusion process, 274 genes, in three clusters, showed initial upregulation followed by downregulation; functional analysis suggested association with apoptotic processes and neuronal death regulation. PPI network analysis identified Esr1, Igf-1, Edn1, Hmox1, Serpine1, and Spp1 as key hub genes. qRT-PCR validated these trends. The present study provides a comprehensive transcriptomic profile of an in vitro OGD/R process. Key hub genes and pathways were identified, offering potential targets for neuroprotection after hypoxic ischemia.

In vivo mapping of cellular resolution neuropathology in brain ischemia with diffusion MRI.

Noninvasive mapping of cellular pathology can provide critical diagnostic and prognostic information. Recent advances in diffusion magnetic resonance imaging enabled in vivo examination of tissue microstructures well beyond the imaging resolution. Here, we proposed to use diffusion time-dependent diffusion kurtosis imaging (tDKI) to simultaneously assess cellular morphology and transmembrane permeability in hypoxic-ischemic (HI) brain injury. Through numerical simulations and organoid imaging, we demonstrated the feasibility of capturing effective size and permeability changes using tDKI. In vivo MRI of HI-injured mouse brains detected a shift of the tDKI peak to longer diffusion times, suggesting swelling of the cellular processes. Furthermore, we observed a faster decrease of the tDKI tail, reflecting increased transmembrane permeability associated with up-regulated water exchange or necrosis. Such information, unavailable from a single diffusion time, can predict salvageable tissues. Preliminary applications of tDKI in patients with ischemic stroke suggested increased transmembrane permeability in stroke regions, illustrating tDKI's potential for detecting pathological changes in the clinics.

Dietary LPC-Bound n-3 LCPUFA Protects against Neonatal Brain Injury in Mice but Does Not Enhance Stem Cell Therapy.

Neonatal hypoxic-ischemic (HI) brain injury is a prominent cause of neurological morbidity, urging the development of novel therapies. Interventions with n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs) and mesenchymal stem cells (MSCs) provide neuroprotection and neuroregeneration in neonatal HI animal models. While lysophosphatidylcholine (LPC)-bound n-3 LCPUFAs enhance brain incorporation, their effect on HI brain injury remains unstudied. This study investigates the efficacy of oral LPC-n-3 LCPUFAs from Lysoveta following neonatal HI in mice and explores potential additive effects in combination with MSC therapy. HI was induced in 9-day-old C57BL/6 mice and Lysoveta was orally supplemented for 7 subsequent days, with or without intranasal MSCs at 3 days post-HI. At 21-28 days post-HI, functional outcome was determined using cylinder rearing, novel object recognition, and open field tasks, followed by the assessment of gray (MAP2) and white (MBP) matter injury. Oral Lysoveta diminished gray and white matter injury but did not ameliorate functional deficits following HI. Lysoveta did not further enhance the therapeutic potential of MSC therapy. In vitro, Lysoveta protected SH-SY5Y neurons against oxidative stress. In conclusion, short-term oral administration of Lysoveta LPC-n-3 LCPUFAs provides neuroprotection against neonatal HI by mitigating oxidative stress injury but does not augment the efficacy of MSC therapy.

Effect of Early Inhibition of Toll-Like Receptor 4 on Hippocampal Plasticity in a Neonatal Rat Model of Hypoxic-Ischemic Brain Damage.

Hippocampal plasticity is closely related to physiological brain functions such as learning and memory. However, the effect of toll-like receptor 4 (TLR4) activation on hippocampal plasticity after neonatal hypoxic-ischaemic brain damage (HIBD) remains unclear. In our study, seven-day-old rat pups were randomly categorised into three groups: control, hypoxic-ischemia (HI), and HI + TAK-242 (TAK-242). The pups were ligated in the left common carotid artery and then subjected to hypoxia to establish the neonatal HIBD model.The expression of the TLR4 in the left hippocampus of the HI group was increased compared to the control group, while TAK-242 reduced the expression level at 3 days after HIBD. Additionally, TAK-242 reversed the increased Zea-Longa score, increased the left/right hippocampal weight ratio, and increased the number of Nissl-positive neurons in the hippocampal CA1 region compared to HI group at 3 days after HIBD. Pre-injection of TAK-242 alleviated the decrease in PSD95, Aggrecan and NR1, BDNF, CREB, and pCREB expression in the hippocampus at 24 h after HIBD. It also alleviated the decrease in PSD95, BDNF, and NR2A/NR1 expression in the hippocampus at 7 days after HIBD. Furthermore, Pre-injection of TAK-242 alleviated the decrease in NR2A/NR1 expression at 21 days after HIBD. Finally,TAK-242 increased the percentage of third-grade dendritic mushroom spines processes in the basal and apical segments of neurons in the hippocampal CA1 region at 21 days after HIBD.Therefore, we conclude that preinhibition of TLR4 prior to neonatal HIBD improved the plasticity of the hippocampus.

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, invitro-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.

Incidence and clinical analysis of asymptomatic intracranial hemorrhage in neonates with cerebral hypoxic-ischaemic risk based on multisequence MR images

The incidence and clinical distribution of intracranial haemorrhage (ICH) in neonates at risk of cerebral hypoxia–ischaemia have not been reported in specific studies. Based on conventional magnetic resonance imaging (MRI) versus susceptibility weighted imaging (SWI), this study aimed to analyse the occurrence of asymptomatic ICH in newborns with or without risk of cerebral hypoxia–ischaemia and to accumulate objective data for clinical evaluations of high-risk neonates and corresponding response strategies. 317 newborns were included. MRI revealed that the overall incidence of ICH was 59.31%. The most common subtype was intracranial extracerebral haemorrhage (ICECH) which included subarachnoid haemorrhage (SAH) and subdural haemorrhage (SDH). ICECH accounted for 92.02% of ICH. The positive detection rate of ICECH by SWI was significantly higher than that by T1WI. The incidence of total ICH, ICECH and SAH was greater among children who were delivered vaginally than among those who underwent caesarean delivery. Asymptomatic neonatal ICH may be a common complication of the neonatal birth process, and SWI may improve the detection rate. Transvaginal delivery and a weight greater than 2500 g were associated with a high incidence of ICECH in neonates. The impact of neonatal cerebral hypoxia–ischaemia risk factors on the occurrence of asymptomatic ICH may be negligible.

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-1β. 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.

Consensus definition and diagnostic criteria for neonatal encephalopathy-study protocol for a real-time modified delphi study.

'Neonatal encephalopathy' (NE) describes a group of conditions in term infants presenting in the earliest days after birth with disturbed neurological function of cerebral origin. NE is aetiologically heterogenous; one cause is peripartum hypoxic ischaemia. Lack of uniformity in the terminology used to describe NE and its diagnostic criteria creates difficulty in the design and interpretation of research and complicates communication with families. The DEFINE study aims to use a modifiedDelphi approach to form a consensus definition for NE, and diagnostic criteria. Directed by an international steering group, we will conduct a systematic review of the literature to assess the terminology used in trials of NE, and with their guidance perform an online Real-time Delphi survey to develop a consensus diagnosis and criteria for NE. A consensus meeting will be held to agree on the final terminology and criteria, and the outcome disseminated widely. A clear and consistent consensus-based definition of NE and criteria for its diagnosis, achieved by use of a modifiedDelphi technique, will enable more comparability of research results and improved communication among professionals and with families. The terms Neonatal Encephalopathy and Hypoxic Ischaemic Encephalopathy tend to be used interchangeably in the literature to describe a term newborn with signs of encephalopathy at birth. This creates difficulty in communication with families and carers, and between medical professionals and researchers, as well as creating difficulty with performance of research. The DEFINE project will use a Real-time Delphi approach to create a consensus definition for the term 'Neonatal Encephalopathy'. A definition formed by this consensus approach will be accepted and utilised by the neonatal community to improve research, outcomes, and parental experience.

Ultrasonic vocalization emission is altered following neonatal hypoxic-ischemic brain injury in mice

Neonatal hypoxic-ischemic (HI) brain injury leads to cognitive impairments including social communication disabilities. Current treatments do not sufficiently target these impairments, therefore new tools are needed to examine social communication in models for neonatal brain injury. Ultrasonic vocalizations (USVs) during early life show potential as a measurement for social development and reflect landmark developmental stages in neonatal mice. However, changes in USV emission early after HI injury have not been found yet. Our current study examines USV patterns and classes in the first 3 days after HI injury. C57Bl/6 mice were subjected to HI on postnatal day (P)9 and USVs were recorded between P10 and P12. Audio files were analyzed using the VocalMat automated tool. HI-injured mice emitted less USVs, for shorter durations, and at a higher frequency compared to control (sham-operated) littermates. The HI-induced alterations in USVs were most distinct at P10 and in the frequency range of 50–75 kHz. At P10 HI-injured mouse pups also produced different ratios of USV class types compared to control littermates. Moreover, alterations in the duration and frequency were specific to certain USV classes in HI animals compared to controls. Injury in the striatum and hippocampus contributed most to alterations in USV communication after HI. Overall, neonatal HI injury leads to USV alterations in newborn mice which could be used as a tool to study early HI-related social communication deficits.

Mesenchymal Stromal Cell therapy for Hypoxic Ischemic Encephalopathy: Future directions for combination therapy with hypothermia and/or melatonin

Hypoxic ischemic encephalopathy (HIE) remains a leading cause of neonatal mortality and lifelong disability across the world. While therapeutic hypothermia (HT) is beneficial, it is only partially protective and adjuvant treatments that further improve outcomes are urgently needed. In high-income countries where HT is standard care, novel treatments are tested in conjunction with HT. Mesenchymal stromal cells (MSC) represent a paradigm shift in brain protection, uniquely adapting to the host cellular microenvironment. MSC have low immunogenicity and potent paracrine effects stimulating the host tissue repair and regeneration and reducing inflammation and apoptosis. Preclinical studies in perinatal brain injury suggest that MSC are beneficial after hypoxia-ischemia (HI) and most preclinical studies of MSC with HT show protection. Preclinical and early phase clinical trials have shown that allogenic administration of MSC to neonates with perinatal stroke and HIE is safe and feasible but further safety and efficacy studies of HT with MSC in these populations are needed. Combination therapies that target all stages of the evolution of injury after HI (eg HT, melatonin and MSC) show promise for improving outcomes in HIE.

Brain metabolism after therapeutic hypothermia for murine hypoxia-ischemia using hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy.

Hypoxic-ischemic encephalopathy (HIE) is a common neurological syndrome in newborns with high mortality and morbidity. Therapeutic hypothermia (TH), which is standard of care for HIE, mitigates brain injury by suppressing anaerobic metabolism. However, more than 40% of HIE neonates have a poor outcome, even after TH. This study aims to provide metabolic biomarkers for predicting the outcomes of hypoxia-ischemia (HI) after TH using hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy. Postnatal day 10 (P10) mice with HI underwent TH at 1 h and were scanned at 6-8 h (P10), 24 h (P11), 7 days (P17), and 21 days (P31) post-HI on a 14.1-T NMR spectrometer. The metabolic images were collected, and the conversion rate from pyruvate to lactate and the ratio of lactate to pyruvate in the injured left hemisphere (kPL(L) and Lac/Pyr(L), respectively) were calculated at each timepoint. The outcomes of TH were determined by the assessments of brain injury on T2-weighted images and behavioral tests at later timepoint. kPL(L) and Lac/Pyr(L) over time between the good-outcome and poor-outcome groups and across timepoints within groups were analyzed. We found significant differences in temporal trends of kPL(L) and Lac/Pyr(L) between groups. In the good-outcome group, kPL(L) increased until P31 with a significantly higher value at P31 compared with that at P10, while the level of Lac/Pyr(L) at P31 was notably higher than those at all other timepoints. In the poor-outcome group, kPL(L) and Lac/Pyr(L) increased within 24 h. The kPL(L) value at P11 was considerably higher compared with P10. Discrete temporal changes of kPL(L) and Lac/Pyr(L) after TH between the good-outcome and poor-outcome groups were seen as early as 24 h after HI, reflecting various TH effects on brain anaerobic metabolism, which may provide insights for early screening for response to TH.

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

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. 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. 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)-1β, 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. ALO ameliorated HIBD in neonatal mice by inhibiting the neuroinflammatory response mediated by TLR4 signaling.

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.

Advancements in understanding the role of ferroptosis in hypoxia-associated brain injury: a narrative review.

Ferroptosis, a form of programmed cell death driven by lipid peroxidation and dependent on iron ions, unfolds through a sophisticated interplay of multiple biological processes. These include perturbations in iron metabolism, lipid peroxidation, aberrant amino acid metabolism, disruptions in hypoxia-inducible factor-prolyl hydroxylase (HIF-PHD) axis, and endoplasmic reticulum (ER) stress. Recent studies indicate that ferroptosis may serve as a promising therapeutic target for hypoxia-associated brain injury such as hypoxic-ischemic brain damage (HIBD) and cerebral ischemia-reperfusion injury (CIRI). HIBD is a neonatal disease that can be fatal, causing death or mental retardation in newborns. HIBD is a kind of diffuse brain injury, which is characterized by apoptosis of nerve cells and abnormal function and structure of neurons after cerebral hypoxia and ischemia. At present, there are no fundamental prevention and treatment measures for HIBD. The brain is the most sensitive organ of the human body to hypoxia. Cerebral ischemia will lead to the damage of local brain tissue and its function, and CIRI will lead to a series of serious consequences. We hope to clarify the mechanism of ferroptosis in hypoxia-associated brain injury, inhibit the relevant targets of ferroptosis in hypoxia-associated brain injury to guide clinical treatment, and provide guidance for the subsequent treatment of disease-related drugs. Our research incorporated data on "ferroptosis", "neonatal hypoxic ischemia", "hypoxic ischemic brain injury", "hypoxic ischemic encephalopathy", "brain ischemia-reperfusion injury", and "therapeutics", which were sourced from Web of Science, PubMed, and comprehensive reviews and articles written in English. This review delineates the underlying mechanisms of ferroptosis and the significance of these pathways in hypoxia-associated brain injury, offering an overview of therapeutic strategies for mitigating ferroptosis. Ferroptosis involves dysregulation of iron metabolism, lipid peroxidation, amino acid metabolism, dysregulation of HIF-PHD axis and endoplasmic reticulum stress (ERS). By reviewing the literature, we identified the involvement of the above processes in HIBD and CIRI, and summarized a series of therapeutic measures for HIBD and CIRI by inhibiting ferroptosis. We hope this study would provide guidance for the clinical treatment of HIBD and CIRI in the future.