METTL3 inhibits microglial pyroptosis in neonatal hypoxia-ischemia encephalopathy by regulating GPR39 expression in an m6A-HuR-dependent manner.

BACKGROUND: Neonatal hypoxia-ischemia encephalopathy (HIE) is a significant reason for neonatal mortality and prolonged disability. We have previously revealed that GPR39 activation attenuates neuroinflammation in a neonatal HIE rat model. This study aimed to investigate whether GPR39 affected microglial pyroptosis post-HIE. METHODS: A neonatal rat model of HIE and a microglia cell model of oxygen-glucose deprivation (OGD) were established. Neuronal loss and cerebral infarction were assessed by using TTC, H&E staining, and Nissl staining. Pyroptosis was evaluated with western blot, LDH assay kit, ELISA, and flow cytometry. Total m6A level and GPR39 m6A modification were determined using m6A dot blot and MeRIP. The interaction between METTL3/HuR/GSK3ß and GPR39 was analyzed by performing molecular interaction experiments. GPR39 mRNA stability was examined with actinomycin D. RESULTS: The level of GPR39 was increased in neonatal HIE rats and OGD-treated microglia. Brain injury and neuronal loss were significantly increased in the HIE model when GPR39 was knocked down. GPR39 knockdown aggravated NLRP3 inflammasome-mediated microglial pyroptosis. METTL3 upregulated GPR39 expression in an m6A-dependent manner. METTL3 enhanced the interaction of HuR and GPR39. In OGD-exposed microglia, METTL3 elevated GPR39 expression and mRNA stability, which declined after HuR depletion. METTL3 knockdown promoted microglial pyroptosis, which was reversed by GPR39 agonist. Furthermore, microglial pyroptosis was inhibited by GPR39 upregulation, but the outcome was reverted by GSK3ß activator SNP. CONCLUSION: METTL3 inhibits microglial pyroptosis in neonatal HIE via regulating m6A-HuR dependent stabilization of GPR39, which contributes to therapeutics development for neonatal HIE.

Emerging ferroptosis inhibitors as a novel therapeutic strategy for the treatment of neonatal hypoxic-ischemic encephalopathy.

Neonatal hypoxia-ischemia encephalopathy (NHIE), an oxygen deprivation-mediated brain injury due to birth asphyxia or reduced cerebral blood perfusion, often leads to lifelong sequelae, including seizures, cerebral palsy, and mental retardation. NHIE poses a significant health challenge, as one of the leading causes of neonatal morbidity and mortality globally. Despite this, available therapies are limited. Numerous studies have recently demonstrated that ferroptosis, an iron-dependent non-apoptotic regulated form of cell death characterized by lipid peroxidation (LPO) and iron dyshomeostasis, plays a role in the genesis of NHIE. Moreover, recently discovered compounds have been shown to exert potential therapeutic effects on NHIE by inhibiting ferroptosis. This comprehensive review summarizes the fundamental mechanisms of ferroptosis contributing to NHIE. We focus on various emerging therapeutic compounds exhibiting characteristics of ferroptosis inhibition and delineate their pharmacological benefits for the treatment of NHIE. This review suggests that pharmacological inhibition of ferroptosis may be a potential therapeutic strategy for NHIE.

Creatine in the fetal brain: A regional investigation of acute global hypoxia and creatine supplementation in a translational fetal sheep model.

Background: Creatine supplementation during pregnancy is a promising prophylactic treatment for perinatal hypoxic brain injury. Previously, in near-term sheep we have shown that fetal creatine supplementation reduces cerebral metabolic and oxidative stress induced by acute global hypoxia. This study investigated the effects of acute hypoxia with or without fetal creatine supplementation on neuropathology in multiple brain regions. Methods: Near-term fetal sheep were administered continuous intravenous infusion of either creatine (6 mg kg-1 h-1) or isovolumetric saline from 122 to 134 days gestational age (dGA; term is approx. 145 dGA). At 131 dGA, global hypoxia was induced by a 10 min umbilical cord occlusion (UCO). Fetuses were then recovered for 72 h at which time (134 dGA) cerebral tissue was collected for either RT-qPCR or immunohistochemistry analyses. Results: UCO resulted in mild injury to the cortical gray matter, thalamus and hippocampus, with increased cell death and astrogliosis and downregulation of genes involved in regulating injury responses, vasculature development and mitochondrial integrity. Creatine supplementation reduced astrogliosis within the corpus callosum but did not ameliorate any other gene expression or histopathological changes induced by hypoxia. Of importance, effects of creatine supplementation on gene expression irrespective of hypoxia, including increased expression of anti-apoptotic (BCL-2) and pro-inflammatory (e.g., MPO, TNFa, IL-6, IL-1ß) genes, particularly in the gray matter, hippocampus, and striatum were identified. Creatine treatment also effected oligodendrocyte maturation and myelination in white matter regions. Conclusion: While supplementation did not rescue mild neuropathology caused by UCO, creatine did result in gene expression changes that may influence in utero cerebral development.

The assessment of general movements in term and late-preterm infants diagnosed with neonatal encephalopathy, as a predictive tool of cerebral palsy by 2 years of age-a scoping review.

BACKGROUND: The General Movements Assessment is a non-invasive and cost-effective tool with demonstrated reliability for identifying infants at risk for cerebral palsy. Early detection of cerebral palsy allows for the implementation of early intervention and is associated with better functional outcomes. No review to date has summarized the utility of the General Movements Assessment to predict cerebral palsy in term and late-preterm infants diagnosed with neonatal encephalopathy. METHODS: We conducted a scoping review involving infants born greater than or equal to 34 weeks gestational age to identify all available evidence and delineate research gaps. We extracted data on sensitivity, specificity, and positive and negative predictive values and described the strengths and limitations of the results. We searched five databases (MEDLINE, Embase, PsychINFO, Scopus, and CINAHL) and the General Movements Trust website. Two reviewers conducted all screening and data extraction independently. The articles were categorized according to key findings, and a critical appraisal was performed. RESULTS: Only three studies, a cohort and two case series, met all of the inclusion criteria. The total number of participants was 118. None of the final eligible studies included late-preterm neonates. All three studies reported on sensitivity, specificity, and positive predictive and negative predictive values. An abnormal General Movement Assessment at 3-5 months has a high specificity (84.6-98%) for cerebral palsy with a similarly high negative predictive value (84.6-98%) when it was normal. Absent fidgety movements, in particular, are highly specific (96%) for moderate to severe cerebral palsy and carry a high negative predictive value (98%) when normal. In the time period between term and 4-5 months post-term, any cramped synchronized movements had results of 100% sensitivity and variable results for specificity, positive predictive value, and negative predictive value. CONCLUSIONS: A normal General Movements Assessment at 3 months in a term high-risk infant is likely associated with a low risk for moderate/severe cerebral palsy. The finding of cramped synchronized General Movements is a strong predictor for the diagnosis of cerebral palsy by 2 years of age in the term population with neonatal encephalopathy. The deficit of high-quality research limits the applicability, and so the General Movements Assessment should not be used in isolation when assessing this population. SYSTEMATIC REVIEW REGISTRATION: Title registration with Joanna Briggs Institute. URL: http://joannabriggswebdev.org/research/registered_titles.aspx .

Dexmedetomidine Use in Infants Undergoing Cooling Due to Neonatal Encephalopathy (DICE Trial): A Randomized Controlled Trial: Background, Aims and Study Protocol.

Background: Neonatal hypoxia-ischemia encephalopathy (HIE) is the leading cause of neonatal death and poor neurodevelopmental outcomes worldwide. Therapeutic hypothermia (TH), while beneficial, still leaves many HIE treated infants with lifelong disabilities. Furthermore, infants undergoing TH often require treatment for pain and agitation which may lead to further brain injury. For instance, morphine use in animal models has been shown to induce neuronal apoptosis. Dexmedetomidine is a potent α2-adrenergic receptor agonist that may be a better alternative to morphine for newborns with HIE treated with TH. Dexmedetomidine provides sedation, analgesia, and prevents shivering but does not suppress ventilation. Importantly, there is increasing evidence that dexmedetomidine has neuroprotective properties. Even though there are limited data on pharmacokinetics (PK), safety and efficacy of dexmedetomidine in infants with HIE, it has been increasingly administered in many centers. Objectives: To review the current approach to treatment of pain, sedation and shivering in infants with HIE undergoing TH, and to describe a new phase II safety and pharmacokinetics randomized controlled trial that proposes the use of dexmedetomidine vs. morphine in this population. Methods: This article presents an overview of the current management of pain and sedation in critically ill infants diagnosed with HIE and undergoing TH for 72 h. The article describes the design and methodology of a randomized, controlled, unmasked multicenter trial of dexmedetomidine vs. morphine administration enrolling 50 (25 per arm) neonates ≥36 weeks of gestation with moderate or severe HIE undergoing TH and that require pain/sedation treatment. Results and Conclusions: Dexmedetomidine may be a better alternative to morphine for the treatment of pain and sedation in newborns with HIE treated with TH. There is increasing evidence that dexmedetomidine has neuroprotective properties in several preclinical studies of injury models including ischemia-reperfusion, inflammation, and traumatic brain injury as well as adult clinical trials of brain trauma. The Dexmedetomidine Use in Infants undergoing Cooling due to Neonatal Encephalopathy (DICE) trial will evaluate whether administration of dexmedetomidine vs. morphine is safe, establish dexmedetomidine optimal dosing by collecting opportunistic PK data, and obtain preliminary neurodevelopmental data to inform a large Phase III efficacy trial with long term neurodevelopment impairment as the primary outcome.

The general movements assessment in term and late-preterm infants diagnosed with neonatal encephalopathy, as a predictive tool of cerebral palsy by 2 years of age: a scoping review protocol.

BACKGROUND: Prediction of long-term neurodevelopmental outcomes remains an elusive goal for neonatology. Clinical and socioeconomic markers have not proven to be adequately reliable. The limitation in prognostication includes those term and late-preterm infants born with neonatal encephalopathy. The General Movements Assessment tool by Prechtl has demonstrated reliability for identifying infants at risk for neuromotor impairment. This tool is non-invasive and cost-effective. The purpose of this study is to identify the published literature on how this tool applies to the prediction of cerebral palsy in term and late-preterm infants diagnosed with neonatal encephalopathy and so detect the research gaps. METHODS: We will conduct a systematic scoping review for data on sensitivity, specificity, positive, and negative predictive value and describe the strengths and limitations of the results. This review will consider studies that included infants more than or equal to 34 + 0 weeks gestational age, diagnosed with neonatal encephalopathy, with a General Movements Assessment done between birth to six months of life and an assessment for cerebral palsy by at least 2 years of age. Experimental and quasi-experimental study designs including randomized controlled trials, non-randomized controlled trials, before and after studies, interrupted time-series studies and systematic reviews will be considered. Case reports, case series, case control, and cross-sectional studies will be included. Text, opinion papers, and animal studies will not be considered for inclusion in this scoping review as this is a highly specific and medical topic. Studies in the English language only will be considered. Studies published from at least 1970 will be included as this is around the time when the General Movements Assessment was first introduced in neonatology as a potential predictor of neuromotor outcomes. We will search five databases (MEDLINE, Embase, PsychINFO, Scopus, and CINAHL). Two reviewers will conduct all screening and data extraction independently. The articles will be categorized according to key findings and a critical appraisal performed. DISCUSSION: The results of this review will guide future research to improve early identification and timely intervention in infants with neonatal encephalopathy at risk of neuromotor impairment. SYSTEMATIC REVIEW REGISTRATION: Title registration with Joanna Briggs Institute https://joannabriggs.org/ebp/systematic_review_register .

Ganoderic acid A alleviates hypoxia-induced apoptosis, autophagy, and inflammation in rat neural stem cells through the PI3K/AKT/mTOR pathways.

Effects of ganoderic acid A (GAA), a lanostane triterpene, on hypoxia-ischemia encephalopathy (HIE) remain unclear. We aimed to figure out the specific role of GAA in hypoxia-treated neural stem cells (NSCs) as well as the regulatory mechanisms. Primary rat NSCs were incubated under hypoxia to simulate HIE. Viability and apoptosis of hypoxia-injured NSCs were measured by cell counting kit-8 and flow cytometry assays, respectively. Proteins related to apoptosis, autophagy, and the PI3K/AKT/mTOR pathways were evaluated by Western blot analysis. LY294002 and rapamycin were added to inhibit the PI3K/AKT pathway and mTOR pathway, respectively. Enzyme-linked immunosorbent assay was carried out to test the release of proinflammatory cytokines. We found that hypoxia-induced decrease of cell viability, increases of apoptotic cells and autophagy, and the release of IL-6, IL-1ß, and TNF-α were all attenuated by GAA stimulation. Activation of caspases induced by hypoxia was alleviated by GAA. Furthermore, we found that inhibition of the PI3K/AKT pathway eliminated the effects of GAA on apoptosis and proinflammatory cytokines release in hypoxia-injured NSCs. Meanwhile, inhibition of the mTOR pathway abrogated the effects of GAA on cell autophagy in hypoxia-injured NSCs. In conclusion, GAA alleviated hypoxia-induced injury in NSCs might be through activating the PI3K/AKT and mTOR pathways.

Effect of GABAB receptor antagonists (CGP 35348 and CGP 55845) on serum interleukin 6 and 18 concentrations in albino mice following neonatal hypoxia ischemia insult.

Interleukin (IL) 6 and 18 plays an important role in inflammatory response following hypoxia ischemia encephalopathy (HIE). Present study was designed to demonstrate the effect of two GABAB receptor antagonists (CGP 35348 and 55845), respectively, on the serum IL6 and IL 18 concentrations in albino mice. Albino mice pups (of both genders) were subjected to Murine model of hypoxia-ischemia encephalopathy on postnatal day 10 (right common carotid artery was ligated followed by 8% hypoxia for 25 minutes). After neonatal brain damage and following weaning, mice were divided in three groups, in gender specific manner, and fed on normal rodent diet till they were 13 week old. At this time point, group 1 received intraperitonial saline solution (control group), group 2 was supplemented with CGP 35348 (1mg/ml solvent/Kg body weight) and group 3 with CGP 55845 (1mg/ml solvent/Kg body weight), intraperitonially, for 12 days and IL 6 and 18 concentrations were determined in serum by ELISA. It was observed that CGP 35348 supplementation resulted in reduced interlukin-6 and interlukin-18 concentrations in male albino mice. While CGP 55845 supplementation increased IL-6 and IL-18 concentrations in female albino mice following HIE. Our results are indicating that GABAB receptor antagonist's supplementation affects IL concentrations in albino mice in a gender specific manner following neonatal brain damage and can be further explored for the treatments of hypoxia ischemia associated neurological ailments.

Neonatal hypoxic-ischemic encephalopathy reduces c-Fos activation in the rat hippocampus: evidence of a long-lasting effect.

The effect of neonatal hypoxic-ischemic encephalopathy (HIE) on maturation of nociceptive pathways has been sparsely explored. To investigate whether neonatal HIE alters neuronal activity, nociceptive behavior, and serum neuroplasticity mediators (brain-derived neurotrophic factor [BDNF] and tumor necrosis factor-α [TNF]) in the short, medium, and long term. Neonate male Wistar rats were randomized to receive a brain insult that could be either ischemic (left carotid artery ligation [LCAL]), hypoxic (8% oxygen chamber), hypoxic-ischemic (LCAL and hypoxic chamber), sham-ischemic, or sham-hypoxic. Neuronal activity (c-Fos activation at region CA1 and dentate gyrus of the hippocampus), nociceptive behavior (von Frey, tail-flick, and hot-plate tests), neuroplasticity mediators (BDNF, TNF), and a cellular injury marker (lactase dehydrogenase [LDH]) were assessed in blood serum 14, 30, and 60 days after birth. Neonatal HIE persistently reduced c-Fos activation in the ipsilateral hippocampal region CA1; however, contralateral c-Fos reduction appeared only 7 weeks after the event. Neonatal HIE acutely reduced the paw withdrawal threshold (von Frey test), but this returned to normal by the 30th postnatal day. Hypoxia reduced serum LDH levels. Serum neuroplasticity mediators increased with age, and neonatal HIE did not affect their ontogeny. Neonatal HIE-induced reduction in neuronal activity occurs acutely in the ipsilateral hippocampal region CA1 and persists for at least 60 days, but the contralateral effect of the insult is delayed. Alterations in the nociceptive response are acute and self-limited. Serum neuroplasticity mediators increase with age, and remain unaffected by HIE.

Restoration of tissue damage, and never activity after hypoxia-ischemia by implantation of peripheral blood mononuclear cells.

Hypoxia-ischemia (HI) encephalopathy is a frequent cause of disability and mortality with limited therapeutic options. Here, we collected peripheral blood mononuclear cells (PB-MNCs) from healthy donors and labeled them with CM-DiI before implanting these cells by tail-vein injection into rats at day 3 after hypoxia-ischemia (HI). For immune-suppression the animals received daily injections of cyclosporine throughout the experiment, commencing 24h before cell transplantation. Then we observed the PB-MNCs by fluorescent microscopy, examined motor function of rats by rotarod and cylinder tests, measured the lesion volume using image-pro plus software, and analyzed the apoptosis of neural cells in HI rats by tunnel assay. The results showed PB-MNCs could survive in the brain of hosts, migrate to the damage area and express neural marker. In addition, The HI rats that received PB-MNCs showed a reduction in motor function impairment, lesion volume and neural cell apoptosis. To better understand the mechanism of cell migration, PB-MNCs were also injected into normal rats via tail-vein. The expression of stromal cell-derived factor-1 (SDF-1) in the brain of normal and HI rats was measured by RT- PCR and western-blot, while the response of PB-MNCs in vitro to HI or normal brain extracts were measured by cell migration assay. Collectively these data suggest that the migration of PB-MNCs is directed to the damaged brain through an SDF-1-dependent pathway. Our results suggest that intravenous transplantation of PB-MNCs may be a feasible candidate for HI therapy.

Hesperidin pretreatment protects hypoxia-ischemic brain injury in neonatal rat.

Neonatal hypoxia-ischemic encephalopathy (HIE) remains a major cause of brain damage, leading to high disability and mortality rates in neonates. In vitro studies have shown that hesperidin, a flavanone glycoside found abundantly in citrus fruits, acts as an antioxidant. Although hesperidin has been considered as a potential treatment for HIE, its effects have not been fully evaluated. In this study, the protective effect of hesperidin pretreatment against hypoxia-ischemic (HI) brain injury and possible signal pathways were investigated using in vivo and in vitro models. In vivo HI model employed unilateral carotid ligation in postnatal day 7 rat with exposure to 8% hypoxia for 2.5h, whereas in vitro model employed primary cortical neurons of neonatal rats subjected to oxygen and glucose deprivation for 2.5h. Hesperidin pretreatment significantly reduced HI-induced brain tissue loss and improved neurological outcomes as shown in 2,3,5-triphenyltetrazolium chloride monohydrate staining and foot-fault results. The neuroprotective effects of hesperidin are likely the results of preventing an increase in intracellular reactive oxygen species and lipid peroxide levels. Hesperidin treatment also activated a key survival signaling kinase, Akt, and suppressed the P-FoxO3 level. Hesperidin pretreatment protected neonatal HIE by reducing free radicals and activating phosphorylated Akt.

Apelin-36, a potent peptide, protects against ischemic brain injury by activating the PI3K/Akt pathway.

Apelin is an endogenous ligand of G protein-coupled receptor-apelin and angiotensin-1-like receptor (APJ). The biological effects of apelin-APJ system are reported in multiple systems including cardiovascular, endocrinal, and gastrointestinal system. Previous studies had shown that apelin-13 is a potential protective agent on cardiac ischemia; however, the role of apelin in the central nervous system remained unknown. In this study, we investigated therapeutic effects of apelin-36, a long form of apelin, in ischemic brain injury models. We found that apelin-36 reduced cerebral infarct volume in the middle cerebral artery occlusion (MCAO) model and the neonatal hypoxic/ischemic (H/I) injury model. Apelin-36 improved neurological deficits in the MCAO model and promoted long-term functional recovery after H/I brain injury. We further explored the protective mechanisms of apelin-36 on H/I brain injury. We clearly demonstrated that apelin-36 significantly reduced the levels of cleaved caspase-3 and Bax, two well-established apoptotic markers after H/I injury, indicating the anti-apoptotic activity of apelin-36 in ischemic injury. Since apelin-36 increased the level of phosphorylated Akt after H/I injury, we treated neonates with a specific PI3K inhibitor LY294002. We found that LY294002 decreased the phosphorylated Akt level and attenuated protective effects of apelin-36 on apoptosis. These suggested that the PI3K/Akt pathway was at least in part involved in the anti-apoptotic mechanisms of apelin-36. Our findings demonstrated that apelin-36 was a promising therapeutic agent on the treatment of ischemic brain injury.