Hypoxic-ischemic Brain Injury In Neonatal Rats Research Articles

TMEM175 downregulation participates in impairment of the autophagy related lysosomal dynamics following neonatal hypoxic-ischemic brain injury.

The mechanism underlying long-term cognitive impairment caused by neonatal hypoxic-ischemic brain injury (HIBI) remains unclear. Autophagy is a closely related mechanism and may play a role in this process. We aimed to investigate the role of lysosomal transmembrane protein 175 (TMEM175) in the autophagy-lysosome pathway in neonatal rats with HIBI. A neonatal rat model of HIBI was established, hypoxia was induced, followed by left common carotid artery ligation. Expression levels of TMEM175 and the corresponding proteins involved in autophagy flux and the endolysosomal system fusion process were measured. Rats were administered TMEM175 plasmid via intracerebroventricular injection to induce overexpression. Brain damage and cognitive function were then assessed. TMEM175 was downregulated in the hippocampal tissue, and the autophagy-lysosome pathway was impaired following HIBI in neonatal rats. Overexpression of TMEM175 significantly mitigated neuronal injury and improved long-term cognitive and memory function in neonatal rats with HIBI. We found that improvement in the autophagy-lysosome pathway and endolysosomal system homeostasis, which are TMEM175 related, occurred via regulation of lysosomal membrane dynamic fusion. TMEM175 plays a critical role in maintaining the autophagy-lysosome pathway and endolysosomal homeostasis, contributing to the amelioration of neuronal injury and impaired long-term cognitive function following neonatal HIBI.

Sevoflurane post-conditioning alleviated hypoxic-ischemic brain injury in neonatal rats by inhibiting endoplasmic reticulum stress-mediated autophagy via IRE1 signalings

Post-conditioning with sevoflurane, a volatile anesthetic, has been proved to be neuroprotective against hypoxic-ischemic brain injury (HIBI). Our previous research showed that autophagy is over-activated in a neonatal HIBI rat model, and inhibition of autophagy confers neuroprotection. There is increasing recognition that autophagy can be stimulated by activating endoplasmic reticulum (ER) stress. Herein, we purposed to explore: i) the association of ER stress with autophagy in the setting of neonatal HIBI; and ii) the possible roles of ER stress-triggered autophagy, as well as IRE1 signaling in the neuroprotection of sevoflurane post-conditioning against neonatal HIBI. Seven-day-old rats underwent ligation of the left common artery, and a subsequent 2 h hypoxia (8% O2/92% N2). The association of ER stress with autophagy was examined by ER stress inducer (tunicamycin), 4-PBA (ER stress inhibitor), or 3-MA (autophagy inhibitor). Rats in the sevoflurane post-conditioning groups were treated with 2.4% sevoflurane for 30 min after HIBI stimulation. The roles of ER stress-mediated autophagy, as well as the IRE1-JNK-beclin1 signaling cascade in the neuroprotection afforded by sevoflurane were explored by ER stress inducer (tunicamycin) and the IRE1 inhibitor (STF-083010). HIBI over-activated ER stress and autophagy in neonatal rats. HIBI-induced autophagy was significantly aggravated by tunicamycin but blocked by 4-PBA; however, HIBI-induced ER stress was not affected by 3-MA. Sevoflurane post-conditioning significantly alleviated ER stress, autophagy, cell apoptosis, and cognitive impairments, which were remarkably abolished by tunicamycin. Also, tunicamycin blocked sevoflurane-induced downregulation of IRE1-JNK-beclin1 signaling pathway. Whereas, IRE1 inhibitor could reverse the effects of tunicamycin. ER stress contributes to autophagy induced by HIBI. Furthermore, sevoflurane post-conditioning significantly protects against HIBI in neonatal rats by inhibiting ER stress-mediated autophagy via IRE1-JNK-beclin1 signaling cascade.

Post-Treatment Sevoflurane Protects Against Hypoxic-Ischemic Brain Injury in Neonatal Rats by Downregulating Histone Methyltransferase G9a and Upregulating Nuclear Factor Erythroid 2-Related Factor 2 (NRF2).

BackgroundPerinatal hypoxia and subsequent reduction of cerebral blood flow leads to neonatal hypoxic-ischemic brain injury (HIBI), resulting in severe disability and even death. Preconditioning or post-conditioning with sevoflurane protects against cerebral injury. This study investigated the mechanism of sevoflurane in HIBI.Material/MethodsThe HIBI model of neonatal rats was established and the model rats were post-treated with sevoflurane. The oxygen-glucose deprivation (OGD) cell model was established, and the OGD cells were transfected with NRF2-siRNA plasmid and post-treated with sevoflurane. The Morris water maze test was used to detect the motor activity, spatial learning, and memory ability of HIBI rats. Histological stainings were performed to observe the area of cerebral infarction, record the number of neurons in the hippocampus, and assess neuron apoptosis. The levels of inflammatory factors were detected by ELISA. The protein levels of histone methyltransferase G9a and histone H3 lysine 9 (H3K9me2) were detected by western blot assay. The apoptosis was detected by flow cytometry.ResultsSevoflurane post-treatment significantly shortened the escape latency of HIBI neonatal rats, increased the density of neurons, reduced the area of cerebral infarction, and decreased the levels of inflammatory factors and neuronal apoptosis. Sevoflurane post-treatment decreased G9a and H3K9me2 levels, and G9a level was negatively correlated with NRF2 level. NRF2 silencing reversed the alleviation of sevoflurane post-treatment on OGD-induced cell injury.ConclusionsSevoflurane post-treatment promotes NRF2 expression by inhibiting G9a and H3K9me2, thus alleviating HIBI in neonatal rats.

Sevoflurane Post-Conditioning Ameliorates Neuronal Deficits and Axon Demyelination After Neonatal Hypoxic Ischemic Brain Injury: Role of Microglia/Macrophage.

Microglia/macrophages have been identified to be highly polarized after ischemia. Interestingly, the polarization of these microglia/macrophages varies immensely under differing disease conditions. Post-conditioning using sevoflurane, a volatile anesthetic, could provide long-term neuroprotection to neonatal rats after hypoxic-ischemic brain injury (HIBI). Thus, the current study aimed at investigating the effects of sevoflurane post-conditioning (SPC) on microglia/macrophage polarization after HIBI induction in neonatal rats. Additionally, we aimed at identifying the underpinning mechanisms specifically related to autophagy and lysosomal protease enzyme, cathepsin B. To develop a HIBI model, 7-day-old Sprague-Dawley rats underwent left common carotid artery ligation followed by 2h of hypoxia. The role of microglia/macrophages in the neuroprotection conferred by SPC was examined by left-side intra-cerebroventricular injection with adenovirus vector carrying catB-GFP or rapamycin. The number of interleukin (IL)-1β+ cells, cathepsin B+ cells, light chain 3B positive (LC3B+) cells among ionized calcium binding adaptor molecule 1(Iba1+)cells to investigate microglia polarization, neuronal apoptosis to assess neuronal death in the acute phase were tested at 24h after HIBI. Behavioral tests including suspension test, Morris water maze tests were performed to investigate the long-term effects of SPC, at 21 to 34days post HIBI. Nissl staining and myelin basic protein (MBP) immunostaining to assess the long-term neuronal and myelin damage were performed at 34days after HIBI. Based on the obtained results post HIBI, we observed the cells that were positive for IL-1β, cathepsin B, and LC3B among Iba1 positive cell population in the hippocampus were significantly decreased after SPC treatment. SPC significantly attenuated the HIBI-induced increase in neuronal apoptosis, improved long-term cognitive function, and attenuated HI-induced decrease of Nissl-positive cells and MBP expression. However, these trends were reversed by injection of adenovirus vector carrying catB-GFP and rapamycin. SPC attenuated microglia polarization towards neurotoxic phenotypes, alleviates neuronal death and axon demyelination after HIBI in neonatal rats by regulating microglia autophagy and cathepsin B expression, and therefore provided long-term cognitive, learning and memory protection.

Sevoflurane post-conditioning alleviates neonatal rat hypoxic-ischemic cerebral injury via Ezh2-regulated autophagy.

Background: When neonatal rats suffer hypoxic-ischemic brain injury (HIBI), autophagy is over-activated in the hippocampus, and inhibition of autophagy provides neuroprotection. The aim of this study was to investigate the possible roles of autophagy and Ezh2-regulated Pten/Akt/mTOR pathway in sevoflurane post-conditioning (SPC)-mediated neuroprotection against HIBI in neonatal rats.Methods: Seven-day-old Sprague–Dawley rats underwent left common artery ligation followed by 2 h hypoxia as described in the Rice–Vannucci model. The roles of autophagy and the Ezh2-regulated Pten/Akt/mTOR signaling pathway in the neuroprotection conferred by SPC were examined by left-side intracerebroventricular injection with the autophagy activator rapamycin and the Ezh2 inhibitor GSK126.Results: SPC was neuroprotective against HIBI through the inhibition of over-activated autophagy in the hippocampus as characterized by the rapamycin-induced reversal of neuronal density, neuronal morphology, cerebral morphology, and the expression of the autophagy markers, LC3B-II and Beclin1. SPC significantly increased the expression of Ezh2, H3K27me3, pAkt, and mTOR and decreased the expression of Pten induced by HI. The Ezh2 inhibitor, GSK126, significantly reversed the SPC-induced changes in expression of H3K27me3, Pten, pAkt, mTOR, LC3B-II, and Beclin1. Ezh2 inhibition also reversed SPC-mediated attenuation of neuronal loss and behavioral improvement in the Morris water maze.Conclusion: These results indicate that SPC inhibits excessive autophagy via the regulation of Pten/Akt/mTOR signaling by Ezh2 to confer neuroprotection against HIBI in neonatal rats.

Autophagy activation involved in hypoxic-ischemic brain injury induces cognitive and memory impairment in neonatal rats.

Hypoxic-ischemic brain injury (HIBI) in neonates can lead to lifelong cognitive and memory impairment, but protective strategies are lacking at present. It has been demonstrated that autophagy plays a critical role in HIBI, while the function of autophagy in cognitive and memory impairment induced by HIBI in neonates has not been tested. In this study, we tested the impact of autophagy on the impairment of cognitive function and memory in HIBI neonatal rats by using a Morris water maze and investigated its possible mechanisms, which were established as HIBI model by ligating the left common carotid artery in neonatal rats, followed by 2-h hypoxia. The expression of microtubule-associated protein 1 light chain 3 (LC3)-II increased in HI group 24h after HI in neonatal rats, while Sequestosome 1 (P62/SQSTM1), phosphorylated cAMP-response element-binding protein (p-CREB) decreased (compared with the sham group, p<0.05), which were shown in the same left hippocampus CA3 region by immunofluorescence analysis. Brain injury of neonatal rats was aggravated significantly at 7day after HI, coinciding with the results of Morris water maze. An autophagy inhibitor, 3-methyladenine (3-MA) pretreatment significantly attenuated the increase of LC3II and the loss of P62/SQSTM1 and p-CREB, ameliorated neuronal death, and improved the results of Morris water maze. Our results demonstrate that HIBI in neonatal rats induced excessive autophagy flux, which aggravated brain injury and induced cognitive and memory impairment during adolescence. Inhibition of autophagy reversed the results partly and improved the function of spatial learning and memory by attenuating the reduction of p-CREB. The use of autophagy modulators in the immature brain would create new opportunities for protective strategies clinically in the future.

Neuroprotective effect of memantine combined with topiramate in hypoxic–ischemic brain injury

Glutamate receptor-mediated neurotoxicity is a major mechanism contributing to hypoxic–ischemic brain injury (HIBI). Memantine is a safe non-competitive NMDA receptor blocker characterized by its low affinity and fast unblocking kinetics. Topiramate is an AMPA/KA receptor blocker and use-dependent sodium channel blocker with several other neuroprotective actions and little neurotoxicity. We hypothesized that the coadministration of memantine and topiramate would be highly effective to attenuate HIBI in neonatal rats. Seven-day-old Sprague–Dawley rat pups were subjected to right common carotid artery ligation and hypoxia for 2 h, and then were randomly and blindly assigned to one of four groups: vehicle, memantine, topiramate and combination group. Brain injury was evaluated by gross damage and weight deficit of the right hemisphere at 22d after hypoxic-ischemia (HI) and by neurofunctional assessment (foot-fault test) at 21d post-HI. Acute neuronal injury was also evaluated by microscopic damage grading at 72 h post-HI. Results showed the combination of memantine and topiramate improved both pathological outcome and performance significantly. The drug-induced apoptotic neurodegeneration was assessed by TUNEL staining at 48 h post-HI and the result showed no elevated apoptosis in all observed areas. The result of the experiment indicates the combination therapy is safe and highly effective to reduce brain damage after HIBI.