Brain Damage In Neonatal Rats Research Articles

GSK-3β inhibitor TWS119 promotes neuronal differentiation after hypoxic-ischemic brain damage in neonatal rats.

Brain injury in preterm infants is a major cause of disability and mortality in children. GSK-3β is a common pathogenic factor for cognitive dysfunction and involves in neuronal proliferation and differentiation. However, GSK-3β affected neuronal differentiation and its molecular pathogenesis after hypoxic-ischemic brain damage in neonatal rats remains unclear. This study investigated the effects of GSK-3β inhibitor (TWS119) on cell cycle regulatory proteins, a neuronal differentiation factor (CEND1), maturation neurons, T-box brain transcription factor 1 (TBR1)-positive neurons to clarify the mechanisms of hypoxic-ischemic brain damage in neonatal rats. We used hypoxic-ischemic Sprague-Dawley neonatal rats with brain damage as models. These rats were used for investigating the effect of GSK-3β on cell cycle regulatory proteins, neuronal differentiation factor (CEND1), maturation neurons, TBR1-positive neurons by western blot and immunofluorescence. Cyclin D1 (a positive cell cycle regulator) expression decreased, and p21 (a negative cell cycle regulator) expression increased in the TWS119 group compared to the hypoxia-ischemia (HI) group 7 days after HI. Additionally, compared to the HI group, TWS119 treatment up-regulated CEND1 expression and promoted neuronal differentiation and cortex development based on NeuN and TBR1 expression. Our study suggests that the GSK-3β inhibitor TWS119 promotes neuronal differentiation after hypoxic-ischemic brain damage in neonatal rats by inhibiting cell cycle pathway.

Intranasal insulin attenuates hypoxia-ischemia-induced short-term sensorimotor behavioral disturbances, neuronal apoptosis, and brain damage in neonatal rats

There is a significant need for additional therapy to improve outcomes for newborns with acute Hypoxic-ischemic (HI) encephalopathy (HIE). New evidence suggests that insulin could be neuroprotective. This study aimed to investigate whether intranasal insulin attenuates HI-induced brain damage and neurobehavioral dysfunction in neonatal rats. Postnatal day 10 (P10), Sprague-Dawley rat pups were randomly divided into Sham + Vehicle, Sham + Insulin, HI + Vehicle, and HI + Insulin groups with equal male-to-female ratios. Pups either had HI by permanent ligation of the right common carotid artery followed by 90 min of hypoxia (8% O2) or sham surgery followed by room air exposure. Immediately after HI or Sham, pups were given fluorescence-tagged insulin (Alex-546-insulin)/vehicle, human insulin (25 μg), or vehicle in each nare under anesthesia. Shortly after administration, widespread Alex-546-insulin-binding cells were detected in the brain, primarily co-localized with neuronal nuclei-positive neurons on double-immunostaining. In the hippocampus, phospho-Akt was activated in a subset of Alex-546-insulin double-labeled cells, suggesting activation of the Akt/PI3K pathway in these neurons. Intranasal insulin (InInsulin) reduced HI-induced sensorimotor behavioral disturbances at P11. InInsulin prevented HI-induced increased Fluoro-Jade C+ degenerated neurons, cleaved caspase 3+ neurons, and volume loss in the ipsilateral brain at P11. There was no sex-specific response to HI or insulin. The findings confirm that intranasal insulin provides neuroprotection against HI brain injury in P10 rats associated with activation of intracellular cell survival signaling. If further pre-clinical research shows long-term benefits, intranasal insulin has the potential to be a promising non-invasive therapy to improve outcomes for newborns with HIE.

Mechanisms of ferroptosis in hypoxic-ischemic brain damage in neonatal rats

This study was to explore the mechanism of ferroptosis and hypoxic-ischemic brain damage in neonatal rats. The neonatal rat hypoxic-ischemic brain damage (HIBD) model was established using the Rice-Vannucci method and treated with the ferroptosis inhibitor liproxstatin-1. Cognitive assessment was performed through absentee field experiments to confirm the successful establishment of the model. Brain tissue damage was evaluated by comparing regional cerebral blood flow and quantifying tissue staining. Neuronal cell morphological changes in the rats' cortical and hippocampal regions were observed using HE and Nissl staining. ELISA was performed to determine GPX4, GSH and ROS expression levels in the rats' brain tissues, and Western blotting to assess the expression levels of 4-HNE, GPX4, GSS, ACSL4, SLC7A11, SLC3A2, TFRC, FHC, FLC, HIF-1α, and Nrf2 proteins in rat brain tissues. Compared to the Sham group, the HIBD group exhibited a significant decrease in cerebral blood perfusion, reduced brain nerve cells, and disordered cell arrangement. The use of the ferroptosis inhibitor effectively improved brain tissue damage and preserved the shape and structure of nerve cells. The oxidative stress products ROS and 4-HNE in the brain tissue of the HIBD group increased significantly, while the expression of antioxidant indicators GPX4, GSH, SLC7A11, and GSS decreased significantly. Furthermore, the expression of iron metabolism-related proteins TFRC, FHC, and FLC increased significantly, whereas the expression of the ferroptosis-related transcription factors HIF-1α and Nrf2 decreased significantly. Treatment with liproxstatin-1 exhibited therapeutic effects on HIBD and downregulated tissue ferroptosis levels. This study shows the involvement of ferroptosis in hypoxic-ischemic brain damage in neonatal rats through the System Xc−-GSH-GPX4 functional axis and iron metabolism pathway, with the HIF-1α and Nrf2 transcription factors identified as the regulators of ferroptosis involved in the HIBD process in neonatal rats.

Ferrostatin-1 attenuates hypoxic-ischemic brain damage in neonatal rats by inhibiting ferroptosis.

Hypoxic-ischemic brain damage (HIBD) is a type of brain damage that is caused by perinatal asphyxia and serious damages the central nervous system. At present, there is no effective drug for the treatment of this disease. Besides, the pathogenesis of HIBD remains elusive. While studies have shown that ferroptosis plays an important role in HIBD, its role and mechanism in HIBD are yet to be fully understood. The HIBD model of neonatal rats was established using the Rice-Vannucci method. A complete medium of PC12 cells was adjusted to a low-sugar medium, and the oxygen-glucose deprivation model was established after continuous hypoxia for 12 h. Laser Doppler blood flow imaging was used to detect the blood flow intensity after modeling. 2,3,5-triphenyl tetrazolium chloride staining was employed to detect ischemic cerebral infarction in rat brain tissue, and hematoxylin and eosin staining and transmission electron microscopy were used to observe brain injury and mitochondrial damage. Immunofluorescence was applied to monitor the expression of GFAP. Real-time quantitative polymerase chain reaction, western blot, and immunofluorescence were utilized to detect the expression of messenger RNA and protein. The level of reactive oxygen species (ROS) in cells was detected using the ROS detection kit. The results showed that ferrostatin-1 (Fer-1) significantly alleviated the brain injury caused by hypoxia and ischemia. Fer-1 significantly increased the expression of SLC3A2, SLC7A11, ACSL3, GSS, and GPX4 (P<0.05) and dramatically decreased the expressions of GFAP, ACSL4, TFRC, FHC, FLC, 4-HNE, HIF-1α, and ROS (P<0.05). Fer-1 inhibits ferroptosis and alleviates HIBD by potentially targeting the GPX4/ACSL3/ACSL4 axis; however, its specific mechanism warrants further exploration.

Dexmedetomidine suppresses hippocampal astrocyte pyroptosis in cerebral hypoxic-ischemic neonatal rats by upregulating microRNA-148a-3p to inactivate the STAT/JMJD3 axis.

Dexmedetomidine (DEX), a selective α2-adrenoceptor agonist, is an anesthetic and sedative agent and has been reported to confer neuroprotective effects after cerebral hypoxic ischemia (CHI). This study was undertaken to elucidate the mechanisms by which microRNA (miR)-148a-3p is involved in the neuroprotective effect of DEX on hypoxic-ischemic brain damage in neonatal rats. Neonatal rats were exposed to CHI conditions, a miR-148a-3p inhibitor, and DEX. Hippocampal astrocytes were isolated to construct an oxygen-glucose deprivation (OGD) model. qRT-PCR and western blot were utilized to inspect miR-148a-3p, STAT1, STAT3, JMJD3, cleaved-Caspase-1, ASC, NLRP3, GSDMD, and GSDMD-N expression in rats and astrocytes. TUNEL staining was employed to measure astrocyte apoptosis rate, immunofluorescence to inspect cleaved-Caspase-1 and ASC levels, and ELISA to determine IL-1β and IL-18 expression. The target genes of miR-148a-3p were predicted using online software and verified by a dual-luciferase reporter gene assay. A prominent increase in astrocyte apoptosis rate and the expression of pyroptosis- and inflammation-related factors were found in rats with CHI and OGD-treated astrocytes. DEX suppressed astrocyte apoptosis rate and decreased expression of pyroptosis- and inflammation-related factors. Knockdown of miR-148a-3p facilitated astrocyte pyroptosis, indicating that DEX exerted its protective effect by upregulating miR-148a-3p. miR-148a-3p negatively mediated STAT to inactivate JMJD3. Overexpression of STAT1 and STAT3 facilitated pyroptosis in astrocytes, which was negated by the overexpression of miR-148a-3p. DEX inhibited hippocampal astrocyte pyroptosis by upregulating miR-148a-3p to inactivate the STAT/JMJD3 axis, thereby alleviating cerebral damage in neonatal rats with CHI.

Ginsenoside Rb1 inhibits ferroptosis to ameliorate hypoxic-ischemic brain damage in neonatal rats.

Hypoxic ischemic encephalopathy (HIE) is among the leading causes of neonatal mortality, and currently there is no effective treatment. Ginsenoside Rb1 (GsRb1) is one of the principal active components of ginseng, and has protective benefits against oxidative stress, inflammation, hypoxic injury, and so on. However, the role and underlying mechanism of GsRb1 on HIE are unclear. Here, we established the neonatal rat hypoxic-ischemic brain damage (HIBD) model in vivo and the PC12 cell oxygen-glucose deprivation (OGD) model in vitro to investigate the neuroprotective effects of GsRb1 on HIE, and illuminate the potential mechanism. Our results showed that GsRb1 and the ferroptosis inhibitor liproxstatin-1 (Lip-1) could significantly restore System Xc activity and antioxidant levels as well as inhibit lipid oxidation levels and inflammatory index levels of HIBD and OGD models. Taken together, GsRb1 might inhibit ferroptosis to exert neuroprotective effects on HIE through alleviating oxidative stress and inflammation, which will set the foundation for future research on ferroptosis by reducing hypoxic-ischemic brain injury and suggest that GsRb1 might be a promising therapeutic agent for HIE.

Neuroprotective effects of oxymatrine on hypoxic–ischemic brain damage in neonatal rats by activating the Wnt/β-catenin pathway

Neuronal apoptosis is a major pathological process associated with neurological dysfunction in neonates after hypoxic–ischemic brain damage (HIBD). Our previous study demonstrated that oxymatrine (OMT) exerts potential neuroprotective effects on neonatal rats subjected to hypoxic–ischemic insult. However, the underlying molecular mechanism remains unclear. In this study, we investigated the effects of OMT-mediated neuroprotection on neonatal HIBD by attempting to determine its effect on the Wnt/β-catenin signaling pathway and explored the underlying mechanism. Both 7-day-old rat pups and primary hippocampus neurons were used to establish the HIBD and oxygen-glucose deprivation (OGD) injury models, respectively. Our results demonstrated that OMT treatment significantly increased cerebral blood flow and reduced S100B concentration, infarct volume, and neuronal apoptosis in neonatal rats. In vitro, OMT markedly increased cell viability and MMP level and decreased DNA damage. Moreover, OMT improved the mRNA and protein levels of Wnt1 and β-catenin, inhibited the expression of DKK1 and GSK-3β, enhanced the nuclear transfer of β-catenin, and promoted the binding activity of β-catenin with Tcf-4; however, it downregulated the expression of cleaved caspase-3 and cleaved caspase-9. Notably, the introduction of XAV-939 (a Wnt/β-catenin signaling inhibitor) reversed the positive effects of OMT both in vivo and in vitro. Collectively, our findings demonstrated that OMT exerted a neuroprotective effect on neonatal HIBD by inhibiting neuronal apoptosis, which was partly via the activation of the Wnt/β-catenin signaling pathway.

Dihydroartemisinin attenuates hypoxic-ischemic brain damage in neonatal rats by inhibiting oxidative stress

Neonatal hypoxic-ischemic encephalopathy (HIE) induced by perinatal asphyxia is a major cause of neurological disability among infants. Dihydroartemisinin (DHA), derived from artemisinin, well known as an anti-malarial medicine, was proved to be able to inhibit oxidative stress and inflammation. However, whether those functions of DHA play roles in hypoxic-ischemic brain damage (HIBD), an animal model of HIE in patient which also been observed to have oxidative stress and inflammation, is unknown. In this study, we demonstrated that the DHA treatment on newborn rats significantly relieved the neuron loss and motor and cognitive impairment caused by HIBD. One of the underlying mechanisms is that DHA enhanced the anti-oxidant capacity of HIBD rats by up-regulating the total antioxidant capacity (T-AOC), gluathione reductase (GR) and catalase (CAT) while down regulating the pro-oxidative substances including hydrogen peroxide (H2O2), total nitric oxide synthase (T-NOS) and inducible nitric oxide synthase (iNOS). Thus, our study illustrated that DHA could alleviate the damage of brains and improve the cognitive and motor function of HIBD rats by inhibiting oxidative stress, provided an opportunity to interrogate potential therapeutics for affected HIE patients.

Ferroptosis is Involved in Hypoxic-ischemic Brain Damage in Neonatal Rats

Ferroptosis is an iron-dependent form of regulated cell death, which is driven by loss of activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and subsequent accumulation of lipid peroxidation. Ferroptosis is implicated in various diseases involving neuronal injury. However, the role of ferroptosis in hypoxic-ischemic brain damage (HIBD) has not been elucidated. The objectives of this study were to evaluate whether ferroptosis is involved in hypoxic-ischemic brain damage and its mechanisms through the HIBD model. A 7-day-old male Sprague-Dawley neonatal rat HIBD model was established by blocking the left common carotid artery. Laser speckle contrast imaging, immunohistochemical staining, transmission electron microscopy were used to measure the effects of ferroptosis on HIBD. Brain tissue on the damaged side in the HIBD group showed atrophied, even liquefied, glial cells increased, and blood perfusion was significantly reduced. The HIBD group insult significantly increased reactive oxygen species levels, as well as the protein levels of iron metabolism-related proteins transferrin receptor (TFRC), ferritin heavy chain (FHC), and ferritin light chain (FLC), while reducing the levels of Solute Carrier Family 7 Member 11 (SLC7A11), glutathione (GSH), and GPX4. These changes resulted in diminished cellular antioxidant capacity and mitochondrial damage, causing neuronal ferroptosis in the cerebral cortex. We conclude that ferroptosis plays a role in HIBD in neonatal rats. Ferroptosis-related mechanisms such as abnormalities in iron metabolism, amino acid metabolism, and lipid peroxidation regulation play important roles in HIBD.

F-box and WD repeat domain-containing 7 (FBXW7) mediates the hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway to affect hypoxic-ischemic brain damage in neonatal rats

ABSTRACT The aim of this study was to determine whether F-box and WD repeat domain-containing 7 (FBXW7) can mediate the hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway to affect neonatal hypoxic-ischemic brain damage (HIBD) in neonatal rats. HIBD rats were treated with LV-shFBXW7. Cerebral infarct size was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining, while microvessel density (MVD) was evaluated by immunohistochemistry. Learning and memory were tested using the Morris water maze (MWM) test. FBXW7 and HIF-1α/VEGF signaling pathway proteins were measured by Western blotting. Brain microvascular endothelial cells (BMECs) were isolated to establish an oxygen-glucose deprivation (OGD) model to evaluate treatment with FBXW7 siRNA. Cell viability was detected using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, while cell migration was evaluated using a wound healing assay. The tube formation of BMECs was also assessed. The results demonstrated that HIBD rats exhibited increased protein expression of FBXW7, HIF-1α, and VEGF. HIBD rats also displayed increased cerebral infarct size, prolonged escape latency and a decreased number of platform crossings. However, HIBD rats treated with LV-shFBXW7 exhibited reversal of these changes. In vitro experiments showed that BMECs in the OGD group had significantly decreased cell viability, shorter vascular lumen length, and shorter migration distance than cells in the control group. Moreover, silencing FBXW7 promoted proliferation, tube formation and migration of BMECs. Taken together, silencing FBXW7 upregulates the HIF-1α/VEGF signaling pathway to promote the angiogenesis of neonatal HIBD rats after brain injury, reducing infarct volume and improving recovery of nerve function in HIBD rats.

GSK-3β inhibitor TWS119 alleviates hypoxic-ischemic brain damage via a crosstalk with Wnt and Notch signaling pathways in neonatal rats

Preterm infant brain injury is a leading cause of morbidity and disability in survivors of preterm infants. Unfortunately, the effective treatment remains absent. Recent evidence suggests that GSK-3β inhibitor TWS119 has a neuroprotectiverole in adult brain injury by activation of Wnt/β-catenin signaling pathway. However, the role on neonatal brain injury is not yet explored. The study aims to evaluate the effect of TWS119 at 7 d after hypoxic-ischemic brain damage and investigate the mechanism that it regulates Wnt and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats. Three-day-old rats were randomly divided into 3 groups: sham group, HI group and TWS119 group. The neonatal rats were subjected to left carotid artery ligation followed by 2 h of hypoxia (8.0% O2). A single dose of TWS119 (30 mg/kg) was intraperitoneally injected 20 min prior to hypoxia-ischemia (HI). At 7 d after HI, TWS119 improved the tissue structure, reduced cell apoptosis, up-regulated bcl-2 expression, up-regulated the expression of PSD-95 and Synapsin-1. At 24 h after HI, it activated Wnt/β-catenin signaling pathway by up-regulation of β-catenin protein expression and wnt3a/wnt5a/wnt7a mRNA expression. Simultaneously, it suppressed Notch signaling pathway by down-regulation of Notch1 and HES-1 proteins expression. Our study suggested that TWS119 performed a neuroprotective function at 7 d after hypoxic-ischemic brain damage via a crosstalk with Wnt/β-catenin and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats.

Neferine Protects against Hypoxic-Ischemic Brain Damage in Neonatal Rats by Suppressing NLRP3-Mediated Inflammasome Activation.

Hypoxic-ischemic encephalopathy (HIE) is recognized as the main cause of neonatal death, and efficient treatment strategies remain limited. Given the prevalence of HIE and the associated fatality, further studies on its pathogenesis are warranted. Oxidative stress and neuroinflammatory injury are two important factors leading to brain tissue injury and nerve cell loss in HIE. Neferine, an alkaloid extracted from lotus seed embryo, exerts considerable effects against several diseases such as cancers and myocardial injury. In this study, we demonstrated the neuroprotective effect of neferine on HIE and hypothesized that it involves the inhibition of neuronal pyroptosis, thereby ameliorating neurological inflammation and oxidative stress. We demonstrated that the mRNA levels of proteins associated with pyroptosis including caspase-1, the caspase adaptor ASC, gasdermin D, interleukin- (IL-) 18, IL-1β, and some inflammatory factors were significantly increased in neonatal HIBD model rats compared to those in the control group. The increase in these factors was significantly suppressed by treatment with neferine. We stimulated PC12 cells with CoCl2 to induce neuronal HIBD in vitro and investigated the relationship between neferine and pyroptosis by altering the expression of the NLRP3 inflammasome. The overexpression of NLRP3 partially reversed the neuroprotective effect of neferine on HIBD, whereas NLRP3 knockdown further inhibited caspase-1 activation and IL-1β and IL18 expression. In addition, simultaneous alteration of NLRP3 expression induced changes in intracellular oxidative stress levels after HIBD. These findings indicate that neferine ameliorates neuroinflammation and oxidative stress injury by inhibiting pyroptosis after HIBD. Our study provides valuable information for future studies on neferine with respect to neuroinflammation and pyroptosis.

LXA4 protects against hypoxic-ischemic damage in neonatal rats by reducing the inflammatory response via the IκB/NF-κB pathway

Hypoxia and the resultant decreases in cerebral blood flow in the perinatal period can lead to neonatal hypoxic-ischemic (HI) brain injury, which can, in turn, cause severe disability or even death. However, the efficacy of current treatment strategies remains limited. Several studies have demonstrated that lipoxin A4 (LXA4), as one of the earliest types of endogenous lipid mediators, can inhibit the accumulation of neutrophils, arrest inflammation, and promote the resolution of inflammation. However, research on LXA4 in the nervous system has rarely been carried out. In the present study, we sought to investigate the protective effect of LXA4 on HI brain damage in neonatal rats, as well as the underlying mechanisms. Through experiments conducted using an HI animal model, we found that the LXA4 intervention promoted the recovery of neuronal function and tissue structure following brain injury while maintaining the integrity of the blood–brain barrier in addition to reducing cerebral edema, infarct volume, and inflammatory responses. Our results suggest that LXA4 interfered with neuronal oxygen-glucose deprivation insults, reduced the expression of inflammatory factors, inhibited apoptosis, and promoted neuronal survival in vitro. Finally, the LXA4 intervention attenuated HI-induced activation of inhibitor kappa B (IκB) and degradation of nuclear factor-κB (NF-κB). In conclusion, our data suggest that LXA4 exerts a neuroprotective effect against neonatal HI brain damage through the IκB/NF-κB pathway. Our findings will help inform future studies regarding the effects of LXA4 on neuroinflammation, blood–brain barrier integrity, and neuronal apoptosis.

Vof-16 knockout improves the recovery from hypoxic-ischemic brain damage of neonatal rats

Hypoxic-ischemic encephalopathy (HIE) results in high neonatal mortality and severe neurological impairments, and its underlying molecular mechanism underwent extensive investigations. Long non-coding RNA (lncRNA) is considered to be an important regulator on brain development and many neurological diseases. Currently, little is known about the role of Vof-16 (lncRNA) in HIE. We detected the relative expression level of Vof-16 in the cortex and hippocampus of hypoxic-ischemic (HI) models whose successful establishment was verified by TTC staining. Then, Vof-16 knockout rats were generated using the CRISPR/Cas engineering technology to search the specific function of the Vof-16 through a series of behavioral evaluations including Neurological severity scores (NSS), Y-maze test, Morris water maze (MWW) test, open field test, and Rotarod test. The results demonstrated the expression of Vof-16 was substantially up-regulated in the cortex and hippocampus of rats with HI injury. Importantly, Vof-16 knockout facilitated the recovery from long-term HI induced nerve damage and neurobehavioral dysfunctions. In conclusion, this study suggests Vof-16 knockout is a promising treatment target for neonatal HIE.

MicroRNA-9 mediated the protective effect of ferulic acid on hypoxic-ischemic brain damage in neonatal rats.

Neonatal hypoxic-ischemic brain damage (HIBD) is prone to cognitive and memory impairments, and there is no effective clinical treatment until now. Ferulic acid (FA) is found within members of the genus Angelica, reportedly shows protective effects on neuronal damage. However, the protective effects of FA on HIBD remains unclear. In this study, using the Morris water maze task, we herein found that the impairment of spatial memory formation in adult rats exposed to HIBD was significantly reversed by FA treatment and the administration of LNA-miR-9. The expression of miRNA-9 was detected by RT-PCR analyses, and the results shown that miRNA-9 was significantly increased in the hippocampus of neonatal rats following HIBD and in the PC12 cells following hypoxic-ischemic injury, while FA and LNA-miR-9 both inhibited the expression of miRNA-9, suggesting that the therapeutic effect of FA was mainly attributed to the inhibition of miRNA-9 expression. Indeed, the silencing of miR-9 by LNA-miR-9 or FA similarly attenuated neuronal damage and cerebral atrophy in the rat hippocampus after HIBD, which was consistent with the restored expression levels of brain-derived neurotrophic factor (BDNF). Therefore, our findings indicate that FA treatment may protect against neuronal death through the inhibition of miRNA-9 induction in the rat hippocampus following hypoxic-ischemic damage.

Retraction: Involvement of the JNK/FOXO3a/Bim Pathway in Neuronal Apoptosis after Hypoxic-Ischemic Brain Damage in Neonatal Rats.

Retraction: Involvement of the JNK/FOXO3a/Bim Pathway in Neuronal Apoptosis after Hypoxic-Ischemic Brain Damage in Neonatal Rats.

Inhibition of miR-200b-3p alleviates hypoxia-ischemic brain damage via targeting Slit2 in neonatal rats

BackgroundBrain damage in premature infants often occurs in very low birth weight infants (VLBW) as a result of hypoxia-ischemia and can lead to cognitive impairment and movement disorders. Many miRNAs have been demonstrated to participate in hypoxia-ischemic brain damage (HIBD). This study was designed to investigate the roles of miR-200b-3p in brain damage of neonatal rats induced by hypoxia-ischemia. Methods and resultsThree-day-old SD rats were used to establish the model of hypoxia-ischemic brain injury mimicking premature infants. RT-qPCR showed that miR-200b-3p was up-regulated in rat brains at the early stage following hypoxia-ischemic treatment. Bioinformatics analysis identified that Slit2 is a target gene of miR-200b-3p and luciferase reporter gene assay confirmed that miR-200b-3p can interact with and target Slit2 mRNA. Inhibition of miR-200b-3p by antagomir increased Slit2 expression at both the mRNA and protein levels in rat brains. TUNEL assay and transmission electron microscopy (TEM) analysis showed decreased numbers of apoptotic neurons in the hypoxia-ischemia-treated animals as a result of administration of miR-200b-3p antagomir. Administration of miR-200b-3p antagomir attenuated spatial and learning memory loss in the animals induced by hypoxia-ischemia as compared to controls. ConclusionOur study has demonstrated that Slit2 is a target gene of miR-200b-3p and that the hypoxia-ischemic brain damage in neonatal rats was alleviated by inhibiting miR-200b-3p via Slit2. miR-200b-3p may be a potential therapeutic target of HIBD for further investigation.

Resatorvid protects against hypoxic-ischemic brain damage in neonatal rats.

Secondary brain damage caused by hyperactivation of autophagy and inflammatory responses in neurons plays an important role in hypoxic-ischemic brain damage (HIBD). Although previous studies have implicated Toll-like receptor 4 (TLR4) and nuclear factor kappa-B (NF-κB) in the neuroinflammatory response elicited by brain injury, the role and mechanisms of the TLR4-mediated autophagy signaling pathway in neonatal HIBD are still unclear. We hypothesized that this pathway can regulate brain damage by modulating neuron autophagy and neuroinflammation in neonatal rats with HIBD. Hence, we established a neonatal HIBD rat model using the Rice-Vannucci method, and injected 0.75, 1.5, or 3 mg/kg of the TLR4 inhibitor resatorvid (TAK-242) 30 minutes after hypoxic ischemia. Our results indicate that administering TAK-242 to neonatal rats after HIBD could significantly reduce the infarct volume and the extent of cerebral edema, alleviate neuronal damage and neurobehavioral impairment, and decrease the expression levels of TLR4, phospho-NF-κB p65, Beclin-1, microtubule-associated protein l light chain 3, tumor necrosis factor-α, and interleukin-1β in the hippocampus. Thus, TAK-242 appears to exert a neuroprotective effect after HIBD by inhibiting activation of autophagy and the release of inflammatory cytokines via inhibition of the TLR4/NF-κB signaling pathway. This study was approved by the Laboratory Animal Ethics Committee of Affiliated Hospital of Yangzhou University, China (approval No. 20180114-15) on January 14, 2018.

Simvastatin preconditioning confers neuroprotection against hypoxia-ischemia induced brain damage in neonatal rats via autophagy and silent information regulator 1 (SIRT1) activation

Previous studies have shown that simvastatin (Sim) has neuroprotective effects in a neonatal model of hypoxia-ischemia (HI)-induced brain injury when administered before but not after HI, pointing to the preconditioning (PC)-like effects of the statin. The present study aimed to gain more insight into the PC-like effect of Sim by studying the role of autophagy and its modulation by mTOR and SIRT1 in neuroprotection. Sim potentiated the autophagy response induced by neonatal HI, as shown by the increased expression of both microtubule-associated protein 1 light chain 3 (LC3) and beclin 1, increased monodansylcadaverine (MDC) labeling, and reduced expression of p62. The autophagy inhibitor 3-methyladenine (3MA) completely blocked the neuroprotective effect of Sim. Two hours after HI, there was a reduction in the activity of mTORC1 and a concomitant increase in that of mTORC2. Sim preconditioning further decreased the activity of mTORC1, but did not affect that of mTORC2. However, 24 h after injury, mTORC2 activity was significantly preserved in Sim-treated rats. Sim preconditioning also prevented the depletion of SIRT1 induced by HI, an effect that was completely blocked by 3MA. These data show that Sim preconditioning may modulate autophagy and survival pathways by affecting mTORC1, mTORC2, and SIRT1 activities. This study provides further preclinical evidence of the PC-like effect of statins in brain tissue, supporting their beneficial effects in improving stroke outcome after prophylactic treatments.

Acoustic Radiation Force Impulse Imaging With Virtual Touch Tissue Quantification Enables Characterization of Mild Hypoxic-Ischemic Brain Damage in Neonatal Rats.

The aim of this study was to investigate whether the measurement of brain tissue stiffness using acoustic radiation force impulse (ARFI) elastography with virtual touch tissue quantification can improve the early detection of neonatal hypoxic-ischemic brain damage in rats. Seven-day-old Sprague-Dawley rats were randomly assigned to 3 groups: the mild asphyxia (n = 30), moderate asphyxia (n = 30), and sham control (n = 10) groups. Rats in the mild and moderate asphyxia groups were exposed to 8% oxygen (hypoxia) for 30 and 60 minutes, respectively, at 1 hour after ligation of the right common carotid artery. An ultrasound diagnostic instrument was used to obtain 2-dimensional ultrasound images, and ARFI with virtual touch tissue quantification was used to measure shear wave velocity preoperatively and at 12, 24, 48, and 72 hours postoperatively. Hematoxylin-eosin staining was used to evaluate brain damage. Two-dimensional ultrasound imaging detected swelling and increased echogenicity at 48 to 72 hours in the mild asphyxia group and at 24 to 72 hours in the moderate asphyxia group. The shear wave velocity substantially increased from 0.65 ± 0.04 m/s preoperatively to 0.78 ± 0.07 m/s at 72 hours in the moderate asphyxia group and from 0.64 ± 0.04 m/s preoperatively to 0.70 ± 0.03 m/s at 72 hours in the mild asphyxia group. The changes in the shear wave velocity coincided with the histopathologic changes in the brain, which included neuronal demyelination, hyperplasia, and necrosis; edema around vascular structures; and hemorrhage in the ependymal and periventricular areas. Shear wave velocity data obtained with the virtual touch tissue quantification technique may be used for early diagnosis of neonatal hypoxic-ischemic brain damage.