Brain Injury In Neonatal Rats Research Articles

Hypaconitine confers protection on ketamine-induced neuronal injury in neonatal rat brain via a mechanism involving PI3K/Akt/Bcl-2 pathway

Purpose: To investigate the neuroprotective effect of hypaconitine against ketamine-induced neuronal injury in the brains of neonatal rats, and the underlying mechanism of action.
 Methods: Seven day-old Sprague-Dawley pups weighing 15.0 to 20.0 g (mean weight = 17.5 ± 2.5 g), and aged 7 days were used for this study. The pups were sacrificed, and their forebrains isolated and used to prepare cell suspensions. The isolated cells were treated with ketamine (100 µM) or varied concentrations of hypaconitine (0.1 – 2 µM) or LY294002 (10 µM). The cells were trypsinized and cultured at 37 °C in 10 % fetal bovine serum (FBS) supplemented Dulbecco's modified Eagle's medium (DMEM) in a humidified incubator containing 5 % CO2. Cell viability was determined using MTT assay, while TUNEL assay was used to determine the extent of apoptosis in the cells. The expressions of pAkt, Bcl-2 and caspase-3 were determined using Western blotting.
 Results: There were only few viable cells in the ketamine-treated group, and cell viability was significantly and dose-dependently increased in hypaconitine-treated groups (p < 0.05). The extent of apoptosis was significantly higher in ketamine-treated cells than in control cells, but treatment with hypaconitine significantly reduced the number of apoptotic cells (p < 0.05). However, in the presence of LY294002 (a PI3K-specific inhibitor), the effect of hypaconitine on neuronal cell apoptosis was significantly reversed (p < 0.05). The expressions of p-Akt and Bcl-2 were significantly down-regulated while the expression of caspase-3 was significantly upregulated in ketamine-treated neuronal cells, when compared with control group (p < 0.05). However, in cells treated with hypaconitine, the expressions of p-Akt and Bcl-2 were significantly upregulated, while the expression of caspase-3 was significantly down-regulated (p < 0.05). Treatment of neuronal cells with hypaconitine in the presence of LY294002 significantly reversed the effect of hypaconitine on the expressions of p-Akt, Bcl-2 and caspase-3 (p < 0.05).
 Conclusion: These results suggest that hypaconitine ameliorates ketamine-induced neuronal injury in neonatal rats via a mechanism involving the PI3K/Akt/Bcl-2 pathway.

Hyperoxia-activated circulating extracellular vesicles induce lung and brain injury in neonatal rats

Hyperoxia-induced lung injury plays a key role in the development of bronchopulmonary dysplasia (BPD), characterized by inflammatory injury and impaired lung development in preterm infants. Although BPD is a predictor of poor neurodevelopmental outcomes, currently it is uncertain how lung injury contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that regulate intercellular and inter-organ communications. Gasdermin D (GSDMD) has emerged as a key executor of inflammasome-mediated cell death and inflammation. In this study, we utilized a neonatal rat model of BPD to assess if hyperoxia stimulates lung release of circulating EVs and if these EVs induce lung and brain injury. We found that hyperoxia-exposed rats had elevated numbers of plasma-derived EVs compared to rats maintained in room air. These EVs also had increased cargos of surfactant protein C, a marker of type II alveolar epithelial cells (AEC), and the active (p30) form of GSDMD. When these EVs were adoptively transferred into normal newborn rats via intravenous injection, they were taken up both by lung and brain tissues. Moreover, EVs from hyperoxic animals induced not only the pathological hallmarks of BPD, but also brain inflammatory injury in recipient rats, as well as inducing cell death in cultured pulmonary vascular endothelial cells and neural stem cells (NSC). Similarly, hyperoxia-exposed cultured AEC-like cells released EVs that also contained increased GSDMD-p30 and these EVs induced pyroptotic cell death in NSC. Overall, these data indicate that hyperoxia-activated circulating EVs mediate a lung to brain crosstalk resulting in brain injury and suggest a mechanism that links lung injury and neurodevelopmental impairment in BPD infants.

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.

Myricitrin attenuates hypoxic-ischemia-induced brain injury in neonatal rats by mitigating oxidative stress and nuclear factor erythroid 2-related factor 2/hemeoxygenase-1/antioxidant response element signaling pathway

Background: Neonatal hypoxic ischemia (HI) is an overwhelming infantile neurological disability causing extreme morbidity and mortality with limited therapies available. Objectives: In this study, we aimed to explore the neuroprotective efficacy of myricitrin and elucidate the mechanism of action of myricitrin through the regulation of nuclear factor erythroid 2-related factor 2 (Nrf2)/hemeoxygenase-1 (HO-1)/antioxidant response element (ARE)-signaling pathways in neonatal rats. Materials and Methods: Seven-day-old neonatal rats were divided into four groups. Sham group served as the control group. After inducing HI through standard operation procedure, two groups of neonatal rats were intraperitoneally administered with 20 and 40 mg/kg bw of myricitrin twice a day for up to 1 week. At the end of the experiment, we assessed the brain infract area, edema, and motor coordination activity by using Rota Rod test. Furthermore, we measured the level of antioxidant enzymes and oxidative stress markers in the brain tissue samples. Real-time quantitative polymerase chain reaction was conducted to detect the inflammatory molecules expression including HO-1 being the activator of Nrf2 where further mediates the transcriptional activation of ARE. Subsequently, the hypothesis was further confirmed by the immunosorbent assay and Western blot analysis. Results: Myricitrin administration ameliorated HI-induced increase in infract volume and edema of the brain, as well as improved neurobehavioral impairments. Interestingly, myricitrin significantly reduced the level of nuclear factor kappa B (NF-κB) p65 and reduced the levels of nuclear fraction of Nrf2-ARE and cytosolic fraction of HO-1 enzyme activation. Conclusion: In summary, myricitrin improved the antioxidant defense mechanism and ameliorated the oxidative stress-associated neurological damage via modulating Nrf2/ARE-dependent HO-1 pathway in neonatal rats.

Neuroprotective effect of apigenin against hypoxic-ischemic brain injury in neonatal rats via activation of the PI3K/Akt/Nrf2 signaling pathway.

Neonatal hypoxic-ischemic (HI) brain injury can lead to mortality and severe long-term disabilities including cerebral palsy and brain injury. However, the treatment options for neonatal hypoxic-ischemic (HI) brain injury are limited. Apigenin is abundantly present in vegetables, celery, and chamomile tea with diverse biological functions, such as anti-inflammatory, anti-apoptotic, antioxidant, and anticancer effects. However, it has not yet been reported whether apigenin exerts a neuroprotective effect against neonatal hypoxic-ischemic (HI) brain injury. In this study, we investigated whether apigenin could ameliorate HI brain injury and explored the associated mechanism using in vivo experiments. We found that apigenin remarkably reduced the infarct volume and ameliorated cerebral edema, decreased inflammatory response, inhibited apoptosis, promoted the recovery of tissue structure, and improved prognosis following HI brain injury. Mechanistically, we found that apigenin exerted a neuroprotective effect against HI brain injury by activating the PI3K/Akt/Nrf2 pathway. In summary, all these results demonstrate that apigenin could be a potential therapeutic approach for neonatal hypoxic-ischemic (HI) brain injury.

Brief inhalation of sevoflurane can reduce glial scar formation after hypoxic-ischemic brain injury in neonatal rats.

Previous studies have demonstrated that sevoflurane postconditioning can provide neuroprotection after hypoxic-ischemic injury and improve learning and memory function in developing rodent brains. The classical Rice-Vannucci model was used to induce hypoxic-ischemic injury, and newborn (postnatal day 7) rats were treated with 2.4% sevoflurane for 30 minutes after hypoxic-ischemic injury. Our results showed that sevoflurane postconditioning significantly improved the learning and memory function of rats, decreased astrogliosis and glial scar formation, increased numbers of dendritic spines, and protected the histomorphology of the hippocampus. Mechanistically, sevoflurane postconditioning decreased expression of von Hippel-Lindau of hypoxia-inducible factor-1α and increased expression of DJ-1. Injection of 1.52 μg of the hypoxia-inducible factor-1α inhibitor YC-1 (Lificiguat) into the left lateral ventricle 30 minutes before hypoxic-ischemic injury reversed the neuroprotection induced by sevoflurane. This finding suggests that sevoflurane can effectively alleviate astrogliosis in the hippocampus and reduce learning and memory impairments caused by glial scar formation after hypoxic-ischemic injury. The underlying mechanism may be related to upregulated DJ-1 expression, reduced ubiquitination of hypoxia-inducible factor-1α, and stabilized hypoxia-inducible factor-1α expression. This study was approved by the Laboratory Animal Care Committee of China Medical University, China (approval No. 2016PS337K) on November 9, 2016.

Involvement of CXCL1/CXCR2 During Microglia Activation Following Inflammation-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Rats.

Background: Microglia are key mediators of inflammation during perinatal brain injury. As shown experimentally after inflammation-sensitized hypoxic ischemic (HI) brain injury, microglia are activated into a pro-inflammatory status 24 h after HI involving the NLRP3 inflammasome pathway. The chemokine (C-X-C motif) ligand 1 (CXCL1), and its cognate receptor, CXCR2, have been shown to be involved in NLRP3 activation, although their specific role during perinatal brain injury remains unclear. In this study we investigated the involvement of CXCL1/CXCR2 in brain tissue and microglia and brain tissue after inflammation-sensitized HI brain injury of newborn rats.Methods: Seven-day old Wistar rat pups were either injected with vehicle (NaCl 0.9%) or E. coli lipopolysaccharide (LPS), followed by left carotid ligation combined with global hypoxia (8% O2 for 50 min). Pups were randomized into four different treatment groups: (1) Sham group (n = 21), (2) LPS only group (n = 20), (3) Veh/HI group (n = 39), and (4) LPS/HI group (n = 42). Twenty-four hours post hypoxia transcriptome and gene expression analysis were performed on ex vivo isolated microglia cells in our model. Additionally protein expression was analyzed in different brain regions at the same time point.Results: Transcriptome analyses showed a significant microglial upregulation of the chemokine CXCL1 and its receptor CXCR2 in the LPS/HI group compared with the other groups. Gene expression analysis showed a significant upregulation of CXCL1 and NLRP3 in microglia cells after inflammation-sensitized hypoxic-ischemic brain injury. Additionally, protein expression of CXCL1 was significantly upregulated in cortex of male pups from the LPS/HI group.Conclusion: These results indicate that the CXCL1/CXCR2 pathway may be involved during pro-inflammatory microglia activation following inflammation-sensitized hypoxic-ischemic brain injury in neonatal rats. This may lead to new treatment options altering CXCR2 activation early after HI brain injury.

Midazolam contributes to neuroprotection against hypoxia/reoxygenation-induced brain injury in neonatal rats via regulation of EAAT2

Excitotoxicity is one of the main mechanisms related to hypoxia/reoxygenation (H/R) injury. Excitatory amino acid transporter (EAAT)2 mainly distributes on astrocytes and plays an important role on glutamate reuptake and glutamate homeostasis. Midazolam has a neuroprotective effect in some neuropathological conditions. The present study aimed to detect the role of EAAT2 in the neuroprotective effect of midazolam in neonatal rat brain subjected to H/R. Pretreatment with midazolam reversed H/R-induced apoptosis and downregulation of EAAT2 mRNA and protein expression in the hippocampus. Pretreatment with dihydrokainic acid (a selective inhibitor of EAAT2) exacerbated apoptosis, and thus inhibited the neuroprotective effect of midazolam against H/R injury. We demonstrated for the first time that dysregulation of EAAT2 expression may be related to the neural injury induced by H/R in rat pups, and pretreatment with midazolam attenuated apoptosis and improved learning and memory partly due to regulating EAAT2 expression.

Resveratrol Relieves Hyperoxia-Induced Brain Injury in Neonatal Rats by Activating Sirt1.

Neonatal rats with hyperoxia-induced brain injury were treated with resveratrol to investigate its protective effects through analyzing changes in reactive oxygen species (ROS), Sirt1, p53, and acetylated p53 levels. Neonatal rats were randomly divided into hyperoxia and resveratrol intervened groups. Rats in both groups were placed in a hyperoxia chamber for 7 days to induce hyperoxia-induced brain injury. The rats in the resveratrol intervened group were administered resveratrol 60 μg/g body weight daily, whereas those in the hyperoxia group were administered a dimethyl sulfoxide-based solvent. Brain tissues were collected, and hematoxylin and eosin (H&E) and TUNEL staining, ROS measurements, real time-polymerase chain reaction, and western blotting were performed. H&E and TUNEL staining revealed increased cell damage and apoptosis in brain tissue from hyperoxia-exposed animals compared with the findings in animals in the resveratrol intervened group. Real time-polymerase chain reaction and western blotting identified increases in Sirt1 expression and decreases in p53 expression in the resveratrol intervened group. In addition, acetylated p53 protein expression was lower in the intervened group than in the hyperoxia group. Resveratrol alleviated brain apoptosis induced by hyperoxia in neonatal rats by upregulating Sirt1-mediated pathways, suggesting its potentially beneficial role in the treatment of brain injury induced by hyperoxia.

Pharmacokinetics of Inter-Alpha Inhibitor Proteins and Effects on Hemostasis After Hypoxic-Ischemic Brain Injury in Neonatal Rats

Hypoxic-ischemic (HI) brain injury is a leading cause of long-term neurodevelopmental morbidities in neonates. Human plasma-derived Inter-Alpha Inhibitor Proteins (hIAIPs) are neuroprotective after HI brain injury in neonatal rats. The light chain (bikunin) of hIAIPs inhibits proteases involved in the coagulation of blood. Newborns exposed to HI can be at risk for significant bleeding in the brain and other organs. The objectives of the present study were to assess the pharmacokinetics (PK) and the duration of bleeding after intraperitoneal (IP) administration of hIAIPs in HI-exposed male and female neonatal rats. HI was induced with the Rice-Vannucci method in postnatal (P) day-7 rats. After the right common carotid artery ligation, rats were exposed to 90 min of 8% oxygen. hIAIPs (30 mg/kg, IP) were given immediately after Sham or HI exposure in the PK study and serum was collected 1, 6, 12, 24, or 36 h after the injections. Serum hIAIP concentrations were measured with a competitive ELISA. ADAPT5 software was used to fit the pooled PK data considering first-order absorption and disposition. hIAIPs (60 mg/kg, IP) were given in the bleeding time studies at 0, 24 and 48 h after HI with tail bleeding times measured 72 h after HI. IP administration yielded significant systemic exposure to hIAIPs with PK being affected markedly including primarily faster absorption and reduced elimination as a result of HI and modestly of sex-related differences. hIAIP administration did not affect bleeding times after HI. These results will help to inform hIAIP dosing regimen schedules in studies of neuroprotection in neonates exposed to HI.

Pterostilbene induces Nrf2/HO-1 and potentially regulates NF-κB and JNK-Akt/mTOR signaling in ischemic brain injury in neonatal rats.

Hypoxic-ischemic (HI) brain injury has a high occurrence rate of 1-4 per 1000 live births and is the leading cause of neurological disabilities. Despite the improvement in neonatal care, the effectiveness of current therapeutic strategies is limited, and thus, additional therapies with better results are of much needed. Pterostilbene is a stilbenoid possessing numerous preventive and therapeutic properties. The current study aimed to assess whether pterostilbene exerted protective effects in neonatal rats against experimentally induced ischemic brain injury. Pterostilbene was administered via oral gavage from postnatal day 3 to day 8. Rat pups that were seven-day-old were exposed to hypoxic-ischemic insult via ligation of the common carotid artery and hypoxic environment exposure. Pterostilbene treatment reduced neuronal loss and infarct volume. Pterostilbene administration regulated the NF-κB pathway, and the levels of inflammatory mediators (Nitric oxide, TNF-α, IL-1β, and IL-6) were reduced. HI-induced oxidative stress was significantly reduced by pterostilbene, as presented by decreased production of malondialdehyde and reactive oxygen species. Levels of glutathione were enhanced by pterostilbene. Pterostilbene regulated Nrf2/HO-1 and JNK expression and activated the PI3K/Akt-mTOR signals. These findings suggest that pterostilbene is a candidate compound for the treatment of neonatal HI.

Treatment With Liraglutide Exerts Neuroprotection After Hypoxic-Ischemic Brain Injury in Neonatal Rats via the PI3K/AKT/GSK3β Pathway.

Neonatal hypoxic–ischemic (HI) brain injury is a detrimental disease, which results in high mortality and long-term neurological deficits. Nevertheless, the treatment options for this disease are limited. Thus, the aim of the present study was to assess the role of liraglutide in neonatal HI brain injury in rats and investigate the associated mechanisms. The results showed that treatment with liraglutide significantly reduced infarct volume and ameliorated cerebral edema, decreased inflammatory response, promoted the recovery of tissue structure, and improved prognosis following HI brain injury. Moreover, treatment with liraglutide inhibited apoptosis and promoted neuronal survival both in the rat model and following oxygen-glucose deprivation (OGD) insult. LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), partially reversed these therapeutic effects, suggesting that the PI3K/protein kinase B (Akt) pathway was involved. In conclusion, our data revealed that treatment with liraglutide exerts neuroprotection after neonatal HI brain injury via the PI3K/Akt/glycogen synthase kinase-3β (GSK3β) pathway and may be a promising therapy for this disease.

Synaptic Injury in the Thalamus Accompanies White Matter Injury in Hypoxia/Ischemia-Mediated Brain Injury in Neonatal Rats.

The broad spectrum of disabilities caused by white matter injury (WMI) cannot be explained simply by hypomyelination. Synaptic injury in the thalamus may be related to disabilities in WMI survivors. Neuronal injury in the thalamus has been found most commonly in autopsy cases of preterm WMI. We hypothesized that hypoxia/ischemia (HI) in neonatal rats results in synaptic abnormalities in the thalamus that contribute to disabilities in WMI survivors. We examined changes in synapses in a neonatal rat model of HI-induced WMI. Right common carotid artery ligation and hypoxia (8% oxygen for 2.5 hours (h)) were performed in three-day-old Sprague-Dawley rats. We found HI rats performed worse in the Morris water maze test than sham rats, suggesting long-term cognition impairment after HI injury. A loss of synapses in the thalamus accompanied by hypomyelination and oligodendrocytes (OLs) reduction was observed. At the ultrastructural level, reductions in active zone (AZ) length and postsynaptic density (PSD) thickness were detected at 2 weeks after HI exposure. Furthermore, increased expression of synaptophysin and PSD-95 in both groups was observed from 3 days (d) to 21 d after hypoxic/ischemic (HI) injury. PSD-95 expression was significantly lower in HI rats than in sham rats from 14 d to 21 d after HI injury, and synaptophysin expression was significantly lower in HI rats from 7 d to 14 d after HI injury. However, no significant difference in synaptophysin expression was observed between HI rats and sham rats at 21 d after HI injury. The results demonstrated synaptic abnormalities in the thalamus accompanied by hypomyelination in WMI in response to HI exposure, which may contribute to the diverse neurological defects observed in WMI patients. Although synaptic reorganization occurred as a compensatory response to HI injury, the impairments in synaptic transmission were not reversed.

Erythropoietin induces synaptogenesis and neurite repair after hypoxia ischemia-mediated brain injury in neonatal rats.

The memory dysfunction is one of the disastrous outcomes for perinatal hypoxia ischemia. Erythropoietin (EPO) has been demonstrated as a neuroprotective agent with multiple effects in a series of neurological diseases. We hypothesized that disruption of neural network including synapses and neurites would contribute to memory dysfunction induced by hypoxia ischemia. The aim of the present study was to elucidate the involvement of EPO on synaptogenesis and neurite repair following perinatal hypoxia ischemia. Using a neonatal hypoxia ischemia rat model, we found that EPO rescued hypoxia ischemia-induced decrease of synaptic proteins including Synapsin1 and PSD95 rather than GluR1 in the cortex and hippocampus. In addition, EPO reduced the expression of APP (an axonal injury marker), induced the expression of microtubule-associated protein MAP-2 (a dendritic marker), and restored axonal density after hypoxia ischemia. These changes contributed to improving electrophysiological properties of synapses and spatial memory performance. In summary, our data revealed an important role of EPO in synaptogenesis and neurite repair, providing a new insight into cellular mechanisms underlying cognitive and memory dysfunction associated with perinatal hypoxia ischemia.

Association between CIRP expression and hypoxic-ischemic brain injury in neonatal rats.

The role of cold inducible RNA-binding protein (CIRP) in mediating ischemic brain injury in neonatal rats under chronic hypobaric hypoxia was investigated. The neonatal rat model of chronic hypobaric hypoxia and the cell culture model of SH-SY5Y cells exposed to hypoxia (1% O2) were constructed. The expression of CIRP and hypoxia-inducible factor-1α (HIF-1α) was detected after hypoxic exposure, and the apoptosis-related proteins were analyzed via terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and western blot analysis to detect neuronal apoptosis. Moreover, the effects of CIRP overexpression on HIF-1α and neuronal apoptosis were identified. Chronic hypobaric hypoxia can lead to HIF-1α expression and neuronal apoptosis in the body. CIRP was induced at early exposure (3 d/7 d). However, the CIRP level in the hypoxic group was obviously lower than that in the control group with the prolongation of exposure time (21 d). In addition, the knockdown of HIF-1α significantly reduced the neuronal apoptosis under hypoxic conditions, indicating that HIF-1α may promote apoptosis during exposure. The overexpression of CIRP significantly inhibited the upregulation of HIF-1α during hypoxia and the HIF-1α-mediated neuronal apoptosis. Results of the current study showed that, CIRP is involved in the ischemic brain injury induced by chronic hypoxia through downregulation of HIF-1α expression.

Quercetin attenuates hypoxia-ischemia induced brain injury in neonatal rats by inhibiting TLR4/NF-κB signaling pathway

Neonatal hypoxic ischemia (HI) is a kind of brain damage that occurs when an infant's brain does not receive enough oxygen and blood. The unrepairable damage leads to newborn death and short/long term brain dysfunctions. Due to the complicated causes and the variety of brain damages, there is no definitive treatment of neonatal HI. In this study, we set up a HI injury model of newborn rat and administrated Quercetin (Que) to treat rat pups before and after HI injury. We performed immunohistochemistry, quantitative PCR and immunoblot experiments to examine whether Que. has a role in attenuating brain injury after HI. We found that Que. treatment could clearly attenuate cortical cell apoptosis, as well as suppress apoptosis marker Bax, and activate anti-apoptosis marker Bcl-2. Moreover, Que. treatment decreased the number of cortical cells microgliosis and astrogliosis induced by HI injury. Furthermore, Que. treatment decreased cortical inflammation. Finally, it is suggested that Que. played a neuroprotective function on HI brain injury via inhibiting the TLR4/NF-κB signaling pathway. From these results, we conclude that Que. treatment may be a used as a therapeutic drug to prevent and decrease the newborn brain damage caused by HI.

Early Pro-inflammatory Microglia Activation After Inflammation-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Rats.

Background: Perinatal asphyxia, leading to neonatal encephalopathy, is one of the leading causes for child mortality and long-term morbidities. Neonatal encephalopathy rates are significantly increased in newborns with perinatal infection. Therapeutic hypothermia is only neuroprotective in 50% of cooled asphyxiated newborns. As shown experimentally, cooling has failed to be neuroprotective after inflammation-sensitized hypoxic ischemic (HI) brain injury. Microglia are thought to be key players after inflammation-sensitized HI brain injury. We performed this study investigating early microglia phenotype polarization in our newborn animal model of inflammation-sensitized HI brain injury, better understanding the underlying pathophysiological processes.Methods: Seven days old Wistar rat pups were injected with either vehicle (NaCl 0.9%) or E. coli lipopolysaccharide (LPS), followed by left carotid ligation combined with global hypoxia inducing a mild unilateral hypoxic-ischemic injury. Pups were randomized to (1) Sham group (n = 41), (2) LPS only group (n = 37), (3) Veh/HI group (n = 56), and (4) LPS/HI group (n = 79). On postnatal days 8 and 14 gene-expression analysis or immunohistochemistry was performed describing early microglia polarization in our model.Results: We confirmed that LPS pre-sensitization significantly increases brain area loss and induced microglia activation and neuronal injury after mild hypoxia-ischemia. Additionally, we show that microglia upregulate pro-inflammatory genes involving NLRP-3 inflammasome gene expression 24 h after inflammation-sensitized hypoxic-ischemic brain injury.Conclusion: These results demonstrate that microglia are early key mediators of the inflammatory response following inflammation-sensitized HI brain injury and that they polarize into a predominant pro-inflammatory phenotype 24 h post HI. This may lead to new treatment options altering microglia phenotype polarization early after HI brain injury.

Echinacoside Alleviates Hypoxic-Ischemic Brain Injury in Neonatal Rat by Enhancing Antioxidant Capacity and Inhibiting Apoptosis.

Hypoxic-ischemic brain damage (HIBD) is a leading cause of death and disability in neonatal or perinatal all over the world, seriously affecting children, families and society. Unfortunately, only few satisfactory therapeutic strategies have been developed. It has been demonstrated that Echinacoside (ECH), the major active component of Cistanches Herba, exerts many beneficial effects, including antioxidative, anti-apoptosis, and neuroprotective in the traditional medical practice in China. Previous research has demonstrated that ECH plays a protective effect on ischemic brain injury. This study aimed to investigate whether ECH provides neuroprotection against HIBD in neonatal rats. We subjected 120 seven-day-old Sprague-Dawley rats to cerebral hypoxia-ischemia (HI) and randomly divided into the following groups: sham group, HI group and ECH (40, 80 and 160mg/kg, intraperitoneal) post-administration group. After 48h of HI, 2,3,5-Triphenyltetrazolium chloride, Hematoxylin-Eosin and Nissl staining were conducted to evaluate the extent of brain damage. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities, total antioxidant capacity (T-AOC), and malondialdehyde (MDA) production were assessed to determine the antioxidant capacity of ECH. TUNEL staining and Western blot analysis was performed to respectively estimate the extent of brain cell apoptosis and the expression level of the apoptosis-related proteins caspase-3, Bax, and Bcl-2. Results showed that ECH remarkably reduced the brain infarct volume and ameliorated the histopathological damage to neurons. ECH post-administration helped recovering the antioxidant enzyme activities and decreasing the MDA production. Furthermore, ECH treatment suppressed neuronal apoptosis in the rats with HIBD was by reduced TUNEL-positive neurons, the caspase-3 levels and increased the Bcl-2/Bax ratio. These results suggested that ECH treatment was beneficial to reducing neuronal damage by attenuating oxidative stress and apoptosis in the brain under HIBD.

Neuroprotective effects of inter-alpha inhibitor proteins after hypoxic-ischemic brain injury in neonatal rats

Hypoxic-ischemic (HI) brain injury is one of the most common neurological problems occurring in the perinatal period. Hypothermia is the only approved intervention for neonatal HI encephalopathy. However, this treatment is only partially protective, has a narrow therapeutic time window after birth and only can be used to treat full-term infants. Consequently, additional therapies are critically needed. Inflammation is an important contributing factor to the evolution of HI brain injury in neonates. Inter-alpha Inhibitor Proteins (IAIPs) are immunomodulatory proteins with anti-inflammatory properties. We have previously shown that IAIPs reduce neuronal cell death and improve behavioral outcomes when given after carotid artery ligation, but before hypoxia in male neonatal rats. The objective of the current study was to investigate the neuroprotective effects of treatment with IAIPs given immediately or 6 h after HI in both male and female neonatal rats. HI was induced with the Rice-Vannucci method in postnatal (P) day 7 rats. After ligation of the right common carotid artery, P7 rats were exposed to 90 min of hypoxia (8% oxygen). Human plasma-derived IAIPs or placebo (phosphate buffered saline) was given at zero, 24, and 48 h after HI. Brains were perfused, weighed and fixed 72 h after HI at P10. In a second, delayed treatment group, the same procedure was followed except that IAIPs or placebo were given at 6, 24 and 48 h after HI. Separate sham-operated, placebo-treated groups were exposed to identical protocols but were not exposed to carotid artery ligation and remained in room air. Rat sex was recorded. The effects of IAIPs on HI brain injury were examined using histopathological scoring and immunohistochemical analyses of the brain and by using infarct volume measurements on frozen tissue of the entire brain hemispheres ipsilateral and contralateral to HI injury. IAIPs given immediately after HI improved (P < 0.050) histopathological brain injury across and within the cingulate, caudate/putamen, thalamus, hippocampus and parietal cortex in males, but not in females. In contrast, IAIPs given immediately after HI reduced (P < 0.050) infarct volumes of the hemispheres ipsilateral to HI injury in similarly both the males and females. Treatment with IAIPs also resulted in higher (P < 0.050) brain weights compared with the placebo-treated HI group, reduced (P < 0.050) neuronal and non-neuronal cell death in the cortex and total hemisphere, and also increased the total area of oligodendrocytes determined by CNPase in the ipsilateral hemisphere and corpus callosum (P < 0.050) of male, but not female subjects exposed to HI. Delayed treatment with IAIPs 6 h after HI did not improve histopathological brain injury in males or females, but resulted in higher (P < 0.050) brain weights compared with the placebo-treated HI males. Therefore, treatment with IAIPs immediately after HI improved brain weights and reduced neuropathological brain injury and cell death in male rats, and reduced infarct volume in both male and female neonatal rats. We conclude that IAIPs exert neuroprotective effects after exposure to HI in neonatal rats and may exhibit some sex-related differential effects.

FGF21 promotes functional recovery after hypoxic-ischemic brain injury in neonatal rats by activating the PI3K/Akt signaling pathway via FGFR1/β-klotho

Perinatal asphyxia often results in neonatal cerebral hypoxia-ischemia (HI), which is associated with high mortality and severe long-term neurological deficits in newborns. Currently, there are no effective drugs to mitigate the functional impairments post-HI. Previous studies have shown that fibroblast growth factor 21 (FGF21) has a potential neuroprotective effect against brain injury. However, the effect of FGF21 on neonatal HI brain injury is unclear. In the present study, both in vivo and in vitro models were used to assess whether recombinant human FGF21 (rhFGF21) could exert a neuroprotective effect after HI and explore the associated mechanism. The results showed that the rhFGF21 treatment remarkably reduced the infarct volume, ameliorated the body weight and improved the tissue structure after HI in neonatal rats. In addition, the rhFGF21 treatment lengthened the running endurance times in the rotarod test and decreased the mean escape latencies and increased the number of platform crossings in the Morris water maze test at 21 d post-HI insult. In contrast, the FGFR1 inhibitor PD173074 and PI3K inhibitor LY294002 partially reversed these therapeutic effects. In isolated primary cortical neurons, the rhFGF21 treatment protected primary neurons from oxygen-glucose deprivation (OGD) insult by inhibiting neuronal apoptosis and promoting neuronal survival. Both our in vivo and in vitro results reveal that rhFGF21 could inhibit neuronal apoptosis by activating the PI3K/Akt signaling pathway via FGF21/FGFR1/β-klotho complex formation. Therefore, rhFGF21 may be a promising therapeutic agent for promoting functional recovery after HI-induced neonatal brain injury.