Neonatal Hypoxic-ischemic Brain Research Articles

T lymphocytes characterization and systemic inflammation after hypoxic ischemia in naturel-resuscitation newborn rats

Abstract Background Perinatal hypoxia-ischemia (HI) remains the primary cause of acute neonatal brain injury, leading to a high mortality rate and long-term neurological deficits, such as cerebral palsy of children. In this study we performed investigations of systemic inflammatory cytokines from peripheral blood and T lymphocytes and CD11b+ cells from spleen tissue at acute stage after self-recovery neonatal HI brain injury in rats. Methods HI was induced in 4-day old rat and specimen was collected up to 14 days natural resuscitation to investigate expression of immune associated with cell-markers, CD3, CD4, CD8 and CD11b, and multiple inflammation cytokines (IL-1β, IL-6, TNF-α, L-8 and IL-10). We assessed quantity changes of T cell populations and CD11b from the spleen by flow cytometry. We measured cytokines concentration from peripheral blood by ELISA after recovery HI rats. Results At 14d after recovery HI rats, FACS analysis showed a marked decrease in the number of CD3, CD4 and CD8 (p<0.001), CD11b+ cells was no relative increase of (p = 0.14). IL-1β, IL-6 and TNF-α was higher than control group in peripheral blood (p=0.0012, 0.001 and 0.003 respectively) and IL-8 and IL-10 were lower than control group (p=0.0042 and 0.018 respectively). CONCLUSIONS/SIGNIFICANCE Our findings demonstrate inhibition of systemic immune responses is changes in self-recovery stage after hypoxic ischemic neonatal. The short term immune inhibition observed is of general importance for future studies of the immune response after hypoxic-ischemia as most previous studies have focused on the first few days after damage, while the effects of the late immunocompromise may be remain existed.

Significant Role of microRNA-182-5p in Neonatal Hypoxic-Ischemic Injury and Related Mechanism

The purpose of current study was to explore the role and mechanism of microRNA-182-5p (miR182-5p) in neonatal hypoxic ischemic brain damage (HIBD). First, we established a hypoxic-ischemic (HI) rat model and assessed the neurological function of the rats using the Zea Longa score. Then, the level of miR-182-5p in brain tissue of neonatal rats was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Findings revealed that miR-182-5p was significantly down-regulated in the brain tissue of HI rat model. Next, we studied the target gene of miR-182-5p by using TargetScan and dual luciferase reporter assay. Results showed that CASP2 was a direct target gene of miR-182-5p, and the level of CASP2 was significantly up-regulated in the brain tissue of HI rat model. Immediately thereafter, we established an oxygen and glucose deprivation (OGD) cell model of primary cortical neurons, and demonstrated the changes of miR182-5p in cells treated with OGD by qRT-PCR. Finally, to determine the function of miR-182-5p in OGD subjected neuronal cells, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and flow cytometry (FCM) assays were used to study cell viability and apoptosis. The study found that compared with the OGD group, miR-182-5p mimic significantly increased nerve cell viability, reduced cell apoptosis and decreased cleaved-Caspase3/7/8 protein expression, however, all these changes were significantly reversed by overexpression of the CASP2 gene. Taken together, miR-182-5p might protect the nerve cells from ischemia and hypoxia by targeting CASP2, thereby playing a protective role in hypoxic ischemic encephalopathy, which might be a new effective target for neonatal hypoxic ischemic brain damage treatment.

Serum 24S-hydroxycholesterol predicts long-term brain structural and functional outcomes after hypoxia-ischemia in neonatal mice

The major pathway of brain cholesterol turnover relies on its hydroxylation into 24S-hydroxycholesterol (24S-HC) using brain-specific cytochrome P450 46A1 (CYP46A1). 24S-HC produced exclusively in the brain normally traverses the blood-brain barrier to enter the circulation to the liver for excretion; therefore, the serum 24S-HC level is an indication of cholesterol metabolism in the brain. We recently reported an upregulation of CYP46A1 following hypoxia-ischemia (HI) in the neonatal mouse brain and a correlation between serum 24S-HC levels and acute brain damage. Here, we performed a longitudinal study to investigate whether the serum 24S-HC concentrations predict long-term brain structural and functional outcomes. In postnatal day 9 mice subjected to HI, the serum 24S-HC levels increased at 6 h and 24 h after HI and correlated with the infarct volumes measured histologically or by T2-weighted MRI. The 24 h levels were associated with white matter volume loss quantified by MBP immunostaining and luxol fast blue staining. The animals with higher serum 24S-HC at 6 h and 24 h corresponded to those with more severe motor and cognitive deficits at 35-40 days after HI. These data suggest that 24S-HC could be a novel and early blood biomarker for severity of neonatal HI brain damage and associated functional impairments.

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.

Overexpression of apoptosis inducing factor aggravates hypoxic-ischemic brain injury in neonatal mice

Apoptosis inducing factor (AIF) has been shown to be a major contributor to neuron loss in the immature brain after hypoxia-ischemia (HI). Indeed, mice bearing a hypomorphic mutation causing reduced AIF expression are protected against neonatal HI. To further investigate the possible molecular mechanisms of this neuroprotection, we generated an AIF knock-in mouse by introduction of a latent transgene coding for flagged AIF protein into the Rosa26 locus, followed by its conditional activation by a ubiquitously expressed Cre recombinase. Such AIF transgenic mice overexpress the pro-apoptotic splice variant of AIF (AIF1) at both the mRNA (5.9 times higher) and protein level (2.4 times higher), but not the brain-specific AIF splice-isoform (AIF2). Excessive AIF did not have any apparent effects on the phenotype or physiological functions of the mice. However, brain injury (both gray and white matter) after neonatal HI was exacerbated in mice overexpressing AIF, coupled to enhanced translocation of mitochondrial AIF to the nucleus as well as enhanced caspase-3 activation in some brain regions, as indicated by immunohistochemistry. Altogether, these findings corroborate earlier studies demonstrating that AIF plays a causal role in neonatal HI brain injury.

Properdin: A Novel Target for Neuroprotection in Neonatal Hypoxic-Ischemic Brain Injury.

Background: Hypoxic-ischemic (HI) encephalopathy is a major cause of neonatal mortality and morbidity, with a global incidence of 3 per 1,000 live births. Intrauterine or perinatal complications, including maternal infection, constitute a major risk for the development of neonatal HI brain damage. During HI, inflammatory response and oxidative stress occur, causing subsequent cell death. The presence of an infection sensitizes the neonatal brain, making it more vulnerable to the HI damage. Currently, therapeutic hypothermia is the only clinically approved treatment available for HI encephalopathy, however it is only partially effective in HI alone and its application in infection-sensitized HI is debatable. Therefore, there is an unmet clinical need for the development of novel therapeutic interventions for the treatment of HI. Such an alternative is targeting the complement system. Properdin, which is involved in stabilization of the alternative pathway convertases, is the only known positive regulator of alternative complement activation. Absence of the classical pathway in the neonatal HI brain is neuroprotective. However, there is a paucity of data on the participation of the alternative pathway and in particular the role of properdin in HI brain damage.Objectives: Our study aimed to validate the effect of global properdin deletion in two mouse models: HI alone and LPS-sensitized HI, thus addressing two different clinical scenarios.Results: Our results indicate that global properdin deletion in a Rice-Vannucci model of neonatal HI and LPS-sensitized HI brain damage, in the short term, clearly reduced forebrain cell death and microglial activation, as well as tissue loss. In HI alone, deletion of properdin reduced TUNEL+ cell death and microglial post-HI response at 48 h post insult. Under the conditions of LPS-sensitized HI, properdin deletion diminished TUNEL+ cell death, tissue loss and microglial activation at 48 h post-HI.Conclusion: Overall, our data suggests a critical role for properdin, and possibly also a contribution in neonatal HI alone and in infection-sensitized HI brain damage. Thus, properdin can be considered a novel target for treatment of neonatal HI brain damage.

Phytoestrogen coumestrol attenuates brain mitochondrial dysfunction and long-term cognitive deficits following neonatal hypoxia–ischemia

IntroductionNeonatal Hypoxia-Ischemia (HI) is a major cause of morbidity and mortality, and is frequently associated with short and long-term neurologic and cognitive impairments. The HI injury causes mitochondrial damage leading to increased production of reactive oxygen species (ROS). Phytoestrogens are non-steroidal plant substances structurally and functionally similar to estrogen. Coumestrol is a potent isoflavonoid with a protective effect against ischemic brain damage in adult rats. Our aim was to determine if coumestrol treatment following neonatal HI attenuates the long-term cognitive deficits induced by neonatal HI, as well as to investigate one possible mechanism underlying its potential effect. MethodsOn the 7th postnatal day, male Wistar rats were submitted to the Levine-Rice HI model. Intraperitoneal injections of 20 mg/kg of coumestrol, or vehicle, were administered immediately pre-hypoxia or 3 h post-hypoxia. At 12 h after HI the mitochondrial status and ROS levels were determined. At 60th postnatal day the cognitive deficits were revealed in the Morris water maze reference and working spatial memories. Following behavioral analysis, histological assessment was performed and reactive astrogliosis was measured by GFAP expression. ResultsResults demonstrate that both pre- and post-HI administration of coumestrol were able to counteract the long-term cognitive and morphological impairments caused by HI, as well as to block the late reactive astrogliosis. The pre-HI administration of coumestrol was able to prevent the early mitochondrial dysfunction in the hippocampus of injured rat pups. ConclusionPresent data suggest that coumestrol exerts protection against experimental neonatal brain hypoxia-ischemia through, at least in part, early modulation of mitochondrial function.

Redox-Dependent Loss of Flavin by Mitochondrial Complex I in Brain Ischemia/Reperfusion Injury.

Aims: Brain ischemia/reperfusion (I/R) is associated with impairment of mitochondrial function. However, the mechanisms of mitochondrial failure are not fully understood. This work was undertaken to determine the mechanisms and time course of mitochondrial energy dysfunction after reperfusion following neonatal brain hypoxia-ischemia (HI) in mice. Results: HI/reperfusion decreased the activity of mitochondrial complex I, which was recovered after 30 min of reperfusion and then declined again after 1 h. Decreased complex I activity occurred in parallel with a loss in the content of noncovalently bound membrane flavin mononucleotide (FMN). FMN dissociation from the enzyme is caused by succinate-supported reverse electron transfer. Administration of FMN precursor riboflavin before HI/reperfusion was associated with decreased infarct volume, attenuation of neurological deficit, and preserved complex I activity compared with vehicle-treated mice. In vitro, the rate of FMN release during oxidation of succinate was not affected by the oxygen level and amount of endogenously produced reactive oxygen species. Innovation: Our data suggest that dissociation of FMN from mitochondrial complex I may represent a novel mechanism of enzyme inhibition defining respiratory chain failure in I/R. Strategies preventing FMN release during HI and reperfusion may limit the extent of energy failure and cerebral HI injury. The proposed mechanism of acute I/R-induced complex I impairment is distinct from the generally accepted mechanism of oxidative stress-mediated I/R injury. Conclusion: Our study is the first to highlight a critical role of mitochondrial complex I-FMN dissociation in the development of HI-reperfusion injury of the neonatal brain. Antioxid. Redox Signal. 31, 608-622.

Hydrogen inhalation protects hypoxic-ischemic brain damage by attenuating inflammation and apoptosis in neonatal rats.

Hypoxic–ischemic brain damage (HIBD) is one of the leading causes of brain injury in infant with high risk of mortality and disability; therefore, it is important to explore more feasible and effective treatment strategies. Here, we assessed the neuroprotective effects of different hydrogen inhalation times for the treatment of HIBD. We induced hypoxia–ischemia in Sprague–Dawley rats (postnatal day 7, both sexes), followed by treatment with hydrogen inhalation for 30, 60, or 90 min. Morphological brain injury was assessed by Nissl and TUNEL staining. Acute inflammation was evaluated by examining the expression of interleukin-1β (IL-1β) and NF-κB p65, as well as Iba-1 immunofluorescence in the brain. Neural apoptosis was evaluated by examining the expression of P-JNK and p53 as well as NeuN immunofluorescence. Neurobehavioral function of rats was evaluated by Morris water maze test at 36 days after surgery. The results showed that hypoxia–ischemia injury induced the inflammatory response of microglia; however, these changes were inhibited by hydrogen inhalation. The inhibitory effects became more apparent as the treatment duration increased ( P < 0.05). Furthermore, hypoxia–ischemia induced neuronal damage and increased the expression of the apoptotic factors, P-JNK, and p53, which were attenuated by hydrogen inhalation ( P < 0.05). Hypoxia–ischemia caused long-term spatial memory deficits during brain maturation, which were ameliorated by hydrogen inhalation ( P < 0.01). In conclusion, hypoxia–ischemia induced severe long-term damage to the brain, which could be alleviated by hydrogen inhalation in a time-dependent manner. Impact statement Oxidative stress is known to be involved in the main pathological progression of neonatal hypoxic–ischemic brain damage (HIBD). Hydrogen (H2) is an antioxidant that can be used to treat HIBD; however, the mechanism by which hydrogen may be used as a promising treatment for neonates with HIBD is not very clear. This study demonstrated that inhaled H2 is neuroprotective against HIBD in SpragueDawley rats by inhibiting the brain’s inflammatory response and neuronal apoptosis or damage and protecting against spatial memory decline. Further, this study showed that inhaled H2 has potential as a therapeutic approach for HIBD. This is relevant to clinical treatment protocols when hypoxia–ischemia is suspected in neonates.

Mild Neonatal Brain Hypoxia-Ischemia in Very Immature Rats Causes Long-Term Behavioral and Cerebellar Abnormalities at Adulthood.

Systemic hypoxia-ischemia (HI) often occurs during preterm birth in human. HI induces injuries to hinder brain cells mainly in the ipsilateral forebrain structures. Such HI injuries may cause lifelong disturbances in the distant regions, such as the contralateral side of the cerebellum. We aimed to evaluate behavior associated with the cerebellum, to acquire cerebellar abundant metabolic alterations using in vivo 1H magnetic resonance spectroscopy (1H MRS), and to determine GFAP, NeuN, and MBP protein expression in the left cerebellum, in adult rats after mild early postnatal HI on the right forebrain at day 3 (PND3). From PND45, HI animals exhibited increased locomotion in the open field while there is neither asymmetrical forelimb use nor coordination deficits in the motor tasks. Despite the fact that metabolic differences between two cerebellar hemispheres were noticeable, a global increase in glutamine of HI rats was observed and became significant in the left cerebellum compared to the sham-operated group. Furthermore, increases in glutamate, glycine, the sum of glutamate and glutamine and total choline, only occurred in the left cerebellum of HI rats. Remarkably, there were decreased expression of MBP and NeuN but no detectable reactive astrogliosis in the contralateral side of the cerebellum of HI rats. Taken together, the detected alterations observed in the left cerebellum of HI rats may reflect disequilibrium in the glutamate-glutamine cycle and a delay in the return of glutamine from astrocytes to neurons from hypoxic-ischemic origin. Our data provides in vivo evidence of long-term changes in the corresponding cerebellum following mild neonatal HI in very immature rats, supporting the notion that systemic HI could cause cell death in the cerebellum, a distant region from the expected injury site.HIGHLIGHTS-Neonatal hypoxia-ischemia (HI) in very immature rats induces hyperactivity toward adulthood.-1H magnetic resonance spectroscopy detects long-term cerebellar metabolic changes in adult rats after neonatal HI at postnatal day 3.-Substantial decreases of expression of neuronal and myelin markers in adult rats cerebellum after neonatal cortical mild HI.

Pretreatment of Grape Seed Proanthocyanidin Extract Exerts Neuroprotective Effect in Murine Model of Neonatal Hypoxic-ischemic Brain Injury by Its Antiapoptotic Property.

Grape seed proanthocyanidin extract (GSPE), an active component extracted from the grape, has been reported to demonstrate antioxidant, anti-inflammatory, anticancer, and antiapoptosis effects. However, little is known about the role of GSPE on neonatal hypoxic-ischemic (HI) brain injury. The aim of this study was to evaluate the neuroprotective effect of GSPE pretreatment on neonatal HI brain injury in mice. A modified Rice-Vannucci method was performed to induce neonatal HI brain injury in the 7-day-old mouse pups pretreated with GSPE or vehicle. The infarct volumes were determined by TTC staining. TUNEL staining was used to detect cells apoptosis, and the expressions of apoptosis-related proteins: bax, bcl2, and cleaved caspase-3 were assayed by Western blot. Behavioral tests were also conducted to assess the functional recovery after injury. We showed that the brain damage and neurobehavioral outcomes improvement was observed in GSPE pretreated group. GSPE was proved to suppress apoptosis through inhibition of bax and cleaved caspase-3 expression. It demonstrates that GSPE could alleviate brain damage maybe through its antiapoptotic activity in a neonatal HI brain injury model, and GSPE has the potential to be a new drug for effective prevention of this disorder.

Neuromodulatory Effect of NLRP3 and ASC in Neonatal Hypoxic Ischemic Encephalopathy

Background: Following birth asphyxia there is a robust inflammatory response. NLRP3 is a receptor of the innate immune system. Upon activation, NLRP3 forms an inflammasome together with ASC and procaspase-1 to mediate release of IL-1β and IL-18. NLRP3 has previously been shown to be upregulated following neonatal hypoxic-ischemic (HI) brain injury in mice, but with no early effect on brain injury. Objective: We aimed to evaluate if deficiency of NLRP3 or ASC protects against neonatal HI brain damage 7 days after hypoxia-ischemia. Methods: C57BL/6J, NLRP3^–/–, and ASC^–/– mice were subjected to unilateral common carotid artery ligation followed by hypoxia at P9. Brain infarction, apoptosis, and microglial response were evaluated, as well as total RNA sequencing and examination of plasma levels of systemic proinflammatory cytokines. Results: NLRP3^–/– mice showed significantly increased brain infarction volumes compared to wild-type (Wt) mice, while ASC^–/– mice showed reduced brain infarction volumes after neonatal hypoxia-ischemia. The amount of activated microglia was increased in NLRP3^–/– mice, while decreased in ASC^–/– mice compared to Wt mice. Total RNA sequencing showed an impaired inflammatory transcriptional response in the hippocampus of NLRP3^–/– mice. Plasma levels of IL-1β and IL-18 were not affected, but TNF was lower in NLRP3^–/– and ASC^–/– mice compared to Wt mice. Conclusion: ASC deficiency is neuroprotective in neonatal HI brain damage in mice, while NLRP3 deficiency increases brain damage.

Human Umbilical Cord Therapy Improves Long-Term Behavioral Outcomes Following Neonatal Hypoxic Ischemic Brain Injury.

Background: Hypoxic ischemic (HI) insult in term babies at labor or birth can cause long-term neurodevelopmental disorders, including cerebral palsy (CP). The current standard treatment for term infants with hypoxic ischemic encephalopathy (HIE) is hypothermia. Because hypothermia is only partially effective, novel therapies are required to improve outcomes further. Human umbilical cord blood cells (UCB) are a rich source of stem and progenitor cells making them a potential treatment for neonatal HI brain injury. Recent clinical trials have shown that UCB therapy is a safe and efficacious treatment for confirmed cerebral palsy. In this study, we assessed whether early administration of UCB to the neonate could improve long-term behavioral outcomes and promote brain repair following neonatal HI brain injury.Methods: HI brain injury was induced in postnatal day (PND) 7 rat pups via permanent ligation of the left carotid artery, followed by a 90 min hypoxic challenge. UCB was administered intraperitoneally on PND 8. Behavioral tests, including negative geotaxis, forelimb preference and open field test, were performed on PND 14, 30, and 50, following brains were collected for assessment of neuropathology.Results: Neonatal HI resulted in decreased brain weight, cerebral tissue loss and apoptosis in the somatosensory cortex, as well as compromised behavioral outcomes. UCB administration following HI improved short and long-term behavioral outcomes but did not reduce long-term histological evidence of brain injury compared to HI alone. In addition, UCB following HI increased microglia activation in the somatosensory cortex compared to HI alone.Conclusion: Administration of a single dose of UCB cells 24 h after HI injury improves behavior, however, a single dose of cells does not modulate pathological evidence of long-term brain injury.

An Inhibitor of the Mitochondrial Permeability Transition Pore Lacks Therapeutic Efficacy Following Neonatal Hypoxia Ischemia in Mice

Neonatal hypoxic ischemic (HI) brain injury causes lifelong neurologic disability. Therapeutic hypothermia (TH) is the only approved therapy that partially mitigates mortality and morbidity. Therapies specifically targeting HI-induced brain cell death are currently lacking. Intracellular calcium dysregulation, oxidative stress, and mitochondrial dysfunction through the formation of the mitochondrial permeability transition pore (mPTP) are drivers of HI cellular injury. GNX-4728, a small molecule direct inhibitor of the mPTP that increases mitochondrial calcium retention capacity, is highly effective in adult neurodegenerative disease models and could have potential as a therapy in neonatal HI. A dose of GNX-4728, equivalent to that used in animal models, 300 mg/kg, IP was highly toxic in p10 mice. We then tested the hypothesis that acute administration of 30 mg/kg, IP of GNX-4728 immediately after HI in a neonatal mouse model would provide neuroprotection. This non-lethal lower dose of GNX-4728 (30 mg/kg, IP) improved the respiratory control ratio of neonatal female HI brain tissue but not in males. Brain injury, assessed histologically with a novel metric approach at 1 and 30 days after HI, was not mitigated by GNX-4728. Our work demonstrates that a small molecule inhibitor of the mPTP has i) an age related toxicity, ii) a sex-related brain mitoprotective profile after HI but iii) this is not sufficient to attenuate forebrain HI neuropathology.

HIF1α Signaling in the Endogenous Protective Responses after Neonatal Brain Hypoxia-Ischemia

Hypoxia-inducible factor 1α (HIF1α) is a key regulator of oxygen homeostasis, and its target genes mediate adaptive, protective, and pathological processes. The role of HIF1α in neuronal survival is controversial and the brain maturation stage is important in determining its function in brain ischemia or hypoxia-ischemia (HI). In this study, we used neuron-specific HIF1α knockout mice at postnatal day 9 (P9), and immature cortical neurons (days 7–8 in vitro) treated with the HIF1α inhibitor 2-methoxyestradiol (2ME2) or stabilizer dimethyloxalylglycine (DMOG), to examine the function of neuronal HIF1α in neonatal HI in vivo (Vannucci model) and in vitro (oxygen glucose deprivation, OGD). Inhibition of HIF1α with 2ME2 in primary neurons or deletion of neuronal HIF1α in P9 mice increased both necrotic and apoptotic cell death following HI, as evaluated by the protein levels of 145/150-kDa and 120-kDa spectrin breakdown products 24 h after HI. DMOG attenuated neuronal death right after OGD. Acute pharmacological manipulation of HIF1α synchronously regulated the expression of its targets, vascular endothelial growth factor (VEGF) and erythropoietin (Epo), in the same manner. The in vivo findings agree with our previous data using the same HIF1α-deficient mice at an earlier age. This study confirms the role of neuronal HIF1α signaling in the endogenous protective responses following HI in the developing brain.

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.

Severe-combined immunodeficient rats can be used to generate a model of perinatal hypoxic-ischemic brain injury to facilitate studies of engrafted human neural stem cells.

Cerebral palsy (CP) encompasses a group of non-progressive brain disorders that are often acquired through perinatal hypoxic-ischemic (HI) brain injury. Injury leads to a cascade of cell death events, resulting in lifetime motor and cognitive deficits. There are currently no treatments that can repair the resulting brain damage and improve functional outcomes. To date, preclinical research using neural precursor cell (NPC) transplantation as a therapy for HI brain injury has shown promise. To translate this treatment to the clinic, it is essential that human-derived NPCs also be tested in animal models, however, a major limitation is the high risk of xenograft rejection. A solution is to transplant the cells into immune-deficient rodents, but there are currently no models of HI brain injury established in such a cohort of animals. Here, we demonstrate that a model of HI brain injury can be generated in immune-deficient Prkdc knockout (KO) rats. Long-term deficits in sensorimotor function were similar between KO and wildtype (WT) rats. Interestingly, some aspects of the injury were more severe in KO rats. Additionally, human induced pluripotent stem cell derived (hiPSC)-NPCs had higher survival at 10 weeks post-transplant in KO rats when compared to their WT counterparts. This work establishes a reliable model of neonatal HI brain injury in Prkdc KO rats that will allow for future transplantation, survival, and long-term evaluation of the safety and efficacy of hiPSC-NPCs for neonatal brain damage. This model will enable critical preclinical translational research using human NPCs.

Peripheral myeloid cells contribute to brain injury in male neonatal mice

BackgroundNeonatal brain injury is increasingly understood to be linked to inflammatory processes that involve specialised CNS and peripheral immune interactions. However, the role of peripheral myeloid cells in neonatal hypoxic-ischemic (HI) brain injury remains to be fully investigated.MethodsWe employed the Lys-EGFP-ki mouse that allows enhanced green fluorescent protein (EGFP)-positive mature myeloid cells of peripheral origin to be easily identified in the CNS. Using both flow cytometry and confocal microscopy, we investigated the accumulation of total EGFP+ myeloid cells and myeloid cell subtypes: inflammatory monocytes, resident monocytes and granulocytes, in the CNS for several weeks following induction of cerebral HI in postnatal day 9 mice. We used antibody treatment to curb brain infiltration of myeloid cells and subsequently evaluated HI-induced brain injury.ResultsWe demonstrate a temporally biphasic pattern of inflammatory monocyte and granulocyte infiltration, characterised by peak infiltration at 1 day and 7 days after hypoxia-ischemia. This occurs against a backdrop of continuous low-level resident monocyte infiltration. Antibody-mediated depletion of circulating myeloid cells reduced immune cell accumulation in the brain and reduced neuronal loss in male but not female mice.ConclusionThis study offers new insight into sex-dependent central-peripheral immune communication following neonatal brain injury and merits renewed interest in the roles of granulocytes and monocytes in lesion development.

Evaluation of 3K3A-Activated Protein C to Treat Neonatal Hypoxic Ischemic Brain Injury in the Spiny Mouse.

Neonatal hypoxic ischemic encephalopathy (HIE) resulting from intrapartum asphyxia is a global problem that causes severe disabilities and up to 1 million deaths annually. A variant form of activated protein C, 3K3A-APC, has cytoprotective properties that attenuate brain injury in models of adult stroke. In this study, we compared the ability of 3K3A-APC and APC (wild-type (wt)) to attenuate neonatal brain injury, using the spiny mouse (Acomys cahirinus) model of intrapartum asphyxia. Pups were delivered at 38 days of gestation (term = 39days), with an intrapartum hypoxic insult of 7.5min (intrapartum asphyxia cohort), or immediate removal from the uterus (control cohort). After 1h, pups received a subcutaneous injection of 3K3A-APC or wild-type APC (wtAPC) at 7mg/kg, or vehicle (saline). At 24h of age, pups were killed and brain tissue was collected for measurement of inflammation and cell death using RT-qPCR and histopathology. Intrapartum asphyxia increased weight loss, inflammation, and apoptosis/necrosis in the newborn brain. 3K3A-APC administration maintained body weight and ameliorated an asphyxia-induced increase of TGFβ1 messenger RNA expression in the cerebral cortex, immune cell aggregation in the corpus callosum, and cell death in the deep gray matter and hippocampus. In the cortex, 3K3A-APC appeared to exacerbate the immune response to the hypoxic ischemic insult. While wtAPC reduced cell death in the corpus callosum and hippocampus following intrapartum asphyxia, it increased markers of neuro-inflammation and cell death in control pups. These findings suggest 3K3A-APC administration may be a useful therapy to reduce cell death and neonatal brain injury associated with HIE.

High-mobility group box-1 translocation and release after hypoxic ischemic brain injury in neonatal rats

Inflammation contributes to neonatal brain injury. Pro-inflammatory cytokines represent key inflammatory meditators in neonatal hypoxic-ischemic (HI) brain injury. The high mobility group box-1 (HMGB1) protein is a nuclear protein with pro-inflammatory cytokine properties when it is translocated from the nucleus and released extracellularly after stroke in adult rodents. We have previously shown that HMGB1 is translocated from the nucleus to cytosolic compartment after ischemic brain injury in fetal sheep. In the current study, we utilized the Rice-Vannucci model to investigate the time course of HMGB1 translocation and release after HI injury in neonatal rats. HMGB1 was located in cellular nuclei of brains from sham control rats. Nuclear to cytoplasmic translocation of HMGB1 was detected in the ipsilateral-HI hemisphere as early as zero h after HI, and released extracellularly as early as 6 h after HI. Immunohistochemical double staining detected HMGB1 translocation mainly in neurons along with release from apoptotic cells after HI. Serum HMGB1 increased at 3 h and decreased by 24 h after HI. In addition, rat brains exposed to hypoxic injury alone also exhibited time dependent HMGB1 translocation at 3, 12 and 48 h after hypoxia. Consequently, HMGB1 responds similarly after HI injury in the brains of neonatal and adult subjects. We conclude that HMGB1 is sensitive early indicator of neonatal HI and hypoxic brain injury.