Neonatal Hypoxic-ischemic Brain Research Articles

C-Type Natriuretic Peptide Ameliorates Vascular Injury and Improves Neurological Outcomes in Neonatal Hypoxic-Ischemic Brain Injury in Mice.

C-type natriuretic peptide (CNP) is an important vascular regulator that is present in the brain. Our previous study demonstrated the innate neuroprotectant role of CNP in the neonatal brain after hypoxic-ischemic (HI) insults. In this study, we further explored the role of CNP in cerebrovascular pathology using both in vivo and in vitro models. In a neonatal mouse HI brain injury model, we found that intracerebroventricular administration of recombinant CNP dose-dependently reduces brain infarct size. CNP significantly decreases brain edema and immunoglobulin G (IgG) extravasation into the brain tissue, suggesting a vasculoprotective effect of CNP. Moreover, in primary brain microvascular endothelial cells (BMECs), CNP dose-dependently protects BMEC survival and monolayer integrity against oxygen-glucose deprivation (OGD). The vasculoprotective effect of CNP is mediated by its innate receptors NPR2 and NPR3, in that inhibition of either NPR2 or NPR3 counteracts the protective effect of CNP on IgG leakage after HI insult and BMEC survival under OGD. Of importance, CNP significantly ameliorates brain atrophy and improves neurological deficits after HI insults. Altogether, the present study indicates that recombinant CNP exerts vascular protection in neonatal HI brain injury via its innate receptors, suggesting a potential therapeutic target for the treatment of neonatal HI brain injury.

Umbilical cord blood therapy modulates neonatal hypoxic ischemic brain injury in both females and males

Preclinical and clinical studies have shown that sex is a significant risk factor for perinatal morbidity and mortality, with males being more susceptible to neonatal hypoxic ischemic (HI) brain injury. No study has investigated sexual dimorphism in the efficacy of umbilical cord blood (UCB) cell therapy. HI injury was induced in postnatal day 10 (PND10) rat pups using the Rice-Vannucci method of carotid artery ligation. Pups received 3 doses of UCB cells (PND11, 13, 20) and underwent behavioural testing. On PND50, brains were collected for immunohistochemical analysis. Behavioural and neuropathological outcomes were assessed for sex differences. HI brain injury resulted in a significant decrease in brain weight and increase in tissue loss in females and males. Females and males also exhibited significant cell death, region-specific neuron loss and long-term behavioural deficits. Females had significantly smaller brains overall compared to males and males had significantly reduced neuron numbers in the cortex compared to females. UCB administration improved multiple aspects of neuropathology and functional outcomes in males and females. Females and males both exhibited injury following HI. This is the first preclinical evidence that UCB is an appropriate treatment for neonatal brain injury in both female and male neonates.

Pyk2 inhibition attenuates hypoxic-ischemic brain injury in neonatal mice.

Newborns suffering from hypoxia-ischemia (HI) brain injury still lack effective treatment. Proline-rich tyrosine kinase 2 (Pyk2) is a non-receptor tyrosine kinase, which is highly correlated with transient ischemic brain injury in adult. In this study, we investigated the role of Pyk2 in neonatal HI brain injury. HI was induced in postnatal day 7 mouse pups by unilateral common carotid artery ligation followed by hypoxic exposure. Pyk2 interference lentivirus (LV-Pyk2 shRNA) was constructed and injected into unilateral cerebral ventricle of neonatal mice before HI. Infarct volume, pathological changes, and neurological behaviors were assessed on postnatal day 8-14. We showed that the phosphorylation level of Pyk2 was significantly increased in neonatal brain after HI, whereas LV-Pyk2 shRNA injection significantly attenuated acute HI brain damage and improved neurobehavioral outcomes. In oxygen-glucose deprivation-treated cultured cortical neurons, Pyk2 inhibition significantly alleviated NMDA receptor-mediated excitotoxicity; similar results were also observed in neonatal HI brain injury. We demonstrated that Pyk2 inhibition contributes to the long-term cerebrovascular recovery assessed by laser speckle contrast imaging, but cognitive function was not obviously improved as evaluated in Morris water maze and novel object recognition tests. Thus, we constructed lentiviral LV-HIF-Pyk2 shRNA, through which HIF-1α promoter-mediated interference of Pyk2 would occur during the anoxic environment. Intracerebroventricular injection of LV-HIF-Pyk2 shRNA significantly improved long-term recovery of cognitive function in HI-treated neonatal mice. In conclusion, this study demonstrates that Pyk2 interference protects neonatal brain from hypoxic-ischemic injury. HIF-1α promoter-mediated hypoxia conditional control is a useful tool to distinguish between hypoxic period and normal period. Pyk2 is a promising drug target for potential treatment of neonatal HI brain injury.

Endothelial-specific insulin receptor substrate-1 overexpression worsens neonatal hypoxic-ischemic brain injury via mTOR-mediated tight junction disassembly

Hypoxic-ischemic (HI) encephalopathy is the major cause of mortality and disability in newborns. The neurovascular unit is a major target of acute and chronic brain injury, and therapies that protect simultaneously both neurons and vascular endothelial cells from neonatal HI injury are in demand. Insulin receptors and its key downstream molecule-insulin receptor substrate −1 (IRS-1) are potential neuroprotective targets and expressed both in neuron and endothelial cells. To investigate whether IRS-1 can act similarly in neurons and vascular endothelial cells in protecting neurovascular units and brain form HI injury, we found that neuron-specific IRS-1 transgenic rats showed reduced neurovascular injury and infarct volumes, whereas endothelial-specific IRS-1 transgenic rats showed increased blood-brain barrier (BBB) disruption and exaggerated neurovascular injury after neonatal HI brain injury. Endothelial-specific IRS-1 overexpression increased vascular permeability and disassembled the tight junction protein (zonula occludens-1) complex. Inhibition of mammalian target of rapamycin (mTOR) by rapamycin preserved tight junction proteins and attenuated BBB leakage and neuronal apoptosis after HI in the endothelial-specific IRS-1 transgenic pups. Together, our findings suggested that neuronal and endothelial IRS-1 had opposite effects on the neurovascular integrity and damage after neonatal HI brain injury and that endothelial IRS-1 worsens neurovascular integrity after HI via mTOR-mediated tight junction protein disassembly.

Biphalin, a dimeric opioid peptide, reduces neonatal hypoxia-ischemia brain injury in mice by the activation of PI3K/Akt signaling pathway

Previous studies have demonstrated that the activation of delta opioid receptors is neuroprotective against neonatal hypoxia-ischemia (HI) brain injury. The aim of this study was to investigate the neuroprotective effects of biphalin, a dimeric opioid peptide, in a mouse model of neonatal HI and the underlying mechanisms. On postnatal day 10, mouse pups were subjected to unilateral carotid artery ligation followed by 1 h of hypoxia (10 % O2 in N2). For treatment, biphalin (5 mg/kg, 10 mg/kg, 20 mg/kg) was administered intraperitoneally immediately after HI. The opioid antagonist naloxone or phosphatidylinositol-3-kinase inhibitor Ly294002 was administered to determine the underlying mechanisms. Infarct volume, brain edema, phosphorylated Akt and apoptosis-related proteins levels were evaluated by using a combination of 2,3,5-triphenyltetrazolium chloride staining, brain water content and Western blotting at 24 h after HI. The long-term effects of biphalin were evaluated by brain atrophy measurement, Nissl staining and neurobehavioral tests at 3 weeks post-HI. Biphalin (10 mg/kg) significantly reduced the infarct volume and ameliorated brain edema. Biphalin also had long-term protective effects against the loss of ipsilateral brain tissue and resulted in improvements in neurobehavioral outcomes. However, naloxone or Ly294002 abrogated the neuroprotective effects of biphalin. Furthermore, biphalin treatment significantly preserved phosphorylated Akt expression, increased Bcl-2 levels, and decreased Bax and cleaved caspase 3 levels after HI. These effects were also reversed by naloxone and Ly294002 respectively. In conclusion, biphalin protects against HI brain injury in neonatal mice, which might be through activation of the opioid receptor/phosphatidylinositol-3-kinase/Akt signaling pathway.

Efferocytosis Mediated Modulation of Injury after Neonatal Brain Hypoxia-Ischemia.

Neonatal brain hypoxia-ischemia (HI) is a leading cause of morbidity and long-term disabilities in children. While we have made significant progress in describing HI mechanisms, the limited therapies currently offered for HI treatment in the clinical setting stress the importance of discovering new targetable pathways. Efferocytosis is an immunoregulatory and homeostatic process of clearance of apoptotic cells (AC) and cellular debris, best described in the brain during neurodevelopment. The therapeutic potential of stimulating defective efferocytosis has been recognized in neurodegenerative diseases. In this review, we will explore the involvement of efferocytosis after a stroke and HI as a promising target for new HI therapies.

Neuronal apoptosis can be prevented by the combined therapy with melatonin and hypothermia in a neonatal rat model of hypoxic-ischemic encephalopathy.

IntroductionBirth hypoxia is a leading cause of perinatal mortality and neurological morbidity, resulting in central nervous system injury. Cerebral hypoxia and ischemia can produce a severe brain damage following a typical pattern, defined by selective vulnerability of the brain regions. The neonates are most prone to hypoxic-ischemic injuries due to the lack of efficient antioxidant defense. Neonatal hypoxia–ischemia (HI) in a 7-day-old rat HI model can produce cell death by apoptotic or necrotic mechanisms. The degree of apoptotic or necrotic mechanisms responsible for cell death in neonatal hypoxia–ischemia are not very clear as yet. The form of neuronal death may also depend on the severity of ischemic injury. Necrosis predominates in more severe cases, whereas apoptosis occurs in areas with milder ischemic injury. A human study demonstrated apoptotic and necrotic forms of cell death after hypoxic injury, whereas in some brains from stillbirths, only apoptotic figures were observed. The expression of activated caspase-3 reflects the role of apoptosis in neonatal hypoxic ischemic brain injury.ObjectivesThe aim of this study was to evaluate the possible neuroprotective effect of melatonin and hypothermia in hypoxic-ischemic encephalopathy in newborn rats. Local damages induced by hypoxia and ischemia were assessed by evaluating the changes in terms of histology and apoptosis.MethodsThe experiment was conducted on 20 newborn Wistar rats premedicated for seven days with melatonin in a dose of 20 mg/kg/day. On the 7th postnatal day (P7), the newborn rats were exposed to ischemia (by clamping the right carotid artery) and hypobaric hypoxia (8% O2 for 90 minutes) and some groups to hypothermia.ResultsIn this experimental model of neonatal encephalopathy, melatonin, in a dose of 20 mg/kg/day has neuroprotective effect by reducing the number of cells expressing apoptosis in Cornu Ammonis (CA) (Ammon’s Horn) CA1, CA2, CA3 and dentate gyrus of the hippocampus when combined with hypothermia.ConclusionThe results of this study prove that melatonin is protective in ischemic-hypoxic brain injuries, but the protection is conditioned in most of the brain regions (excepting cerebral cortex) by conjugation with post-injury hypothermia treatment.

Inhibition of Colony Stimulating Factor 1 Receptor Suppresses Neuroinflammation and Neonatal Hypoxic-Ischemic Brain Injury.

Hypoxic-ischemic (HI) brain injury is a major cause of neonatal death or lifetime disability without widely accepted effective pharmacological treatments. It has been shown that the survival of microglia requires colony-stimulating factor 1 receptor (CSF1R) signaling and microglia participate in neonatal HI brain injury. We therefore hypothesize that microglia depletion during a HI insult period could reduce immature brain injury. In this study, CD1 mouse pups were treated with a CSF1R inhibitor (PLX3397, 25 mg/kg/daily) or a vehicle from postnatal day 4 to day 11 (P4–11), and over 90% of total brain microglia were deleted at P9. Unilateral hemisphere HI injury was induced at P9 by permanently ligating the left common carotid arteries and exposing the pups to 10% oxygen for 30 min to produce moderate left hemisphere injury. We found that the PLX3397 treatment reduced HI brain injury by 46.4%, as evaluated by the percentage of brain infarction at 48 h after HI. Furthermore, CSF1R inhibition suppressed the infiltration of neutrophils (69.7% reduction, p = 0.038), macrophages (77.4% reduction, p = 0.009), and T cells (72.9% reduction, p = 0.008) to the brain, the production of cytokines and chemokines (such as CCL12, CCL6, CCL21, CCL22, CCL19, IL7, CD14, and WISP-1), and reduced neuronal apoptosis as indicated by active caspase-3 labeled cells at 48 h after HI (615.20 ± 156.84/mm2 vs. 1,205.00 ± 99.15/mm2, p = 0.013). Our results suggest that CSF1R inhibition suppresses neuroinflammation and neonatal brain injury after acute cerebral hypoxia-ischemia in neonatal mice.

Sodium 4-Phenylbutyrate Protects Hypoxic-Ischemic Brain Injury via Attenuating Endoplasmic Reticulum Stress in Neonatal Rats.

Neonatal hypoxic-ischemic (HI) brain injury is associated with long-term neurological disorders, and protective strategies are presently scarce. Sodium 4-phenylbutyrate (4-PBA) reportedly acts as a chemical chaperone that alleviates endoplasmic reticulum (ER) stress, which plays a critical role in neurological diseases. The present study aimed to evaluate the neuroprotective effects of 4-PBA on HI-induced neonatal brain injury in a rat model, and to characterize possible underlying mechanisms. The HI brain injury model was established by ligating the left common carotid artery in 7-day-old rats, followed by exposure to 8% oxygen for 2 h. The 4-PBA or vehicle was administered by an intracerebroventricular injection 30 min before HI. The protein expression levels of ER stress markers (GRP78, ATF6, and CHOP) were detected by western blotting at 24 h after HI insult. The activation of cAMP-response element-binding protein (CREB) was evaluated by western blotting and immunofluorescence. TUNEL and Nissl staining were performed to detect the histomorphological changes in the hippocampal neurons at 24 h and 7 days, respectively, after HI injury. From days 29 to 34 after brain HI, rats underwent Morris water maze tests to assess cognitive functioning. The results showed that pretreatment with 4-PBA decreased HI-induced excessive ER stress and neuronal injury. Moreover, CREB activation might be involved in the beneficial effects of 4-PBA on HI-induced learning and memory deficits in rats. In conclusion, the present study suggested a potential therapeutic approach of ER stress inhibition in the treatment of neonatal HI brain injury.

A Review of Plant Extracts and Plant-Derived Natural Compounds in the Prevention/Treatment of Neonatal Hypoxic-Ischemic Brain Injury.

Neonatal hypoxic-ischemic (HI) brain injury is one of the major drawbacks of mortality and causes significant short/long-term neurological dysfunction in newborn infants worldwide. To date, due to multifunctional complex mechanisms of brain injury, there is no well-established effective strategy to completely provide neuroprotection. Although therapeutic hypothermia is the proven treatment for hypoxic-ischemic encephalopathy (HIE), it does not completely chang outcomes in severe forms of HIE. Therefore, there is a critical need for reviewing the effective therapeutic strategies to explore the protective agents and methods. In recent years, it is widely believed that there are neuroprotective possibilities of natural compounds extracted from plants against HIE. These natural agents with the anti-inflammatory, anti-oxidative, anti-apoptotic, and neurofunctional regulatory properties exhibit preventive or therapeutic effects against experimental neonatal HI brain damage. In this study, it was aimed to review the literature in scientific databases that investigate the neuroprotective effects of plant extracts/plant-derived compounds in experimental animal models of neonatal HI brain damage and their possible underlying molecular mechanisms of action.

MicroRNA-210 downregulates TET2 and contributes to inflammatory response in neonatal hypoxic-ischemic brain injury

BackgroundNeonatal hypoxic-ischemic (HI) brain injury is a leading cause of acute mortality and chronic disability in newborns. Our previous studies demonstrated that HI insult significantly increased microRNA-210 (miR-210) in the brain of rat pups and inhibition of brain endogenous miR-210 by its inhibitor (LNA) provided neuroprotective effect in HI-induced brain injury. However, the molecular mechanisms underpinning this neuroprotection remain unclear.MethodsWe made a neonatal HI brain injury model in mouse pups of postnatal day 7 to uncover the mechanism of miR-210 in targeting the ten eleven translocation (TET) methylcytosine dioxygenase 2 that is a transcriptional suppressor of pro-inflammatory cytokine genes in the neonatal brain. TET2 silencing RNA was used to evaluate the role of TET2 in the neonatal HI-induced pro-inflammatory response and brain injury. MiR-210 mimic and inhibitor (LNA) were delivered into the brain of mouse pups to study the regulation of miR-210 on the expression of TET2. Luciferase reporter gene assay was performed to validate the direct binding of miR-210 to the 3′ untranslated region of the TET2 transcript. Furthermore, BV2 mouse microglia cell line was employed to confirm the role of miR-210-TET2 axis in regulating pro-inflammatory response in microglia. Post-assays included chromatin immunoprecipitation (ChIP) assay, co-immunoprecipitation, RT-PCR, brain infarct assay, and neurobehavioral test. Student’s t test or one-way ANOVA was used for statistical analysis.ResultsHI insult significantly upregulated miR-210, downregulated TET2 protein abundance, and increased NF-κB subunit p65 acetylation level and its DNA binding capacity to the interleukin 1 beta (IL-1β) promoter in the brain of mouse pups. Inhibition of miR-210 rescued TET2 protein level from HI insult and miR-210 mimic decreased TET2 protein level in the brain of mouse pups, suggesting that TET2 is a functional target of miR-210. The co-immunoprecipitation was performed to reveal the role of TET2 in HI-induced inflammatory response in the neonatal brain. The result showed that TET2 interacted with NF-κB subunit p65 and histone deacetylase 3 (HDAC3), a co-repressor of gene transcription. Furthermore, TET2 knockdown increased transcriptional activity of acetyl-p65 on IL-1β gene in the neonatal brain and enhanced HI-induced upregulation of acetyl-p65 level and pro-inflammatory cytokine expression. Of importance, TET2 knockdown exacerbated brain infarct size and neurological deficits and counteracted the neuroprotective effect of miR-210 inhibition. Finally, the in vitro results demonstrated that the miR-210-TET2 axis regulated pro-inflammatory response in BV2 mouse microglia cell line.ConclusionsThe miR-210-TET2 axis regulates pro-inflammatory cytokine expression in microglia, contributing to neonatal HI brain injury.

MicroRNAs in the Blood-Brain Barrier in Hypoxic-Ischemic Brain Injury.

Hypoxic-ischemic (HI) brain injury is a leading cause of acute mortality and chronic disability in newborns. Current evidence shows that cerebral microvascular response and compromised blood-brain barrier (BBB) integrity occur rapidly and could primarily be responsible for the brain injury observed in many infants with HI brain injury. MicroRNAs (miRNAs) are a type of highly conserved non-coding RNAs (ncRNAs), which consist of 21-25 nucleotides in length and usually lead to suppression of target gene expression. Growing evidence has revealed that brain-enriched miRNAs act as versatile regulators of BBB dysfunctions in various neurological disorders including neonatal HI brain injury. In the present review, we summarize the current findings regarding the role of miRNAs in BBB impairment after hypoxia/ischemia brain injury. Specifically, we focus on the recent progress of miRNAs in the pathologies of neonatal HI brain injury. These findings can not only deepen our understanding of the role of miRNAs in BBB impairment in HI brain injury, but also provide insight into the development of new therapeutic strategies for preservation of BBB integrity under pathological conditions.

Brain-targeted hypoxia-inducible factor stabilization reduces neonatal hypoxic-ischemic brain injury

Hypoxia-inducible factor-1α (HIF1α) is a major regulator of cellular adaptation to hypoxia and oxidative stress, and recent advances of prolyl-4-hydroxylase (P4H) inhibitors have produced powerful tools to stabilize HIF1α for clinical applications. However, whether HIF1α provokes or resists neonatal hypoxic-ischemic (HI) brain injury has not been established in previous studies.We hypothesize that systemic and brain-targeted HIF1α stabilization may have divergent effects.To test this notion, herein we compared the effects of GSK360A, a potent P4H inhibitor, in in-vitro oxygen-glucose deprivation (OGD) and in in-vivo neonatal HI via intracerebroventricular (ICV), intraperitoneal (IP), and intranasal (IN) drug-application routes. We found that GSK360A increased the erythropoietin (EPO), heme oxygenase-1 (HO1) and glucose transporter 1 (Glut1) transcripts, all HIF1α target-genes, and promoted the survival of neurons and oligodendrocytes after OGD. Neonatal HI insult stabilized HIF1α in the ipsilateral hemisphere for up to 24 h, and either ICV or IN delivery of GSK360A after HI increased the HIF1α target-gene transcripts and decreased brain damage. In contrast, IP-injection of GSK360A failed to reduce HI brain damage, but elevated the risk of mortality at high doses, which may relate to an increase of the kidney and plasma EPO, leukocytosis, and abundant vascular endothelial growth factor (VEGF) mRNAs in the brain. These results suggest that brain-targeted HIF1α-stabilization is a potential treatment of neonatal HI brain injury, while systemic P4H-inhibition may provoke unwanted adverse effects.

Lycopene Exerts Neuroprotective Effects After Hypoxic-Ischemic Brain Injury in Neonatal Rats via the Nuclear Factor Erythroid-2 Related Factor 2/Nuclear Factor-κ-Gene Binding Pathway.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a brain injury caused by perinatal asphyxia and is the main cause of neonatal death and chronic neurological diseases. Protection of neuron after hypoxic-ischemic (HI) brain injury is considered as a potential therapeutic target of HI brain injury. To date, there are no effective medicines for neonatal HI brain injury. Lycopene (Lyc), a member of the carotenoids family, has been reported to have anti-oxidative and anti-inflammatory effects. However, its effects and potential mechanisms in HI brain injury have not yet to be systematically evaluated. In this study, we investigated whether Lyc could ameliorate HI brain injury and explored the associated mechanism both in vivo and in vitro experiments. In vivo study, Lyc significantly reduced infarct volume and ameliorated cerebral edema, decreased inflammatory response, promoted the recovery of tissue structure, and improved prognosis following HI brain injury. In vitro study, results showed that Lyc reduced expression of apoptosis mediators in oxygen-glucose deprivation (OGD)-induced primary cortical neurons. Mechanistically, we found that Lyc-induced Nrf2/NF-κB pathway could partially reversed by Brusatol (an Nrf2 inhibitor), indicated that the Nrf2/NF-κB pathway was involved in the therapy of Lyc. In summary, our findings indicate that Lyc can attenuated HI brain injury in vivo and OGD-induced apoptosis of primary cortical neurons in vitro through the Nrf2/NF-κB signaling pathway.

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.

Early Blood Biomarkers Distinguish Inflammation from Neonatal Hypoxic-Ischemia Encephalopathy.

Neonatal hypoxic-ischemic encephalopathy is the most common cause of neurological disability in infancy. Superimposed inflammation may further worsen neurological outcomes. Reliable biomarkers which are both sensitive to hypoxic-ischemia and inflammation are critically needed. We tested plasma osteopontin (OPN) and glial fibrillary astrocytic protein (GFAP) within the reported therapeutic window (90min after hypoxic-ischemic (HI) injury) in neonatal rats with different HI severity and inflammation. Two different HI severity groups (mild-HI with 75min hypoxia and severe-HI with 150min hypoxia) were established. Inflammation-sensitized HI brain injury induced by lipopolysaccharide (LPS) further increased apoptotic neurons and infarct volumes. In HI alone groups, OPN was significantly decreased (p < 0.001) but GFAP was slightly increased (p < 0.05) at 90min after HI either in mild-HI or severe-HI compared with naïve group. In LPS-sensitized HI groups, both OPN and GFAP were significantly increased either in LPS-mild-HI or LPS-severe-HI groups compared with the naïve group (all p < 0.05). Induced inflammation by LPS exaggerated neonatal HI brain injury. The plasma OPN and GFAP levels may be useful to differentiate HI alone groups from inflammation-sensitized HI groups or naïve group.

Neuroprotective role of Nrf2 on hypoxic-ischemic brain injury in neonatal mice.

Inflammation and oxidative stress play a key role in mediating the pathophysiology of hypoxic-ischemic (HI) brain injury. Nrf2 is a transcriptional factor that contributes to the innate defense of the body against oxidative stress and inflammation. The current study investigated the effect of Nrf2 in neonatal HI brain injury using Nrf2-/- mice. Nrf2-/- and wild-type Nrf2+/+ mice on a C57BL/6J background at postnatal day 9 underwent unilateral common carotid artery ligation, followed by hypoxia. Brain damage was determined by infarct size measurement. Apoptosis was evaluated by measuring the expression of Bax and Bcl-2. The levels of inflammatory cytokines and mediators involved in oxidative stress were measured. Nrf2 knockout exacerbated HI injury-induced brain infarct and cell apoptosis in the brain. Nrf2-/- mice showed increased inflammatory cytokines and MDA, and reduced activities of antioxidant enzymes including CAT, GSH-Px, and SOD. Nrf2-/- mice showed reduced HO-1 expression after HI injury compared with wild-type mice. This study supported a protective effect of Nrf2 in neonatal HI brain injury.

Protective effects of FGF10 on neurovascular unit in a rat model of neonatal hypoxic-ischemic brain injury

Neonatal hypoxic-ischemic (HI) brain injury remains a devastating clinical disease associated with high mortality and lifetime disability. Neonatal HI injury damages the architecture of neurovascular unit (NVU), thus, therapy targeting the NVU may provide effective neuroprotection against HI. This study was designed to investigate whether fibroblast growth factor 10 (FGF10) protected the NVU against HI and afforded observable neuroprotection in a rat model of neonatal HI brain injury. The results showed that FGF10 treatment significantly reduced brain damage post HI, characterized by reduction in brain infarct volume and tissue loss. Further interesting findings showed that FGF10 treatment exerted neuroprotective effects on HI brain injury in neonate rats through protecting the NVU against HI, evidenced by inhibition of neuronal cell apoptosis, suppression of gliosis, and amelioration of blood-brain barrier disruption. Collectively, our study indicates that FGF10 treatment exhibits great potential for protecting NVU against HI and attenuates neonatal brain injury, suggesting a potential novel therapeutic agent to this disease.

Changes in arginase isoforms in a murine model of neonatal brain hypoxia-ischemia.

Background:Arginases (ARG-isoforms, ARG-1/ARG-2) are key regulatory enzymes of inflammation and tissue repair, however their role after neonatal brain hypoxia (H) and hypoxia-ischemia (HI) remains unknown.Methods:C57BL/6-mice subjected to the Vannucci procedure on postnatal day 9 were sacrificed at different timepoints. The degree of brain damage was assessed histologically. ARG spatiotemporal localization was determined via immunohistochemistry. ARG expression was measured by Western blot and activity spectrophotometrically.Results:ARG-isoforms expression increased during neurodevelopment (P9-P17) in the cortex and hippocampus. This was suppressed with H and HI only in the hippocampus. In the cortex, both isoforms increased with H alone and only ARG-2 increased with HI at 3 d. ARG activity during neurodevelopment remained unchanged but increased at 1 d with H and not HI. ARG-1 localized with microglia at the injury site as early as 4 h after injury, while ARG-2 localized with neurons.Conclusions:ARG-isoforms expression increases with age from P9-P17 but is suppressed by injury specifically in the hippocampus and not in the cortex. Both, levels and activity of ARG-isoforms increase with H while ARG-1 immunolabelling is upregulated in the HI cortex. Evidently, ARG-isoforms in brain differ in spatiotemporal localization, expression and activity during neurodevelopment and after injury.

Mean Diffusivity in Striatum Correlates With Acute Neuronal Death but Not Lesser Neuronal Injury in a Pilot Study of Neonatal Piglets With Encephalopathy.

Diffusion MRI is routinely used to evaluate brain injury in neonatal encephalopathy. Although abnormal mean diffusivity (MD) is often attributed to cytotoxic edema, the specific contribution from neuronal pathology is unclear. To determine whether MD from high-resolution diffusion tensor imaging (DTI) can detect variable degrees of neuronal degeneration and pathology in piglets with brain injury induced by excitotoxicity or global hypoxia-ischemia (HI) with or without overt infarction. Prospective. Excitotoxic brain injury was induced in six neonatal piglets by intrastriatal stereotaxic injection of the glutamate receptor agonist quinolinic acid (QA). Three piglets underwent global HI or a sham procedure. Piglets recovered for 20-96 hours before undergoing MRI (n = 9). 3.0T MRI with DTI, T1 - and T2 -weighted imaging. MD, fractional anisotropy (FA), and qualitative T2 injury were assessed in the putamen and caudate. The cell bodies of normal neurons, degenerating neurons (excitotoxic necrosis, ischemic necrosis, or necrosis-apoptosis cell death continuum), and injured neurons with equivocal degeneration were counted by histopathology. Spearman correlations were used to compare MD and FA to normal, degenerating, and injured neurons. T2 injury and neuron counts were evaluated by descriptive analysis. The QA insult generated titratable levels of neuronal pathology. In QA, HI, and sham piglets, lower MD correlated with higher ratios of degenerating-to-total neurons (P < 0.05), lower ratios of normal-to-total neurons (P < 0.05), and greater numbers of degenerating neurons (P < 0.05). MD did not correlate with abnormal neurons exhibiting nascent injury (P > 0.99). Neuron counts were not related to FA (P > 0.30) or to qualitative injury from T2 -weighted MRI. MD is more accurate than FA for detecting neuronal degeneration and loss during acute recovery from neonatal excitotoxic and HI brain injury. MD does not reliably detect nonfulminant, nascent, and potentially reversible neuronal injury. 1 TECHNICAL EFFICACY: Stage 2 J. Magn. Reson. Imaging 2020;52:1216-1226.