Cerebral Hypoxia-ischemia Research Articles

Optimization of an Intranasal Route for the Delivery of Human Neural Stem Cells to Treat a Neonatal Hypoxic-Ischemic Brain Injury Rat Model.

ObjectiveStem cell administration via the intranasal route has shown promise as a new therapy for hypoxic-ischemic encephalopathy (HIE). In this study, we aimed to improve the intranasal delivery of stem cells to the brain.MethodsHuman neural stem cells (hNSCs) were identified using immunofluorescence, morphological, and flow cytometry assays before transplantation, and cell migration capacity was examined using the transwell assay. Cerebral hypoxia-ischemia (HI) was induced in 7-day-old rats, followed by the intranasal transplantation of CM-Dil-labeled hNSCs. We examined various experimental conditions, including preconditioning hNSCs with hypoxia, catheter method, multiple low-dose transplantation, head position, cell appropriate concentration, and volume. Rats were sacrificed 1 or 3 days after the final intranasal administration, and parts of the nasal tissue and whole brain sections were analyzed under a fluorescence microscope.ResultsThe isolated hNSCs met the characteristics of neural stem cells. Hypoxia (5% O2, 24 h) enhanced the surface expression of CXC chemokine receptor 4 (CXCR4) (9.21 ± 1.9% ~ 24.76 ± 2.24%, P < 0.01) on hNSCs and improved migration (toward stromal cell-derived factor 1 [SDF-1], 0.54 ± 0.11% ~ 8.65 ± 1.76%, P < 0.001; toward fetal bovine serum, 8.36 ± 0.81% ~ 21.74 ± 0.85%, P < 0.0001). Further improvement increased the number of surviving cell distribution with increased uniformity on the olfactory epithelium and allowed the cells to stay in the nasal cavity for at least 72 h, but they did not survive for longer than 48 h. Optimization of pre-transplantation conditions augmented the success rate of intranasally delivered cells to the brain (0–41.6%). We also tentatively identified that hNSCs crossed the olfactory epithelium into the tissue space below the lamina propria, with cerebrospinal fluid entering the cribriform plate into the subarachnoid space, and then migrated toward injured areas along the brain blood vessels.ConclusionThis study offers some helpful advice and reference for addressing the problem of repeatability in the intranasal delivery of stem cells.

Effects of delayed HIF-1α expression in astrocytes on myelination following hypoxia-ischaemia white matter injury in immature rats.

BackgroundThe underlying cause of neurological sequelae after immature cerebral hypoxia-ischaemia (HI) white matter injury is impaired myelination. Previous studies have indicated that astrocyte activation is closely related to impaired myelination. However, the mechanism of reactive gliosis in white matter injury post-HI remains poorly understood.MethodsStudies using adult ischaemic animal models demonstrated that hypoxia inducible factor-1α (HIF-1α) expression was involved in the formation of reactive astrocytes. Here, we investigated the temporal expression of HIF-1α and its impact on reactive gliosis and further myelination using a perinatal HI white matter injury model induced in rats at postnatal day 3. The temporal pattern of HIF-1α expression post-HI injury was tested by western blotting and immunofluorescence. Rats were treated with a HIF-1α inhibitor at 72 hours post-HI injury. Reactive gliosis and myelination were assessed with western blotting, immunofluorescence and electron microscopy, and neurological functions were examined by behavioural testing.ResultsOur results showed that the expression of HIF-1α was upregulated in neurons at 24 hours and in astrocytes at 7 days post-HI. Inhibiting delayed HIF-1α expression post-HI injury could restrain reactive gliosis, ameliorate hypomyelination, and improve the performance of rats in the Morris water maze test.ConclusionsOur findings suggest that a delayed increase in HIF-1α in astrocytes is involved in glial scar formation and leads to arrested oligodendrocyte maturation, impaired myelination, and long-term neurological function after experimental white matter injury in immature rats.

Creatine transporter deficiency impairs stress adaptation and brain energetics homeostasis.

The creatine transporter (CrT) maintains brain creatine (Cr) levels, but the effects of its deficiency on energetics adaptation under stress remain unclear. There are also no effective treatments for CrT deficiency, the second most common cause of X-linked intellectual disabilities. Herein, we examined the consequences of CrT deficiency in brain energetics and stress-adaptation responses plus the effects of intranasal Cr supplementation. We found that CrT-deficient (CrT–/y) mice harbored dendritic spine and synaptic dysgenesis. Nurtured newborn CrT–/y mice maintained baseline brain ATP levels, with a trend toward signaling imbalance between the p-AMPK/autophagy and mTOR pathways. Starvation elevated the signaling imbalance and reduced brain ATP levels in P3 CrT–/y mice. Similarly, CrT–/y neurons and P10 CrT–/y mice showed an imbalance between autophagy and mTOR signaling pathways and greater susceptibility to cerebral hypoxia-ischemia and ischemic insults. Notably, intranasal administration of Cr after cerebral ischemia increased the brain Cr/N-acetylaspartate ratio, partially averted the signaling imbalance, and reduced infarct size more potently than intraperitoneal Cr injection. These findings suggest important functions for CrT and Cr in preserving the homeostasis of brain energetics in stress conditions. Moreover, intranasal Cr supplementation may be an effective treatment for congenital CrT deficiency and acute brain injury.

Transient Oxygen-Glucose Deprivation Causes Region- and Cell Type-Dependent Functional Deficits in the Mouse Hippocampus In Vitro.

Neurons are highly vulnerable to conditions of hypoxia-ischemia (HI) such as stroke or transient ischemic attacks. Recovery of cognitive and behavioral functions requires re-emergence of coordinated network activity, which, in turn, relies on the well-orchestrated interaction of pyramidal cells (PYRs) and interneurons. We therefore modelled HI in the mouse hippocampus, a particularly vulnerable region showing marked loss of PYR and fast-spiking interneurons (FSIs) after hypoxic-ischemic insults. Transient oxygen-glucose deprivation (OGD) in ex vivo hippocampal slices led to a rapid loss of neuronal activity and spontaneous network oscillations (sharp wave-ripple complexes; SPW-Rs), and to the occurrence of a spreading depolarization. Following reperfusion, both SPW-R and neuronal spiking resumed, but FSI activity remained strongly reduced compared with PYR. Whole-cell recordings in CA1 PYR revealed, however, a similar reduction of both EPSCs and IPSCs, leaving inhibition-excitation (I/E) balance unaltered. At the network level, SPW-R incidence was strongly reduced and the remaining network events showed region-specific changes including reduced ripple energy in CA3 and increased ripple frequency in CA1. Together, our data show that transient hippocampal energy depletion results in severe functional alterations at the cellular and network level. While I/E balance is maintained, synaptic activity, interneuron spiking and coordinated network patterns remain reduced. Such alterations may be network-level correlates of cognitive and functional deficits after cerebral HI.

Oleuropein Activates Neonatal Neocortical Proteasomes, but Proteasome Gene Targeting by AAV9 Is Variable in a Clinically Relevant Piglet Model of Brain Hypoxia-Ischemia and Hypothermia.

Cerebral hypoxia-ischemia (HI) compromises the proteasome in a clinically relevant neonatal piglet model. Protecting and activating proteasomes could be an adjunct therapy to hypothermia. We investigated whether chymotrypsin-like proteasome activity differs regionally and developmentally in the neonatal brain. We also tested whether neonatal brain proteasomes can be modulated by oleuropein, an experimental pleiotropic neuroprotective drug, or by targeting a proteasome subunit gene using recombinant adeno-associated virus-9 (AAV). During post-HI hypothermia, we treated piglets with oleuropein, used AAV-short hairpin RNA (shRNA) to knock down proteasome activator 28γ (PA28γ), or enforced PA28γ using AAV-PA28γ with green fluorescent protein (GFP). Neonatal neocortex and subcortical white matter had greater proteasome activity than did liver and kidney. Neonatal white matter had higher proteasome activity than did juvenile white matter. Lower arterial pH 1 h after HI correlated with greater subsequent cortical proteasome activity. With increasing brain homogenate protein input into the assay, the initial proteasome activity increased only among shams, whereas HI increased total kinetic proteasome activity. OLE increased the initial neocortical proteasome activity after hypothermia. AAV drove GFP expression, and white matter PA28γ levels correlated with proteasome activity and subunit levels. However, AAV proteasome modulation varied. Thus, neonatal neocortical proteasomes can be pharmacologically activated. HI slows the initial proteasome performance, but then augments ongoing catalytic activity. AAV-mediated genetic manipulation in the piglet brain holds promise, though proteasome gene targeting requires further development.

HSP70 inhibition suppressed glioma cell viability during hypoxia/reoxygenation by inhibiting the ERK1/2 and PI3K/AKT signaling pathways.

Heat shock protein 70 (HSP70) can regulate astrocyte viability under hypoxic and ischemic conditions. However, the protective mechanism involved is not completely clear. This study aimed to investigate whether HSP70 protects U87 glioma cells against hypoxic damage via the extracellular signal-regulated kinases 1/2 (ERK1/2) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathways. Lentivirus-mediated HSP70-siRNA was used for HSP70 silencing. U87 glioma cells with lentiviral infection were exposed to hypoxia for 4, 8, 12, and 24h, respectively, followed by a 24-h reoxygenation treatment. A Cell-Counting Kit-8 was then used to evaluate the viability of the U87 glioma cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting were performed to determine the mRNA and protein levels, respectively. The expression of HSP70, p-ERK1/2, p-AKT, and U87 cell viability were increased after 8h of hypoxia/24h of reoxygenation (P < 0.01). However, HSP70 silencing significantly decreased the U87 cell viability after the hypoxia/reoxygenation treatment (P < 0.01). The protein expressions of p-ERK1/2 and p-AKT also decreased in HSP70-silenced U87 cells (P < 0.01). Inconclusion,HSP70 inhibition suppressed the viability of U87 glioma cells during hypoxia/reoxygenation (at least partially) by inhibiting the ERK1/2 and PI3K/AKT signaling pathways. This study may help to understand the molecular mechanisms underlying the progression and development of cerebral hypoxia-ischemia.

Corrigendum: Offspring of rats with cerebral hypoxia-ischemia manifest cognitive dysfunction in learning and memory abilities.

[This corrects the article DOI: 10.4103/1673-5374.276359].

Hydroxycarboxylic Acid Receptor 1 and Neuroprotection in a Mouse Model of Cerebral Ischemia-Reperfusion.

Lactate is an intriguing molecule with emerging physiological roles in the brain. It has beneficial effects in animal models of acute brain injuries and traumatic brain injury or subarachnoid hemorrhage patients. However, the mechanism by which lactate provides protection is unclear. While there is evidence of a metabolic effect of lactate providing energy to deprived neurons, it can also activate the hydroxycarboxylic acid receptor 1 (HCAR1), a Gi-coupled protein receptor that modulates neuronal firing rates. After cerebral hypoxia-ischemia, endogenously produced brain lactate is largely increased, and the exogenous administration of more lactate can decrease lesion size and ameliorate the neurological outcome. To test whether HCAR1 plays a role in lactate-induced neuroprotection, we injected the agonists 3-chloro-5-hydroxybenzoic acid and 3,5-dihydroxybenzoic acid into mice subjected to 30-min middle cerebral artery occlusion. The in vivo administration of HCAR1 agonists at reperfusion did not appear to exert any relevant protective effect as seen with lactate administration. Our results suggest that the protective effects of lactate after hypoxia-ischemia come rather from the metabolic effects of lactate than its signaling through HCAR1.

Moderately Inducing Autophagy Reduces Tertiary Brain Injury after Perinatal Hypoxia-Ischemia.

Recent studies of cerebral hypoxia-ischemia (HI) have highlighted slowly progressive neurodegeneration whose mechanisms remain elusive, but if blocked, could considerably improve long-term neurological function. We previously established that the cytokine transforming growth factor (TGF)β1 is highly elevated following HI and that delivering an antagonist for TGFβ receptor activin-like kinase 5 (ALK5)—SB505124—three days after injury in a rat model of moderate pre-term HI significantly preserved the structural integrity of the thalamus and hippocampus as well as neurological functions associated with those brain structures. To elucidate the mechanism whereby ALK5 inhibition reduces cell death, we assessed levels of autophagy markers in neurons and found that SB505124 increased numbers of autophagosomes and levels of lipidated light chain 3 (LC3), a key protein known to mediate autophagy. However, those studies did not determine whether (1) SB was acting directly on the CNS and (2) whether directly inducing autophagy could decrease cell death and improve outcome. Here we show that administering an ALK5 antagonist three days after HI reduced actively apoptotic cells by ~90% when assessed one week after injury. Ex vivo studies using the lysosomal inhibitor chloroquine confirmed that SB505124 enhanced autophagy flux in the injured hemisphere, with a significant accumulation of the autophagic proteins LC3 and p62 in SB505124 + chloroquine treated brain slices. We independently activated autophagy using the stimulatory peptide Tat-Beclin1 to determine if enhanced autophagy is directly responsible for improved outcomes. Administering Tat-Beclin1 starting three days after injury preserved the structural integrity of the hippocampus and thalamus with improved sensorimotor function. These data support the conclusion that intervening at this phase of injury represents a window of opportunity where stimulating autophagy is beneficial.

SNAP25 protects primary cortical neurons from hypoxic-ischemic injury associated with CREB signal.

Hypoxic-ischemic encephalopathy (HIE) could induce exacerbated changes and unpredictable effects in brain cells, and the mechanism remains unclear. HIE model was established in neonatal rats, Zea-Longa score and TTC staining were used to observe the neurobehavior and brain infarct volume in rats subjected to cerebral hypoxia-ischemia (HI). Primary cortical neurons were then cultured in vitro to establish an oxygen and glucose deprivation model. To determine the role of synaptosomal-associated protein-25 (SNAP25) in HIE, PC12 cells were cultured and effective siRNA fragments were screened, and SNAP25 was transfected into primary neurons. Then, quantitative real-time polymerase chain reaction was used to detect the mRNA expression level and immunofluorescence staining was used to observe the morphological changes of neurons before and after the injury. Finally, the abundance values of SNAP25 and its associated genes were filtered using the NCBI and GeneMANIA, respectively. HI leads to a decrease in neuronal number and an increase in SNAP25 expression. Whereas, the interference of SNAP25 caused marked decrease in neuronal number and the length of neurite. Moreover, the expression levels of CREB and SYP were significantly decreased after interference of SNAP25. SNAP25 exhibited several neuroprotective effects to neuronal protection in neonatal cerebral HI by regulating CREB and SYP.

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.

Up-regulating lncRNA OIP5-AS1 protects neuron injury against cerebral hypoxia-ischemia induced inflammation and oxidative stress in microglia/macrophage through activating CTRP3 via sponging miR-186-5p

BackgroundInflammation and oxidative stress is closely associated with the development of ischemic brain stroke. Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1), a novel identified long non-coding RNA (lncRNA), has been suggested to play an important role in the development of many types of human cancers. However, the functional involvement of OIP5-AS1 in ischemic stroke is still unknown. MethodsQuantitative real-time polymerase chain reaction and /or western blot were conducted to determine the expression profiles of OIP5-AS1, C1q/TNF-related protein 3 (CTRP3) and miR-186-5p in the serum of stroke patients, as well as in the ischemic penumbra of rats with middle cerebral artery occlusion/reperfusion (MCAO/R) injury and microglial cells treated with oxygen glucose deprivation/re-oxygenation (OGD/R). Upon selective regulation of OIP5-AS1 and miR-186-5p, the inflammation and oxidative stress responses in microglia/macrophage as well as neurologic functions in MCAO/R rats were detected. Furthermore, the interactions between OIP5-AS1 and miR-186-5p, miR-186-5p and CTRP3 were investigated by RNA immunoprecipitation (RIP) assay, luciferase report assay and bioinformation anaylsis. ResultsWe observed markedly increased infarct volume, neuronal apoptosis, inflammation and oxidative stress responses in the infarcted lesions of MCAO/R rats, in line with down-regulated levels of OIP5-AS1 and CTRP3 while up-regulated miR-186-5p. Functional studies demonstrated that up-regulation of OIP5-AS1 attenuated infarct volume, neuronal apoptosis, microglia/macrophage inflammation and oxidative stress responses induced by MCAO/R or OGD/R. In terms of mechanism, we revealed that OIP5-AS1-miR-186-5p-CTRP3 axis played a vital role in modulating microglia/macrophage activation and neuronal apoptosis. ConclusionUp-regulating lncRNA OIP5-AS1 protects neuron injury against MCAO/R induced inflammation and oxidative stress in microglia/macrophage through activating CTRP3 via sponging miR-186-5p.

Hypoxia, Oxidative Stress, and Inflammation: Three Faces of Neurodegenerative Diseases.

The cerebral hypoxia-ischemia can induce a wide spectrum of biologic responses that include depolarization, excitotoxicity, oxidative stress, inflammation, and apoptosis, and result in neurodegeneration. Several adaptive and survival endogenous mechanisms can also be activated giving an opportunity for the affected cells to remain alive, waiting for helper signals that avoid apoptosis. These signals appear to help cells, depending on intensity, chronicity, and proximity to the central hypoxic area of the affected tissue. These mechanisms are present not only in a large list of brain pathologies affecting commonly older individuals, but also in other pathologies such as refractory epilepsies, encephalopathies, or brain trauma, where neurodegenerative features such as cognitive and/or motor deficits sequelae can be developed. The hypoxia inducible factor 1α (HIF-1α) is a master transcription factor driving a wide spectrum cellular response. HIF-1α may induce erythropoietin (EPO) receptor overexpression, which provides the therapeutic opportunity to administer pharmacological doses of EPO to rescue and/or repair affected brain tissue. Intranasal administration of EPO combined with other antioxidant and anti-inflammatory compounds could become an effective therapeutic alternative, to avoid and/or slow down neurodegenerative deterioration without producing adverse peripheral effects.

Human umbilical cord mesenchymal stromal cells as an adjunct therapy with therapeutic hypothermia in a piglet model of perinatal asphyxia

Background: With therapeutic hypothermia (HT) for neonatal encephalopathy, disability rates are reduced, but not all babies benefit. Pre-clinical rodent studies suggest mesenchymal stromal cells (MSCs) augment HT protection. Aims:The authors studied the efficacy of intravenous (IV) or intranasal (IN) human umbilical cord-derived MSCs (huMSCs) as adjunct therapy to HT in a piglet model. Methods:A total of 17 newborn piglets underwent transient cerebral hypoxia-ischemia (HI) and were then randomized to (i) HT at 33.5°C 1–13 h after HI (n = 7), (ii) HT+IV huMSCs (30 × 106 cells) at 24 h and 48 h after HI (n = 5) or (iii) HT+IN huMSCs (30 × 106 cells) at 24 h and 48 h after HI (n = 5). Phosphorus-31 and hydrogen-1 magnetic resonance spectroscopy (MRS) was performed at 30 h and 72 h and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and oligodendrocytes quantified. In two further piglets, 30 × 106 IN PKH-labeled huMSCs were administered. Results:HI severity was similar between groups. Amplitude-integrated electroencephalogram (aEEG) recovery was more rapid for HT+IN huMSCs compared with HT from 25 h to 42 h and 49 h to 54 h (P ≤ 0.05). MRS phosphocreatine/inorganic phosphate was higher on day 2 in HT+IN huMSCs than HT (P = 0.035). Comparing HT+IN huMSCs with HT and HT+IV huMSCs, there were increased OLIG2 counts in hippocampus (P = 0.011 and 0.018, respectively), internal capsule (P = 0.013 and 0.037, respectively) and periventricular white matter (P = 0.15 for IN versus IV huMSCs). Reduced TUNEL-positive cells were seen in internal capsule with HT+IN huMSCs versus HT (P = 0.05). PKH-labeled huMSCs were detected in the brain 12 h after IN administration. Conclusions:After global HI, compared with HT alone, the authors saw beneficial effects of HT+IN huMSCs administered at 24 h and 48 h (30 × 106 cells/kg total dose) based on more rapid aEEG recovery, improved 31P MRS brain energy metabolism and increased oligodendrocyte survival at 72 h.

Monocytic Infiltrates Contribute to Autistic-like Behaviors in a Two-Hit Model of Neurodevelopmental Defects.

Growing evidence suggests that early-life interactions among genetic, immune, and environment factors may modulate neurodevelopment and cause psycho-cognitive deficits. Maternal immune activation (MIA) induces autism-like behaviors in offspring, but how it interplays with perinatal brain injury (especially birth asphyxia or hypoxia ischemia [HI]) is unclear. Herein we compared the effects of MIA (injection of poly[I:C] to dam at gestational day 12.5), HI at postnatal day 10, and the combined MIA/HI insult in murine offspring of both sexes. We found that MIA induced autistic-like behaviors without microglial activation but amplified post-HI NFκB signaling, pro-inflammatory responses, and brain injury in offspring. Conversely, HI neither provoked autistic-like behaviors nor concealed them in the MIA offspring. Instead, the dual MIA/HI insult added autistic-like behaviors with diminished synaptic density and reduction of autism-related PSD-95 and Homer-1 in the hippocampus, which were missing in the singular MIA or HI insult. Further, the dual MIA/HI insult enhanced the brain influx of Otx2-positive monocytes that are associated with an increase of perineuronal net-enwrapped parvalbumin neurons. Using CCR2-CreER mice to distinguish monocytes from the resident microglia, we found that the monocytic infiltrates gradually adopted a ramified morphology and expressed the microglial signature genes (Tmem119, P2RY12, and Sall1) in post-MIA/HI brains, with some continuing to express the proinflammatory cytokine TNFα. Finally, genetic or pharmacological obstruction of monocytic influx significantly reduced perineuronal net-enwrapped parvalbumin neurons and autistic-like behaviors in MIA/HI offspring. Together, these results suggest a pathologic role of monocytes in the two-hit (immune plus neonatal HI) model of neurodevelopmental defects.SIGNIFICANCE STATEMENT In autism spectrum disorders (ASDs), prenatal infection or maternal immune activation (MIA) may act as a primer for multiple genetic and environmental factors to impair neurodevelopment. This study examined whether MIA cooperates with neonatal cerebral hypoxia ischemia to promote ASD-like aberrations in mice using a novel two-hit model. It was shown that the combination of MIA and neonatal hypoxia ischemia produces autistic-like behaviors in the offspring, and has synergistic effects in inducing neuroinflammation, monocytic infiltrates, synaptic defects, and perineuronal nets. Furthermore, genetic or pharmacological intervention of the MCP1-CCR2 chemoattractant pathway markedly reduced monocytic infiltrates, perineuronal nets, and autistic-like behaviors. These results suggest reciprocal escalation of immune and neonatal brain injury in a subset of ASD that may benefit from monocyte-targeted treatments.

Gastrodin Regulates the Notch Signaling Pathway and Sirt3 in Activated Microglia in Cerebral Hypoxic-Ischemia Neonatal Rats and in Activated BV-2 Microglia.

In response to hypoxic-ischemic brain damage (HIBD), microglia activation and its mediated inflammation contribute to neuronal damage. Inhibition of over-activated microglia is deemed to be a potential therapeutic strategy. Our previous studies showed that gastrodin efficiently depressed the neuroinflammation mediated by activated microglia in HIBD neonatal rats. The underlying mechanisms through which gastrodin acts on activated microglia have not been fully elucidated. This study is designed to determine whether gastrodin would regulate the Notch signaling pathway and Sirtuin3 (Sirt3), which are implicated in regulating microglia activation. The present results showed that gastrodin markedly suppressed the expression of members of Notch signaling pathway (Notch-1, NICD, RBP-JK and Hes-1) in activated microglia both in vivo and in vitro. Conversely, Sirt3 expression was enhanced. In BV-2 microglia treated with a γ-secretase inhibitor of Notch pathway- DAPT, the expression of RBP-JK, Hes-1, and NICD was suppressed in activated microglia. Treatment with DAPT and gastrodin further decreased NICD and Hes-1 expression. Sirt3 expression was also decreased after DAPT treatment. However, Sirt3 expression in activated BV-2 microglia given a combined DAPT and gastrodin treatment was not further increased. In addition, combination of DAPT and Gastrodin cumulatively decreased tumor necrosis factor-α (TNF-α) expression. The results suggest that gastrodin regulates microglia activation via the Notch signaling pathway and Sirt3. More importantly, interference of the Notch signaling pathway inhibited Sirt3 expression, indicating that Sirt3 is a downstream gene of the Notch signaling pathway. It is suggested that Notch and Sirt3 synergistically regulate microglia activation such as in TNF-α production.

Neuroprotective effect of astrocyte-derived IL-33 in neonatal hypoxic-ischemic brain injury

BackgroundInterleukin-33 (IL-33) is a well-recognized pleiotropic cytokine which plays crucial roles in immune regulation and inflammatory responses. Recent studies suggest that IL-33 and its receptor ST2 are involved in the pathogenesis of neurological diseases. Here, we explore the effect of IL-33/ST2 signaling in neonatal hypoxic-ischemic (HI) brain injury and elucidate the underlying mechanisms of action.MethodsThe brain HI model was established in neonatal C57BL/6 mice by left common carotid artery occlusion with 90 min hypoxia and treated with IL-33 at a dose of 0.2 μg/day i.p. for 3 days. TTC staining and neurobehavioral observation were used to evaluate the HI brain injury. Immunofluorescence and flow cytometry were applied to determine the expression of IL-33 and its receptor ST2 on brain CNS cells and cell proliferation and apoptosis. OGD experiment was used to assay the viability of astrocytes and neurons. RT-qPCR was used to measure the expression of neurotrophic factor-associated genes.ResultsThe expression level of IL-33 was markedly enhanced in astrocytes 24 h after cerebral HI in neonatal mice. Exogenous delivery of IL-33 significantly alleviated brain injury 7 days after HI, whereas ST2 deficiency exacerbated brain infarction and neurological deficits post HI. Flow cytometry analyses demonstrated high levels of ST2 expression on astrocytes, and the expression of ST2 was further elevated after HI. Intriguingly, IL-33 treatment apparently improved astrocyte response and attenuated HI-induced astrocyte apoptosis through ST2 signaling pathways. Further in vitro studies revealed that IL-33-activated astrocytes released a series of neurotrophic factors, which are critical for raising neuronal survival against oxygen glucose deprivation.ConclusionsThe activation of IL-33/ST2 signaling in the ischemic brain improves astrocyte response, which in turn affords protection to ischemic neurons in a glial-derived neurotrophic factor-dependent manner.

Interaction between the autophagy protein Beclin 1 and Na+,K+-ATPase during starvation, exercise, and ischemia.

Autosis is a distinct form of cell death that requires both autophagy genes and the Na+,K+-ATPase pump. However, the relationship between the autophagy machinery and Na+,K+-ATPase is unknown. We explored the hypothesis that Na+,K+-ATPase interacts with the autophagy protein Beclin 1 during stress and autosis-inducing conditions. Starvation increased the Beclin 1/Na+,K+-ATPase interaction in cultured cells, and this was blocked by cardiac glycosides, inhibitors of Na+,K+-ATPase. Increases in Beclin 1/Na+,K+-ATPase interaction were also observed in tissues from starved mice, livers of patients with anorexia nervosa, brains of neonatal rats subjected to cerebral hypoxia-ischemia (HI), and kidneys of mice subjected to renal ischemia/reperfusion injury (IRI). Cardiac glycosides blocked the increased Beclin 1/Na+,K+-ATPase interaction during cerebral HI injury and renal IRI. In the mouse renal IRI model, cardiac glycosides reduced numbers of autotic cells in the kidney and improved clinical outcome. Moreover, blockade of endogenous cardiac glycosides increased Beclin 1/Na+,K+-ATPase interaction and autotic cell death in mouse hearts during exercise. Thus, Beclin 1/Na+,K+-ATPase interaction is increased in stress conditions, and cardiac glycosides decrease this interaction and autosis in both pathophysiological and physiological settings. This crosstalk between cellular machinery that generates and consumes energy during stress may represent a fundamental homeostatic mechanism.

Offspring of rats with cerebral hypoxia-ischemia manifest cognitive dysfunction in learning and memory abilities.

Neonatal hypoxic-ischemic encephalopathy is a serious neurological disease, often resulting in long-term neurodevelopmental disorders among surviving children. However, whether these neurodevelopmental issues can be passed to offspring remains unclear. The right common carotid artery of 7-day-old parental-generation rats was subjected to permanent ligation using a vessel electrocoagulator. Neonatal hypoxic-ischemic rat models were established by subjecting the rats to 8% O2-92% N2 for 2 hours. The results showed that 24 hours after hypoxia and ischemia, pathological damage, cerebral atrophy, liquefaction, and impairment were found, and Zea-Longa scores were significantly increased. The parental-generation rats were propagated at 3 months old, and offspring were obtained. No changes in the overall brain structures of these offspring rats were identified by magnetic resonance imaging. However, the escape latency was longer and the number of platform crossings was reduced among these offspring compared with normal rats. These results indicated that the offspring of hypoxic-ischemic encephalopathy model rats displayed cognitive impairments in learning and memory. This study was approved by the Animal Care & Welfare Committee of Kunming Medical University, China in 2018 (approval No. kmmu2019072).

Quantification of the severity of hypoxic-ischemic brain injury in a neonatal preclinical model using measurements of cytochrome-c-oxidase from a miniature broadband-near-infrared spectroscopy system.

.We describe the development of a miniaturized broadband near-infrared spectroscopy system (bNIRS), which measures changes in cerebral tissue oxyhemoglobin () and deoxyhemoglobin ([HHb]) plus tissue metabolism via changes in the oxidation state of cytochrome-c-oxidase ([oxCCO]). The system is based on a small light source and a customized mini-spectrometer. We assessed the instrument in a preclinical study in 27 newborn piglets undergoing transient cerebral hypoxia-ischemia (HI). We aimed to quantify the recovery of the HI insult and estimate the severity of the injury. The recovery in brain oxygenation (), blood volume (), and metabolism () for up to 30 min after the end of HI were quantified in percentages using the recovery fraction (RF) algorithm, which quantifies the recovery of a signal with respect to baseline. The receiver operating characteristic analysis was performed on bNIRS-RF measurements compared to proton () magnetic resonance spectroscopic (MRS)-derived thalamic lactate/N-acetylaspartate (Lac/NAA) measured at 24-h post HI insult; Lac/NAA peak area ratio is an accurate surrogate marker of neurodevelopmental outcome in babies with neonatal HI encephalopathy. The -RF cut-off threshold of 79% within 30 min of HI predicted injury severity based on Lac/NAA with high sensitivity (100%) and specificity (93%). A significant difference in thalamic Lac/NAA was noticed () between the two groups based on this cut-off threshold of 79% -RF. The severe injury group () had smaller recovery in -RF () and no significant difference was observed in -RF between groups. At 48 h post HI, significantly higher -MRS-measured inorganic phosphate/exchangeable phosphate pool (epp) () and reduced phosphocreatine/epp () were observed in the severe injury group indicating persistent cerebral energy depletion. Based on these results, the bNIRS measurement of the oxCCO recovery fraction offers a noninvasive real-time biomarker of brain injury severity within 30 min following HI insult.