IRE1α inhibition decreased TXNIP/NLRP3 inflammasome activation through miR-17-5p after neonatal hypoxic-ischemic brain injury in rats.

BACKGROUND: The endoplasmic reticulum (ER) is responsible for the control of correct protein folding and protein function which is crucial for cell survival. However, under pathological conditions, such as hypoxia-ischemia (HI), there is an accumulation of unfolded proteins thereby triggering the unfolded protein response (UPR) and causing ER stress which is associated with activation of several stress sensor signaling pathways, one of them being the inositol requiring enzyme-1 alpha (IRE1α) signaling pathway. The UPR is regarded as a potential contributor to neuronal cell death and inflammation after HI. In the present study, we sought to investigate whether microRNA-17 (miR-17), a potential IRE1α ribonuclease (RNase) substrate, arbitrates downregulation of thioredoxin-interacting protein (TXNIP) and consequent NLRP3 inflammasome activation in the immature brain after HI injury and whether inhibition of IRE1α may attenuate inflammation via miR-17/TXNIP regulation. METHODS: Postnatal day 10 rat pups (n = 287) were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O2). STF-083010, an IRE1α RNase inhibitor, was intranasally delivered at 1 h post-HI or followed by an additional one administration per day for 2 days. MiR-17-5p mimic or anti-miR-17-5p inhibitor was injected intracerebroventricularly at 48 h before HI. Infarct volume and body weight were used to evaluate the short-term effects while brain weight, gross and microscopic brain tissue morphologies, and neurobehavioral tests were conducted for the long-term evaluation. Western blots, immunofluorescence staining, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and co-immunoprecipitation (Co-IP) were used for mechanism studies. RESULTS: Endogenous phosphorylated IRE1α expression was significantly increased after HI. Intranasal administration of STF-083010 alleviated brain injury and improved neurological behavior. MiR-17-5p expression was reduced after HI, and this decrease was attenuated by STF-083010 treatment. MiR-17-5p mimic administration ameliorated TXNIP expression, NLRP3 inflammasome activation, caspase-1 cleavage, and IL-1ß production, as well as brain infarct volume. Conversely, anti-miR-17-5p inhibitor reversed IRE1α inhibition-induced decrease in TXNIP expression and inflammasome activation, as well as exacerbated brain injury after HI. CONCLUSIONS: IRE1a-induced UPR pathway may contribute to inflammatory activation and brain injury following neonatal HI. IRE1a activation, through decay of miR-17-5p, elevated TXNIP expression to activate NLRP3 inflammasome and aggravated brain damage.

Neuroprotective Mechanisms of Melatonin in Hemorrhagic Stroke.

Hemorrhagic stroke which consists of subarachnoid hemorrhage and intracerebral hemorrhage is a dominant cause of death and disability worldwide. Although great efforts have been made, the physiological mechanisms of these diseases are not fully understood and effective pharmacological interventions are still lacking. Melatonin (N-acetyl-5-methoxytryptamine), a neurohormone produced by the pineal gland, is a broad-spectrum antioxidant and potent free radical scavenger. More importantly, there is extensive evidence demonstrating that melatonin confers neuroprotective effects in experimental models of hemorrhagic stroke. Multiple molecular mechanisms such as antioxidant, anti-apoptosis, and anti-inflammation, contribute to melatonin-mediated neuroprotection against brain injury after hemorrhagic stroke. This review article aims to summarize current knowledge regarding the beneficial effects of melatonin in experimental models of hemorrhagic stroke and explores the underlying mechanisms. We propose that melatonin is a promising neuroprotective candidate that is worthy of further evaluation for its potential therapeutic applications in hemorrhagic stroke.

Melatonin attenuates neuronal apoptosis through up-regulation of K(+) -Cl(-) cotransporter KCC2 expression following traumatic brain injury in rats.

Traumatic brain injury (TBI) initiates a complex cascade of neurochemical and signaling changes that leads to neuronal apoptosis, which contributes to poor outcomes for patients with TBI. The neuron-specific K(+) -Cl(-) cotransporter-2 (KCC2), the principal Cl(-) extruder in adult neurons, plays an important role in Cl(-) homeostasis and neuronal function. This present study was designed to investigate the expression pattern of KCC2 following TBI and to evaluate whether or not melatonin is able to prevent neuronal apoptosis by modulating KCC2 expression in a Sprague Dawley rat controlled cortical impact model of TBI. The time course study showed decreased mRNA and protein expression of KCC2 in the ipsilateral peri-core parietal cortex after TBI. Double immunofluorescence staining demonstrated that KCC2 is located in the plasma membrane of neurons. In addition, melatonin (10 mg/kg) was injected intraperitoneally at 5 minutes and repeated at 1, 2, 3, and 4 hours after brain trauma, and brain samples were extracted 24 hours after TBI. Compared to the vehicle group, melatonin treatment altered the down-regulation of KCC2 expression in both mRNA and protein levels after TBI. Also, melatonin treatment increased the protein levels of brain-derived neurotrophic factor (BDNF) and phosphorylated extracellular signal-regulated kinase (p-ERK). Simultaneously, melatonin administration ameliorated cortical neuronal apoptosis, reduced brain edema, and attenuated neurological deficits after TBI. In conclusion, our findings suggested that melatonin restores KCC2 expression, inhibits neuronal apoptosis and attenuates secondary brain injury after TBI, partially through activation of BDNF/ERK pathway.

Changes in Brain Swelling and Infarction Volume over Four Days After Hypoxia Ischemia in Neonatal Rats.

The leading cause of morbidity and mortality in infants is hypoxia-ischemia (HI). The current therapies for HI have limited success, in part due to a lack of understanding of HI pathophysiology and underlying mechanisms. Herein, a neonatal rat model of HI was used to examine the changes in brain swelling and infarct volume over 4 days after HI. Forty-four P10 rat pups were sacrificed at 2, 3, or 4 days post-HI. After sacrifice, the brains were removed, sliced, and stained with TTC (2,3,5-triphenyl-2H-tetrazolium chloride). Images of TTC-stained brains were used for measurement of the ipsilateral hemisphere brain volumes and infarct volumes, calculated using standard equations. The hemispheric brain volumes of HI animals in all groups was lower than that of sham animals and decreased as the post-HI sacrifice time increased. The infarct volume of HI animals was larger than that of sham animals. Infarct volumes tended to decrease over the days post-HI. The change in infarct volume is likely the result of a combination of brain growth and repair mechanisms. However, changes in the hemispheric brain volume may include tissue growth and repair mechanism, so also may be a limitation of the current algorithm used for calculating ipsilateral hemisphere brain volume.

Astaxanthin as a Potential Neuroprotective Agent for Neurological Diseases.

Neurological diseases, which consist of acute injuries and chronic neurodegeneration, are the leading causes of human death and disability. However, the pathophysiology of these diseases have not been fully elucidated, and effective treatments are still lacking. Astaxanthin, a member of the xanthophyll group, is a red-orange carotenoid with unique cell membrane actions and diverse biological activities. More importantly, there is evidence demonstrating that astaxanthin confers neuroprotective effects in experimental models of acute injuries, chronic neurodegenerative disorders, and neurological diseases. The beneficial effects of astaxanthin are linked to its oxidative, anti-inflammatory, and anti-apoptotic characteristics. In this review, we will focus on the neuroprotective properties of astaxanthin and explore the underlying mechanisms in the setting of neurological diseases.

Neuroprotective Strategies after Neonatal Hypoxic Ischemic Encephalopathy.

Neonatal hypoxic ischemic encephalopathy (HIE) is a devastating disease that primarily causes neuronal and white matter injury and is among the leading cause of death among infants. Currently there are no well-established treatments; thus, it is important to understand the pathophysiology of the disease and elucidate complications that are creating a gap between basic science and clinical translation. In the development of neuroprotective strategies and translation of experimental results in HIE, there are many limitations and challenges to master based on an appropriate study design, drug delivery properties, dosage, and use in neonates. We will identify understudied targets after HIE, as well as neuroprotective molecules that bring hope to future treatments such as melatonin, topiramate, xenon, interferon-beta, stem cell transplantation. This review will also discuss some of the most recent trials being conducted in the clinical setting and evaluate what directions are needed in the future.

ErbB4 protects against neuronal apoptosis via activation of YAP/PIK3CB signaling pathway in a rat model of subarachnoid hemorrhage.

Neuronal apoptosis is a central pathological process in subarachnoid hemorrhage (SAH)-induced early brain injury. Previous studies indicated that ErbB4 (EGFR family member v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4) is essential for normal development and maintenance of the nervous system. In this study, we explored the neuroprotective effects of ErbB4 and its downstream YAP (yes-associated protein)/PIK3CB signaling pathway in early brain injury after SAH in a rat model using the endovascular perforation method. Rats were neurologically evaluated with the Modified Garcia Scale and beam balance test at 24h and 72h after SAH. An ErbB4 activator Neuregulin 1ß1 (Nrg 1ß1), ErbB4 siRNA and YAP siRNA were used to explore this pathway. The expression of p-ErbB4 and YAP was significantly increased after SAH. Multiple immunofluorescence labeling experiments demonstrated that ErbB4 is mainly expressed in neurons. Activation of ErbB4 and its downstream signals improved the neurological deficits after SAH and significantly reduced neuronal cell death. Inhibition of ErbB4 reduced YAP and PIK3CB expression, and aggravated cell apoptosis. YAP knockdown reduced the PIK3CB level and eliminated the anti-apoptotic effects of ErbB4 activation. These findings indicated that ErbB4 plays a neuroprotective role in early brain injury after SAH, possibly via the YAP/PIK3CB signaling pathway.

Recombinant Gas6 augments Axl and facilitates immune restoration in an intracerebral hemorrhage mouse model.

Axl, a tyrosine kinase receptor, was recently identified as an essential component regulating innate immune response. Suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 are potent Axl-inducible negative inflammatory regulators. This study investigated the role of Axl signaling pathway in immune restoration in an autologous blood-injection mouse model of intracerebral hemorrhage. Recombinant growth arrest-specific 6 (Gas6) and R428 were administrated as specific agonist and antagonist. In vivo knockdown of Axl or suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 by siRNA was applied. After intracerebral hemorrhage, the expression of endogenous Axl, soluble Axl, and Gas6 was increased, whereas the expression of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 was inhibited. Recombinant growth arrest-specific 6 administration alleviated brain edema and improved neurobehavioral performances. Moreover, enhanced Axl phosphorylation with cleavage of soluble Axl (sAxl), and an upregulation of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 were observed. In vivo knockdown of Axl and R428 administration both abolished the effect of recombinant growth arrest-specific 6 on brain edema and also decreased the expression suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3. In vivo knockdown of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 aggravated cytokine releasing despite of recombinant growth arrest-specific 6. In conclusion, Axl plays essential role in immune restoration after intracerebral hemorrhage. And recombinant growth arrest-specific 6 attenuated brain injury after intracerebral hemorrhage, probably by enhancing Axl phosphorylation and production of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3.

Neuroprotective Effects of Valproic Acid on Blood-Brain Barrier Disruption and Apoptosis-Related Early Brain Injury in Rats Subjected to Subarachnoid Hemorrhage Are Modulated by Heat Shock Protein 70/Matrix Metalloproteinases and Heat Shock Protein 70/AKT Pathways.

BACKGROUND: Blood-brain barrier (BBB) disruption and neural apoptosis are thought to promote early brain injury (EBI) after subarachnoid hemorrhage (SAH). Previous studies have demonstrated that valproic acid (VPA) decreased brain injury in a prechiasmatic injection model of SAH in mice. It should be noted that the beneficial effects of VPA and the underlying mechanisms have not been fully elucidated. OBJECTIVE: To characterize the effects of VPA on BBB disruption and neural apoptosis and to determine mechanisms involved in EBI after SAH. METHODS: An endovascular perforation model was used to induce SAH in rats. VPA (300 mg/kg) was promptly administered after SAH induction, and the same dose was given 12 hours later. Quercetin (100 mg/kg), an inhibitor of heat shock protein 70 (HSP70), was injected into the peritoneum 2 hours before SAH induction. Mortality, SAH grades, neurological function, Evans Blue extravasation, brain edema, transmission electron microscopy, Western blot, double fluorescence labeling, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining also were used. RESULTS: VPA treatment decreased BBB disruption and brain edema, attenuated neural apoptosis, and improved neurobehavioral functions in EBI after SAH. Double fluorescence labeling indicated that matrix metallopeptidase 9 (MMP-9) was located predominately in neurons and endothelial cells. VPA upregulated the expression of HSP70, effectively decreased the expression and activity of MMP-9, and reduced claudin-5 and occludin degradation. Meanwhile, VPA also upregulated the expression of phosphorylated Akt and bcl-2. Both the anti-BBB disruption and antiapoptotic effects of VPA were abolished by quercetin. CONCLUSION: VPA prevented BBB disruption and alleviated neural apoptosis after SAH. The action of VPA appeared to be mediated though the HSP70/MMPs and HSP70/Akt pathways. ABBREVIATIONS: BBB, blood-brain barrierEBI, early brain injuryHSP, heat shock proteinMMP, matrix metalloproteinasePBS, phosphate-buffered salineSAH, subarachnoid hemorrhageTUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelingVPA, valproic acid.

Intranasal Administration of Interferon Beta Attenuates Neuronal Apoptosis via the JAK1/STAT3/BCL-2 Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxic-ischemic encephalopathy (HIE) is an injury that often leads to detrimental neurological deficits. Currently, there are no established therapies for HIE and it is critical to develop treatments that provide protection after HIE. The objective of this study was to investigate the ability of interferon beta (IFNß) to provide neuroprotection and reduce apoptosis after HIE. Postnatal Day 10 rat pups were subjected to unilateral carotid artery ligation followed by 2.5 hr of exposure to hypoxia (8% O2). Intranasal administration of human recombinant IFNß occurred 2 hr after HIE and infarct volume, body weight, neurobehavioral tests, histology, immunohistochemistry, brain water content, blood-brain barrier permeability, enzyme-linked immunosorbent assay, and Western blot were all used to evaluate various parameters. The results showed that both IFNß and the Type 1 interferon receptor expression decreases after HIE. Intranasal administration of human recombinant IFNß was able to be detected in the central nervous system and was able to reduce brain infarction volumes and improve neurological behavior tests 24 hr after HIE. Western blot analysis also revealed that human recombinant IFNß treatment stimulated Stat3 and Bcl-2 expression leading to a decrease in cleaved caspase-3 expression after HIE. Positive Fluoro-Jade C staining also demonstrated that IFNß treatment was able to decrease neuronal apoptosis. Furthermore, the beneficial effects of IFNß treatment were reversed when a Stat3 inhibitor was applied. Also an intraperitoneal administration of human recombinant IFNß into the systemic compartment was unable to confer the same protective effects as intranasal IFNß treatment.

G-CSF attenuates neuroinflammation and stabilizes the blood-brain barrier via the PI3K/Akt/GSK-3ß signaling pathway following neonatal hypoxia-ischemia in rats.

OBJECTIVE: Neonatal hypoxia occurs in approximately 60% of premature births and is associated with a multitude of neurological disorders. While various treatments have been developed, translating them from bench to bedside has been limited. We previously showed G-CSF administration was neuroprotective in a neonatal hypoxia-ischemia rat pup model, leading us to hypothesize that G-CSF inactivation of GSK-3ß via the PI3K/Akt pathway may attenuate neuroinflammation and stabilize the blood-brain barrier (BBB). METHODS: P10 Sprague-Dawley rat pups were subjected to unilateral carotid artery ligation followed by hypoxia for 2.5h. We assessed inflammation by measuring expression levels of IKKß, NF-κB, TNF-α, IL-1ß, IL-10, and IL-12 as well as neutrophil infiltration. BBB stabilization was evaluated by measuring Evans blue extravasation, and Western blot analysis of Claudin-3, Claudin-5, ICAM-1, and VCAM-1. MEASUREMENTS AND MAIN RESULTS: First, the time course study showed that p-ß-catenin/ß-catenin, IKKß, and NF-κB expression levels peaked at 48h post-HI. The knockdown of GSK-3ß with siRNA prevented the HI-induced increase of p-ß-catenin/ß-catenin, IKKß, and NF-κB expression levels 48h after HI. G-CSF treatment reduced brain water content and neuroinflammation by downregulating IKKß, NF-κB, TNF-α, IL-1ß, and IL-12 and upregulating IL-10, thereby reducing neutrophil infiltration. Additionally, G-CSF stabilizes the BBB by downregulating VCAM-1 and ICAM-1, as well as upregulating Claudins 3 and 5 in endothelial cells. G-CSFR knockdown by siRNA and Akt inhibition by Wortmannin reversed G-CSF's neuroprotective effects. CONCLUSIONS: We demonstrate G-CSF plays a pivotal role in attenuating neuroinflammation and BBB disruption following HI by inactivating GSK-3ß through the PI3K/Akt pathway.

The evolution of molecular hydrogen: a noteworthy potential therapy with clinical significance.

Studies on molecular hydrogen have evolved tremendously from its humble beginnings and have continued to change throughout the years. Hydrogen is extremely unique since it has the capability to act at the cellular level. Hydrogen is qualified to cross the blood brain barrier, to enter the mitochondria, and even has the ability to translocate to the nucleus under certain conditions. Once in these ideal locations of the cell, previous studies have shown that hydrogen exerts antioxidant, anti-apoptotic, anti-inflammatory, and cytoprotective properties that are beneficial to the cell. Hydrogen is most commonly applied as a gas, water, saline, and can be applied in a variety of other mediums. There are also few side effects involving hydrogen, thus making hydrogen a perfect medical gas candidate for the convention of novel therapeutic strategies against cardiovascular, cerebrovascular, cancer, metabolic, and respiratory diseases and disorders. Although hydrogen appears to be faultless at times, there still are several deficiencies or snares that need to be investigated by future studies. This review article seeks to delve and comprehensively analyze the research and experiments that alludes to molecular hydrogen being a novel therapeutic treatment that medicine desperately needs.

Isoflurane provides neuroprotection in neonatal hypoxic ischemic brain injury.

Isoflurane is a volatile anesthetic that is widely used clinically as an inhalational anesthetic. In recent years, several studies have indicated that isoflurane has neuroprotective properties. This has led to the beneficial effects of isoflurane being analyzed in both cell culture and animal models, including various models of brain injury. Neonatal hypoxia ischemia may be characterized as injury that occurs in the immature brain, resulting in delayed cell death via excitotoxicity and oxidative stress. These adverse events in the developing brain often lead to detrimental neurological defects in the future. Currently, there are no well-established effective therapies for neonatal hypoxia ischemia. In line with this, isoflurane, which displays neuroprotective properties in several paradigms and has been shown to improve neurological deficits caused by brain injuries, has the capability to be an extremely relevant clinical therapy for the resolution of deficits concomitant with neonatal hypoxic ischemic brain injuries. This review therefore seeks to explore and analyze the current information on isoflurane, looking at general isoflurane anesthetic properties, and the protection it confers in different animal models, focusing particularly on neuroprotection as shown in studies with neonatal hypoxic ischemic brain injury.