LncRNA MALAT1 Promoted Neuronal Necroptosis in Cerebral Ischemia-reperfusion Mice by Stabilizing HSP90.

The objective of this research was to investigate the role of lncRNA MALAT1 and HSP90 in the regulation of neuronal necroptosis in mice with cerebral ischemia-reperfusion (CIR). We used male C57BL/6J mice to establish a middle cerebral artery occlusion (MCAO) model and conducted in vitro experiments using the HT-22 mouse hippocampal neuron cell line. The cellular localization of NeuN and MLKL, as well as the expression levels of neuronal necroptosis factors, MALAT1, and HSP90 were analyzed. Cell viability and necroptosis were assessed, and we also investigated the relationship between MALAT1 and HSP90. The results showed that MALAT1 expression increased after MCAO and oxygen-glucose deprivation/re-oxygenation (OGD/R) treatment in both cerebral tissues and cells compared with the control group. The levels of neuronal necroptosis factors and the co-localization of NeuN and MLKL were also increased in MCAO mice compared with the Sham group. MALAT1 was found to interact with HSP90, and inhibition of HSP90 expression led to decreased phosphorylation levels of neuronal necroptosis factors. Inhibition of MALAT1 expression resulted in decreased co-localization levels of NeuN and MLKL, decreased phosphorylation levels of neuronal necroptosis factors, and reduced necroptosis rate in cerebral tissues. Furthermore, inhibiting MALAT1 expression also led to a shorter half-life of HSP90, increased ubiquitination level, and decreased phosphorylation levels of neuronal necroptosis factors in cells. In conclusion, this study demonstrated that lncRNA MALAT1 promotes neuronal necroptosis in CIR mice by stabilizing HSP90.

miR-188-5p silencing improves cerebral ischemia/reperfusion injury by targeting Lin28a.

This report aimed to explore whether miR-188-5p regulated the pathological regulatory network of cerebral ischemia/reperfusion (I/R) injury. We simulated the cerebral I/R injury model with MACO/R and OGD/R treatments. Neuronal viability and apoptosis were assessed. The contents of miR-188-5p and Lin 28a were evaluated. The abundances of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) and pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) were measured. The interaction of miR-188-5p and Lin28a was confirmed. Lin28a silencing was supplemented to determine the delicate regulation of miR-188-5p. We revealed that miR-188-5p was upregulated and Lin28a was downregulated in I/R rats and OGD/R-induced cells. miR-188-5p silencing remarkably reduced the cerebral infarction volume, neurobehavioral score, brain edema, and Evans blue leakage. miR-188-5p silencing enhanced neuronal viability and alleviated apoptosis. The abundance of Bax and cleaved caspase-3 was reduced by miR-188-5p silencing, while Bcl-2 was augmented. miR-188-5p silencing impeded the contents of TNF-α, IL-1ß, and IL-6. miR-188-5p interacted with Lin28a and negatively regulated its expression. Interestingly, extra Lin28a silencing reversed apoptosis and the content of inflammatory cytokines. Our studies confirmed that miR-188-5p silencing alleviated neuronal apoptosis and inflammation by mediating the expression of Lin28a. The crosstalk of miR-188-5p and Lin28a offered a different direction for ischemic stroke therapy.

MiR-181c-5p ameliorates learning and memory in sleep-deprived mice via HMGB1/TLR4/NF-κB pathway.

Sleep deprivation (SD) can lead to cognitive impairment caused by neuroinflammation. MiR-181c-5p/HMGB1 axis plays a part in anti-inflammation effects. However, the mechanism that miR-181c-5p facilitates learning and memory in SD mice remains unclear. So we investigated the role of miR-181c-5p in learning and memory impairment induced by SD. We overexpressed miR-181c-5p in the mice hippocampus by injecting lentivirus vector-miR-181c-5p (LV-miR-181c-5p) particles. Mice were divided into four groups: control (Ctrl), SD, SD + miR-181c-5p and SD + vector. We found that mice in the third group showed ameliorated learning and memory compared with the fourth group. The content of ionized calcium binding adaptor molecule 1 (IBA-1) in the third group was decreased compared with the fourth group. Moreover, the expression levels of HMGB1, TLR4 and p-NF-κB in the hippocampus of overexpressed miR-181c-5p mice were reduced. In total, miR-181c-5p ameliorated learning and memory in SD mice via the HMGB1/TLR4/NF-κB pathway.

Inhibition of Cerebral Ischemia/Reperfusion Injury by MSCs-Derived Small Extracellular Vesicles in Rodent Models: A Systematic Review and Meta-Analysis.

Small extracellular vesicles (sEVs) secreted by mesenchymal stem cells (MSCs) have shown great therapeutic potential in cerebral ischemia-reperfusion injury (CIRI). In this study, we firstly performed a systematic review to evaluate the efficacy of MSCs-derived sEV for experimental cerebral ischemia/reperfusion injury. 24 studies were identified by searching 8 databases from January 2012 to August 2022. The methodological quality was assessed by using the SYRCLE 's risk of bias tool for animal studies. All the data were analyzed using RevMan 5.3 software. As a result, the score of study quality ranged from 3 to 9 in a total of ten points. Meta-analyses showed that MSCs-derived sEVs could effectively alleviate neurological impairment scores, reduced the volume of cerebral infarction and brain water content, and attenuated neuronal apoptosis. Additionally, the possible mechanisms of MSCs-derived sEVs for attenuating neuronal apoptosis were inhibiting microglia-mediated neuroinflammation. Thus, MSCs-derived sEVs might be regarded as a novel insight for cerebral ischemic stroke. However, further mechanistic studies, therapeutic safety, and clinical trials are required. Systematic review registration. PROSPERO CRD42022312227.

Safety and efficacy of glibenclamide combined with rtPA in acute cerebral ischemia with occlusion/stenosis of anterior circulation (SE-GRACE): study protocol for a randomized controlled trial.

BACKGROUND: Thrombolysis with recombinant tissue plasminogen activator (rtPA) improves outcome for patients with acute ischemic stroke (AIS), but many of them still have substantial disability. Glibenclamide (US adopted name, glyburide), a long-acting sulfonylurea, shows promising result in treating AIS from both preclinical and clinical studies. This study investigates the safety and efficacy of glibenclamide combined with rtPA in treating AIS patients. METHODS: This is a prospective, randomized, double-blind, placebo-controlled, multicenter trial with an estimated sample size of 306 cases, starting in January 2018. Patients aged 18 to 74 years, presented with a symptomatic anterior circulation occlusion with a deficit on the NIHSS of 4 to 25 points and treated with intravenous rtPA within the first 4.5 h of their clinical onsets, are eligible for participation in this study. The target time from the onset of symptoms to receive the study drug is of 10 h. Subjects are randomized 1: 1 to receive glibenclamide or placebo with a loading dose of 1.25 mg, followed by 0.625 mg every 8 h for total 5 days. The primary efficacy endpoint is 90-day good outcome, measured as modified Rankin Scale of 0 to 2. Safety outcomes are all-cause 30-day mortality and early neurological deterioration, with a focus on cardiac- and glucose-related serious adverse events. DISCUSSION: This study will provide valuable information about the safety and efficacy of oral glibenclamide for AIS patients treated with rtPA. This would bring benefits to a large number of patients if the agent is proved to be effective. TRIAL REGISTRATION: The trial was registered on September 14th 2017 at www.clinicaltrials.gov having identifier NCT03284463. Registration was performed before recruitment was initiated.

Infection and neuromyelitis optica spectrum disorders. / è§†ç¥žç»è„Šé«“ç‚Žè°±ç³»ç–¾ç— ä¸Žæ„ŸæŸ“.

The pathogenesis of neuromyelitisoptica spectrum disorders (NMOSD) is influenced by a combination of genetic and environmental factors, including infectious agents. Several viral and bacterial infectious diseases are related to the onset and the relapse of NMOSD.The cases of NMOSD with bacterial meningitis have also been reported. Different infections play different role and outcomes in NMOSD.However,the major pathogenesis contains bystander activation, molecular mimicry, and systemic infection triggering independent central nervous system diseases. Viral and bacteria infections are closely related to NMOSD.We recommend that patients with NMOSD and altered mental status need to complete infectious examination, including a lumbar cone puncture.

Hsa_circ_0002468 Regulates the Neuronal Differentiation of SH-SY5Y Cells by Modulating the MiR-561/E2F8 Axis.

BACKGROUND It has been shown that circular RNAs (circRNAs) play a vital role in the regulation of neuronal differentiation; however, the precise role of circRNAs in human neuronal differentiation remains largely unexplored. MATERIAL AND METHODS A dual-luciferase reporter assay was carried out to confirm the targets of hsa_circ_0002468, miR-561, E2F8 (E2F transcription factor 8, a protein coding gene), and miR-561. We detected the expression of hsa_circ_0002468, miR-561, and E2F8 by using quantitative real-time polymerase chain reaction (qRT-PCR) analyses. In addition, we performed the functional experiments by using a BrdU (5-bromo-2'-deoxyuridine) assay and qRT-PCR analyses. RESULTS In this study, we showed that hsa_circ_0002468 can act as a sponge of miR-561 to regulate SH-SY5Y proliferation and differentiation. A bioinformatics analysis showed that hsa_circ_0002468 had a binding site that corresponded to miR-561, which was verified by dual-luciferase reporter assay. The expression of hsa_circ_0002468 was increased during SH-SY5Y differentiation and was inversely correlated with miR-561 expression. Using qRT-PCR analysis, we showed that hsa_circ_0002468 negatively regulated miR-561 in SH-SY5Y cells. Intriguingly, the overexpression of hsa_circ_0002468 increased SH-SY5Y differentiation and reduced SH-SY5Y proliferation; the suppression of hsa_circ_0002468 led to decreased SH-SY5Y differentiation levels and increased SH-SY5Y proliferation levels. Additionally, overexpression of miR-561 rescued the SH-SY5Y proliferation deficiency induced by hsa_circ_0002468 overexpression and abolished the SH-SY5Y differentiation promoted by hsa_circ_0002468. Furthermore, E2F8 was validated as a direct target of miR-561. CONCLUSIONS Our data suggested that hsa_circ_0002468 was a novel circRNA that regulated SH-SY5Y cell proliferation and differentiation via targeting the miR-561/E2F8 axis. Therefore, manipulating hsa_circ_0002468 in SH-SY5Y cells could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders.

Betel quid dependence is associated with functional connectivity changes of the anterior cingulate cortex: a resting-state fMRI study.

OBJECTIVE: It is generally acknowledged that drug dependence is connected with abnormal functional organization in the individual's brain. The present study aimed to identify the anterior cingulate cortex (ACC) abnormality with the cerebral networks involved in betel quid dependence (BQD) by resting-state functional connectivity (rsFC) using functional magnetic resonance imaging (fMRI). METHODS: With fMRI data measured from 33 resting-state BQD individuals and 32 non-addicted and age-, sex-, education-matched healthy controls, we inquired into the BQD-related changes in FC between the regions of ACC with the whole brain involved in BQD individuals using a region of interest vised method, and to identify the relation of the alteration with the severity of BQD and duration. RESULTS: Compared to controls, the BQD group showed increased connectivity from ACC to the regions of the reward network (brainstem including midbrain regions such as the ventral tegmental area and pons, caudate, thalamus) and cerebellum. Decreased connectivity was observed in the BQD group in regions from ACC to the default mode network (medial prefrontal cortex and precuneus) and para Hippocampal/hypothalamus. Specifically, the BQD scale was positively correlated with increased FC of right ACC to left thalamus and left ACC to pons; the durations were negatively correlated with FC of right ACC to left precuneus. CONCLUSION: These disturbances in rsFC from ACC to the reward network and DMN revealed by fMRI may have a key function in providing insights into the neurological pathophysiology underlying BQD-associated executive dysfunction and disinhibition. These findings may contribute to our better understanding of the mechanisms underlying BQD.

Overexpression of MiR-188-5p Downregulates IL6ST/STAT3/NLRP3 Pathway to Ameliorate Neuron Injury in Oxygen-glucose Deprivation/Reoxygenation.

BACKGROUND: CI/R, characterized by ischemic injury following abrupt reestablishment of blood flow, can cause oxidative stress, mitochondrial dysfunction, and apoptosis. We used oxygen-glucose deprivation/reoxygenation (OGD/R) induced injury in HT22 and primary mouse cortical neurons (MCN) as a model for CI/R. OBJECTIVE: This study investigates the role of miR-188-5p in hippocampal neuron cell injury associated with Cerebral Ischemia-Reperfusion (CI/R). METHODS: HT22 and MCN cells were induced by OGD/R to construct an in vitro model of CI/R. Cell apoptosis and proliferation were assessed using flow cytometry and the Cell Counting Kit-8 (CCK8). ELISA was conducted to measure the levels of IL-1ß, IL-6, and TNF-α. Moreover, the interaction between miR-188-5p and IL6ST was investigated using dual luciferase assay, the expression of miR-188-5p, Bax, cleaved-caspase3, IL-6, Bcl-2, IL-1ß, TNF-α, IL6ST, NFκB, NLRP3 and STAT3 was evaluated using RT-qPCR or Western blot, and immunofluorescence was used to analyze the co-expression of p-STAT3 and NLRP3 in neuronal cells. RESULTS: OGD/R reduced proliferation and miR-188-5p levels and increased IL6ST expression, inflammation, and apoptosis in HT22 and MCN cells. Moreover, miR-188-5p was found to bind to IL6ST. Mimics of miR-188-5p reduced apoptosis, lowered the expression of cleaved-caspase3 and Bax proteins, and elevated Bcl-2 protein expression in cells treated with OGD/R. Overexpression of miR-188-5p decreased the levels of NLRP3 and p-STAT3 in the OGD/R group. Furthermore, the overexpression of miR-188-5p reduced IL6ST, p- NFκB/NFκB, p-STAT3/STAT3, and NLRP3 proteins in OGD/R, and these effects could be reversed by IL6ST overexpression. CONCLUSION: Mimics of miR-188-5p were found to inhibit inflammation and the STAT3/NLRP3 pathway via IL6ST, thereby ameliorating injury in HT22 and MCN cells treated with OGD/R in the context of CI/R.

Melatonin attenuates chronic sleep deprivation-induced cognitive deficits and HDAC3-Bmal1/clock interruption.

BACKGROUND AND AIMS: Sleep is predicted as a key modulator of cognition, but the underlying mechanisms are poorly understood. In this study, we investigated the effects of melatonin on chronic rapid eye movement sleep deprivation (CRSD)-induced cognitive impairment and circadian dysfunction in rat models. METHODS: Thirty-six Sprague-Dawley male rats were divided into three groups: CRSD with saline treatment, CRSD with chronic melatonin injection (20 mg/kg/day), and non-sleep-deprived control. The cognitive behavioral tests as well as the expression of clocks and HDAC3 were evaluated in all groups. RESULTS: CRSD significantly reduced recognition index in novel object location, increased escape latency and distance traveling in Morris water maze while melatonin treatment attenuated CRSD-induced hippocampal-dependent spatial learning and memory deficits. Furthermore, the mRNAs of brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1(Bmal1) and circadian locomotor output cycles kaput (Clock) were globally down-regulated by CRSD with constant intrinsic oscillation in both hippocampus and peripheral blood. The protein levels of hippocampal Bmal1, Clock, and HDAC3 were also remarkably down-regulated following CRSD. Melatonin treatment reversed CRSD-induced alterations of Bmal1/Clock and HDAC3 on both mRNA levels and protein levels. CONCLUSIONS: Our data indicate that melatonin treatment attenuates CRSD-induced cognitive impairment via regulating HDAC3-Bmal1/Clock interaction. These findings explore a broader understanding of the relationship between sleep and cognition and provide a potential new therapeutic target for cognitive impairment.

Melatonin upregulates BMAL1 to attenuate chronic sleep deprivation-related cognitive impairment by alleviating oxidative stress.

PURPOSE: To investigate the mechanism underlying the regulatory effect of melatonin on chronic sleep deprivation-related cognitive impairment. METHODS: Chronic sleep deprivation (CSD) model was established using the MMPM method. After the model was established, melatonin receptor agonist and inhibitor were given, respectively. Water maze was conducted to record the escape latency and the duration of crossing the platform of space exploration. The concentration of TNF-α, IL-6, MDA, and SOD was measured by ELISA. Immunofluorescence was used to determine the expression level of CD86 and CD206, while the mRNA expression of Bax, Bcl-2, P65, IκB, and BMAL1 was detected by qPCR. Western blotting assay was utilized to determine the protein expression of Bax, Bcl-2, P65, p-P65, IκB, p-I κB, and BMAL1. RESULTS: Compared with the control, the escape latency was greatly increased on the second and third day, accompanied by the increased expression of TNF-α, IL-6, MDA, and SOD in serum. Furthermore, dramatically upregulated Bax, Bcl-2, P65, IκB, and CD86 were observed in the model group, accompanied by the declined expression level of BMAL1 and CD206. Compared with the model group, the escape latency was declined, the concentration of TNF-α, IL-6, MDA, and SOD was decreased, the expression level of Bax, Bcl-2, P65, IκB, and CD86 was declined, and the level of BMAL1 and CD206 was promoted by the treatment of the melatonin agonist, while the opposite results were observed under the treatment of the melatonin inhibitor. CONCLUSION: Melatonin upregulates BMAL1 to attenuate chronic sleep deprivation-related cognitive impairment by alleviating oxidative stress.

Bone Marrow Mesenchymal Stem Cell Exosomal miR-345-3p Ameliorates Cerebral Ischemia-reperfusion Injury by Targeting TRAF6.

INTRODUCTION: The purpose of this study was to investigate the effects of bone marrow mesenchymal stem cells (BMSCs) exosomal miR-345-3p and tumor necrosis factor receptorassociated factor 6 (TRAF6) on cerebral ischemia reperfusion (CIR) injury. Exosomes (Exos) derived from BMSCs were isolated and identified. PC12 (rat pheochromocytoma) cells were used to establish an oxygen and glucose deprivation/reoxygenation (OGD/R) model. METHODS: Cell counting kit-8, TUNEL staining, lactate dehydrogenase staining, RT-qPCR, and western blotting were utilized for analyzing the functions of miR-345-3p about PC12 cells. Dualluciferase reporter experiment was then to confirm the link between miR-345-3p and TRAF6. Finally, using male SD rats, the middle cerebral artery occlusion (MCAO) model was constructed. Regulation of I/R damage in MCAO rats of miR-345-3p and TRAF6 were further explored in the changes of modified neurological severity score, cerebral infarction pictures, relative infarct volume, and histopathological changes. After OGD/R treatment, neuronal apoptosis was dramatically increased. After treatment with exosomal miR-345-3p, OGD/R-induced neuroapoptosis was dramatically inhibited. Exosomal miR-345-3p inhibited OGD/R-induced neuroapoptosis by downregulating the expression of TRAF6. However, the miR-345-3p inhibitor aggravated the changes caused by OGD/R. RESULTS: The corresponding regulations of miR-345-3p were reversed with TRAF6 overexpression. The animal experiments in vivo further verified that miR-345-3p ameliorated brain I/R injury in MCAO rats by targeting TRAF6. CONCLUSION: This study found that BMSCs-exosomal miR-345-3p protected against CIR injury by decreasing TRAF6.

Safety and efficacy of glibenclamide combined with rtPA in acute cerebral ischemia with occlusion/stenosis of anterior circulation (SE-GRACE): a randomized, double-blind, placebo-controlled trial.

Background: Glibenclamide alleviates brain edema and improves neurological outcomes in experimental models of stroke. We aimed to assess whether glibenclamide improves functional outcomes in patients with acute ischemic stroke treated with recombinant tissue plasminogen activator (rtPA). Methods: In this randomized, double-blind, placebo-controlled trial, patients with acute ischemic stroke were recruited to eight academic hospitals in China. Patients were eligible if they were aged 18-74 years, presented with a symptomatic anterior circulation occlusion with a deficit on the NIHSS of 4-25, and had been treated with rtPA within 4.5 h of symptom onset. We used web-based randomization (1:1) to allocate eligible participants to the glibenclamide or placebo group, stratified according to endovascular treatment and baseline stroke severity. Glibenclamide or placebo was taken orally or via tube feeding at a loading dose of 1.25 mg within 10 h after symptom onset, followed by 0.625 mg every 8 h for 5 days. The primary outcome was the proportion of patients with good outcomes (modified Rankin Scale of 0-2) at 90 days, assessed in all randomly assigned patients who had been correctly diagnosed and had begun study medication. The study is registered with ClinicalTrials.gov, NCT03284463, and is closed to new participants. Findings: Between January 1, 2018, and May 28, 2022, 305 patients were randomly assigned, of whom 272 (142 received glibenclamide and 130 received placebo) were included in the primary efficacy analysis. 103 (73%) patients in the glibenclamide group and 94 (72%) in the placebo group had a good outcome (adjusted risk difference 0.002, 95% CI -0.098 to 0.103; p = 0.96). 12 (8%) patients allocated to glibenclamide and seven (5%) patients allocated to placebo died from any cause at 90 days (p = 0.35). The number and type of adverse events were similar between the two groups. There were no drug-related adverse events and no drug-related deaths. Interpretation: The addition of glibenclamide to thrombolytic therapy did not increase the proportion of patients who achieved good outcomes after stroke compared with placebo, but it did not lead to any safety concerns. Funding: Southern Medical University and Nanfang Hospital.

One-step production of functional branched oligoglucosides with coupled fermentation of Pichia pastoris GS115 and Sclerotium rolfsii WSH-G01.

Endo-ß-1,3-glucanase with high specific activity is a prerequisite for enzymatic preparation of valuable ß-oligoglucosides. Heterologous expression in Pichia pastoris GS115 with error-prone PCR technology was implemented, and the mutant strain 7 N12 was obtained. The mutant endo-ß-1,3-glucanase showed efficient specific activities for degrading curdlan (366 U mg-1) and scleroglucan (274.5 U mg-1). Thereafter, one-step production of functional branched oligoglucosides was established with coupled fermentation of Pichia pastoris and Sclerotium rolfsii. During the fermentation process, the endo-ß-1,3-glucanase secreted by Pichia pastoris GS115 can efficiently hydrolyse scleroglucan metabolized by Sclerotium rolfsii WSH-G01. The maximum yields of ß-oligoglucosides in the shake flasks and 7-L bioreactor reached 1.73 g L-1 and 12.71 g L-1, respectively, with polymerization degrees of 2-17. The successful implementation of heterologous expression with error-prone PCR and the coupled fermentation simplified the multi-step enzymatic ß-oligoglucoside preparation procedures, which makes it a potential strategy for industrial production of functional oligosaccharides.

Efficient endo-ß-1,3-glucanase expression in Pichia pastoris for co-culture with Agrobacterium sp. for direct curdlan oligosaccharide production.

The production of curdlan oligosaccharides, a multifunctional and valuable carbohydrate, by hydrolyzing polysaccharides is of great interest. The endo-ß-1,3-glucanase derived from Trichoderma harzianum was expressed in Pichia pastoris with three commonly used promoters (AOX1, GAP and FLD1). The purified recombinant endo-ß-1,3-glucanase expressed by Pichia pastoris with GAP promoter displayed high specific activity at pH 5.5 and 50 °C. Thereafter, a co-culture system of Pichia pastoris GS115 (GAP promoter) and Agrobacterium sp. was constructed in which Agrobacterium sp.-metabolized curdlan can be directly hydrolyzed by Pichia pastoris-secreted endo-ß-1,3-glucanase to produce functional curdlan oligosaccharides. The co-culture conditions were optimized and the process was carried out in a 7-L bioreactor. The maximum yield of curdlan oligosaccharides reached 18.77 g/L with 3-10 degrees of polymerization. This study presents a novel and easy curdlan oligosaccharide production strategy that can replace traditional sophisticated production procedures and could potentially be implemented for production of other oligosaccharides.

mTOR is involved in stroke-induced seizures and the anti-seizure effect of mild hypothermia.

Stroke is considered an underlying etiology of the development of seizures. Stroke leads to glucose and oxygen deficiency in neurons, resulting in brain dysfunction and injury. Mild hypothermia is a therapeutic strategy to inhibit stroke­induced seizures, which may be associated with the regulation of energy metabolism of the brain. Mammalian target of rapamycin (mTOR) signaling and solute carrier family 2, facilitated glucose transporter member (GLUT)­1 are critical for energy metabolism. Furthermore, mTOR overactivation and GLUT­1 deficiency are associated with genetically acquired seizures. It has been hypothesized that mTOR and GLUT­1 may additionally be involved in seizures elicited by stroke. The present study established global cerebral ischemia (GCI) models of rats. Convulsive seizure behaviors frequently occurred during the first and the second days following GCI, which were accompanied with seizure discharge reflected in the EEG monitor. Expression of phosphor (p)­mTOR and GLUT­1 were upregulated in the cerebral cortex and hippocampus, as evidenced by immunohistochemistry and western blot analyses. Mild hypothermia and/or rapamycin (mTOR inhibitor) treatments reduced the number of epileptic attacks, seizure severity scores and seizure discharges, thereby alleviating seizures induced by GCI. Mild hypothermia and/or rapamycin treatments reduced phosphorylation levels of mTOR and the downstream effecter p70S6 in neurons, and the amount of GLUT­1 in the cytomembrane of neurons. The present study revealed that mTOR is involved in stroke­induced seizures and the anti­seizure effect of mild hypothermia. The role of GLUT­1 in stroke­elicited seizures appears to be different from the role in seizures induced by other reasons. Further studies are necessary in order to elucidate the exact function of GLUT-1 in stroke­elicited seizures.

Mild hypothermia inhibits the Notch 3 and Notch 4 activation and seizure after stroke in the rat model.

Ischemic brain injury is an important cause for seizure. Mild hypothermia of the brain or the whole body is an effective way to remit the post-stroke seizure. Our previous study revealed an implication of Notch 1 and 2 in the post-stroke seizure. This study further investigated the involvement of Notch 3 and 4 in post-stroke seizure and the effect of mild hypothermia on these two factors. A global cerebral ischemia (GCI) model was conducted in Sprague Dawley rats. Seizure activity was evaluated by the frequency of seizure attacks, seizure severity scores, and seizure discharges. Seizures were frequently occurred in the first and the second 24 h after GCI, however active whole-body cooling (mild hypothermia) and DAPT (Notch inhibitor) injection into the hippocampus, alone or in combination, alleviated seizure activity after GCI. Immunohistochemistry and Western blot assays revealed the up-regulation of Notch intracellular domain (NICD) 3 and 4 in the cerebral cortex and hippocampus following GCI, but mild hypothermia and DAPT inhibited the up-regulation of NICD 3 and 4. NF-κB, PPARα, PPARγ, cyclin D1, Sox2 and Pax6 are associated with the pathogenesis of diverse type of seizures. GCI induced NF-κB, cyclin D1, and Pax6 activity, but suppressed PPARγ. These effects of GCI were abolished by both mild hypothermia and DAPT treatment. This indicated the implication of Notch signaling in the effects of GCI. Collectively, mild hypothermia inhibits Notch 3 and Notch 4 activation and seizure after stroke in the rat model. This study adds to the further understanding of the pathogenesis of post-stroke seizures and the protective mechanism of mild hypothermia.

Synergistic effect of mild hypothermia and the Notch inhibitor DAPT against post stroke seizures.

Seizure is a serious complication of stroke, indicating poor prognosis. Notch signaling is associated with neuronal activity. Inhibition of Notch signaling suppresses seizure activity induced by kainic acid. The present study investigated the effect of the Notch inhibitor, DAPT, alone or in combination with mild hypothermia, on post-stroke seizures. A global cerebral ischemia (GCI) model was performed in Sprague Dawley (SD) male rats. Seizure activity was evaluated by the frequency of seizure attacks, seizure severity scores, and seizure discharges. Without any intervention, seizures occurred intensively between 24h and 48h following GCI. Seizure activity was confirmed using EEG monitoring. The expression of Notch intracellular domains (NICD) 1 and 2 were up-regulated in the cerebral cortex and hippocampus following GCI. DAPT was injected into the hippocampus of the rats to inhibit local Notch signaling. Active whole-body cooling was performed to maintain the core temperatures of rats at 33.5°C (mild hypothermia). Mild hypothermia and DAPT synergistically inhibited NICD 1 and 2 up-regulation, and post-stroke seizures. GCI augmented excitatory synaptic neurotransmission by up-regulating glutamate receptor subunits (GluN2A, GluA1) and the cotransporter, NKCC1, but attenuated inhibitory synaptic neurotransmission by down-regulating gamma amino acid, butyric acid (GABA), and the cotransporter, KCC2. DAPT treatment normalized the homeostasis of excitatory and inhibitory synaptic neurotransmission, suggesting that aberrant activation of Notch signaling is involved in post-stroke seizures. The present study adds to the further understanding of the pathogenesis of post-stroke seizures and the improvement of the treatment provided with hypothermia.

Mild hypothermia modulates the expression of nestin and caspase-3 in the sub-granular zone and improves neurological outcomes in rats with ischemic stroke.

We assessed neurological outcomes, infarct volume, and the expression of nestin and caspase-3 in the hippocampal dentate gyrus following middle cerebral artery occlusion (MCAO) followed by reperfusion, with mild hypothermia (MH) treatment at the onset of ischemia in a MCAO rat model. Reperfusion began 2 hours after the MCAO model was set-up. MH treatment began at the onset of ischemia and was maintained for 4 hours. We evaluated neurological deficit score, brain infarct volumes, along with the immunohistochemical staining of nestin and caspase-3 in the sub-granular zone of the injured hemisphere on the 1st, 3rd, 7th, and 14th day after the onset of ischemia. Correlations between the number of nestin-positive (nestin+) cells, caspase-3-positive (caspase-3+) cells with infarct volume, as well as neurological deficit scores, were evaluated by linear regression. MH significantly promoted survival, reduced mortality, improved neurological deficit score, reduced brain infarct volume, increased the number of neural stem/progenitor cells and inhibited neuronal apoptosis in the sub-granular zone of the injured hemisphere. The number of nestin+ cells correlated with neurological deficit score in the normothermic group, and with infarct volume in the hypothermia group except for the first day after the onset of ischemia. The number of caspase-3+ cells correlated with the neurological deficit score but not infarct volume. The neuroprotective effects of MH may be mediated by modulating neural stem/progenitor cells and neuronal apoptotic cells in the sub-granular zone of the injured hemisphere during cerebral ischemia/reperfusion injury.

Altered Spontaneous Brain Activity in Betel Quid Dependence: A Resting-state Functional Magnetic Resonance Imaging Study.

It has been suggested by the first voxel-based morphometry investigation that betel quid dependence (BQD) individuals are presented with brain structural changes in previous reports, and there may be a neurobiological basis for BQD individuals related to an increased risk of executive dysfunction and disinhibition, subjected to the reward system, cognitive system, and emotion system. However, the effects of BQD on neural activity remain largely unknown. Individuals with impaired cognitive control of behavior often reveal altered spontaneous cerebral activity in resting-state functional magnetic resonance imaging and those changes are usually earlier than structural alteration.Here, we examined BQD individuals (n = 33) and age-, sex-, and education-matched healthy control participants (n = 32) in an resting-state functional magnetic resonance imaging study to observe brain function alterations associated with the severity of BQD. Amplitude of low-frequency fluctuation (ALFF) and regional homogeneity (ReHo) values were both evaluated to stand for spontaneous cerebral activity. Gray matter volumes of these participants were also calculated for covariate.In comparison with healthy controls, BQD individuals demonstrated dramatically decreased ALFF and ReHo values in the prefrontal gurus along with left fusiform, and increased ALFF and ReHo values in the primary motor cortex area, temporal lobe as well as some regions of occipital lobe. The betel quid dependence scores (BQDS) were negatively related to decreased activity in the right anterior cingulate.The abnormal spontaneous cerebral activity revealed by ALFF and ReHo calculation excluding the structural differences in patients with BQD may help us probe into the neurological pathophysiology underlying BQD-related executive dysfunction and disinhibition. Diminished spontaneous brain activity in the right anterior cingulate cortex may, therefore, represent a biomarker of BQD individuals.