Ischemia-induced Brain Injury Research Articles

Protection of Gueichih-Fuling-Wan on cerebral ischemia-induced brain injury in rodents is mediated by trans-cinnamaldehyde via inhibition of neuroinflammation and apoptosis

Protection of Gueichih-Fuling-Wan on cerebral ischemia-induced brain injury in rodents is mediated by trans-cinnamaldehyde via inhibition of neuroinflammation and apoptosis

The Effects of IRL-1620 in Post-ischemic Brain Injury: A Systematic Review and Meta-analysis of Experimental Studies.

Sovateltide (IRL-1620), an endothelin B receptor agonist, has previously demonstrated neuroprotective and neuroregenerative effects in animal models of acute ischemic stroke. Recently, clinical trials indicated that it could also be effective in humans with stroke. Here, we systematically investigate whether IRL-1620 may be used for the treatment of ischemia-induced brain injury. A systematic review was performed following the Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines. MEDLINE (PubMed) and Scopus databases were searched for eligible studies up to December 2022. The databases ClinicalTrials.gov and Pharmazz Inc. were screened for unpublished or ongoing trials. Only studies in English were evaluated for eligibility. Meta-analysis of the included studies was also conducted. Finally, seven studies were included in the review, all in animal rat models because of scarcity of clinical trials. Six studies, all in middle cerebral artery occlusion (MCAO) models, were selected for meta-analysis. In the two studies assessing mortality, no deaths were reported in the IRL-1620 group 24h after MCAO, whereas the vehicle group had almost a five times higher mortality risk (risk ratio 5.3, 95% confidence interval 0.7-40.1, I2 = 0%). In all five studies evaluating outcome on day 7 after MCAO, IRL-1620 was associated with statistically significantly lower neurological deficit and improved motor performance compared with the vehicle. Infract volume, differentiation potential of neuronal progenitor cells, and mitochondrial fate also improved with IRL-1620 administration. According to the above, in animal MCAO models, IRL-1620 enhanced neurogenesis and neuroprotection and improved outcome. Future studies are needed to expand our understanding of its effects in human study participants with acute ischemic stroke as well as in other common causes of cerebral ischemia including cardiac arrest.

Uncovering Sex-Specific Epigenetic Regulatory Mechanism Involving H3k9me2 in Neural Inflammation, Damage, and Recovery in the Internal Carotid Artery Occlusion Mouse Model.

Cerebral ischemic stroke is one of the foremost global causes of death and disability. Due to inadequate knowledge in its sequential disease mechanisms, therapeutic efforts to mitigate acute ischemia-induced brain injury are limited. Recent studies have implicated epigenetic mechanisms, mostly histone lysine acetylation/deacetylation, in ischemia-induced neural damage and death. However, the role of lysine methylation/demethylation, another prevalent epigenetic mechanism in cerebral ischemia has not undergone comprehensive investigation, except a few recent reports, including those from our research cohort. Considering the impact of sex on post-stroke outcomes, we studied both male and female mice to elucidate molecular details using our recently developed Internal Carotid Artery Occlusion (ICAO) model, which induces mild to moderate cerebral ischemia, primarily affecting the striatum and ventral hippocampus. Here, we demonstrate for the first time that female mice exhibit faster recovery than male mice following ICAO, evaluated through neurological deficit score and motor coordination assessment. Furthermore, our investigation unveiled that dysregulated histone lysine demethylases (KDMs), particularly kdm4b/jmjd2b are responsible for the sex-specific variance in the modulation of inflammatory genes. Building upon our prior reportage blocking KDMs by DMOG (Dimethyloxalylglycine) and thus preventing the attenuation in H3k9me2 reduced the post-ICAO transcript levels of the inflammatory molecules and neural damage, our present study delved into investigating the differential role of H3k9me2 in the regulation of pro-inflammatory genes in female vis-à-vis male mice underlying ICAO-induced neural damage and recovery. Overall, our results reveal the important role of epigenetic mark H3k9me2 in mediating sex-specific sequential events in inflammatory response, elicited post-ICAO.

THE EFFECTS OF COMBINATION OF RADIOFREQUENCY AND PULSED MAGNETIC FIELD ON CAROTID ARTERIA ISCHEMIA AND REPERFUSION INDUCED BRAIN INJURY: A PRELIMINARY REPORT

Objective
 Cerebrovascular accident due to ischemia (IS)
 mediated by atherosclerotic plaque in the brain
 can trigger inflammation in the cerebral cortex,
 hippocampus and cerebellum tissues. Radiofrequency
 electromagnetic field (RF-EMF) and pulsed magnetic
 field (PMF) applications can increase nitric oxide
 formation from the vascular endothelial layer. The
 aim of this preliminary study is to reduce the damage
 caused by IS in different tissues of the brain by
 magnetic field applications.
 Material and Method
 A total of 9 rats, one rat in each group; sham,
 prophylactic RF, PMF, RF+PMF and therapeutic
 RF-EMF, PMF, RF-EMF+PMF, prophylactic and
 therapeutic RF-EMF+PMF and IS-only groups were
 distinguished. In single or combined applications of
 prophylactic/therapeutic RF-EMF and PMF groups,
 rats were taken to the experimental unit for 30
 minutes of magnetic field exposure before and after
 30 minutes of carotid artery occlusion for IS purposes.
 Histopathological hematoxylin-eosin staining in
 brain tissue (cerebral cortex and hippocampus)
 and cerebellum tissues taken after sacrification;
 With immunohistochemical analysis, brain derived
 neurotrophic factor (BDNF), tumor necrosis factoralpha
 (TNF-α), mammalian target of rapamycin
 (mTOR) and inducible nitric oxide synthase (iNOS)
 expressions were examined.
 Results
 Histopathologically significant hyperemia, edema,
 bleeding and neuronal degeneration were detected
 in the IS group. Additionally, immunohistochemically,
 an increase in TNF-α, mTOR, iNOS and a decrease
 in BDNF staining were observed. Prophylactic and/or
 therapeutic RF-EMF and/or PMF applications reversed
 all these parameters. The greatest improvement
 was observed in the Prophylactic+Therapeutic RFEMF+
 PMF group.
 Conclusion
 As a result, the regression of IS-related inflammation in
 both brain tissue parts and cerebellar tissues with RFEMF
 and PMF is important in terms of the formation
 of neurological deficits, the continuity of learning
 and memory mechanisms, and the preservation of
 balance functions.

Kaempferol-3-O-(2″-O-galloyl-β-D-glucopyranoside): a novel neuroprotective agent from Diospryros kaki against cerebral ischemia-induced brain injury.

Our previous study demonstrated neuroprotective and therapeutic effects of a standardized flavonoid extract from leaves of Diospyros kaki L.f. (DK) on middle cerebral artery occlusion-and-reperfusion (MCAO/R)-induced brain injury and its underlying mechanisms. This study aimed to clarify flavonoid components responsible for the effects of DK using in vitro and in vivo transient brain ischemic models. Organotypic hippocampal slice cultures (OHSCs) subjected to oxygen- and glucose-deprivation (OGD) were performed to evaluate in vitro neuroprotective activity of DK extract and nine isolated flavonoid components. MCAO/R mice were employed to elucidate in vivo neuroprotective effects of the flavonoid component that exhibited the most potent neuroprotective effect in OHSCs. DK extract and seven flavonoids [quercetin, isoquercetin, hyperoside, quercetin-3-O-(2″-O-galloyl-β-D-galactopyranoside), kaempferol, astragalin, and kaempferol-3-O-(2″-O-galloyl-β-D-glucopyranoside) compound (9)] attenuated OGD-induced neuronal cell damage and compound (9) possessed the most potent neuroprotective activity in OHSCs. The MCAO/R mice showed cerebral infarction, massive weight loss, characteristic neurological symptoms, and deterioration of neuronal cells in the brain. Compound (9) and a reference drugs, edaravone, significantly attenuated these physical and neurological impairments. Compound (9) mitigated the blood-brain barrier dysfunction and the change of glutathione and malondialdehyde content in the MCAO mouse brain. Edaravone suppressed the oxidative stress but did not significantly affect the blood-brain barrier permeability. The present results indicated that compound (9) is a flavonoid constituent of DK with a potent neuroprotective activity against transient ischemia-induced brain damage and this action, at least in part, via preservation of blood-brain barrier integrity and suppression of oxidative stress caused by ischemic insult.

Therapeutic effects of a standardized-flavonoid Diospyros kaki L.f. leaf extract on transient focal cerebral ischemia-induced brain injury in mice.

This study aimed to investigate the neuroprotective and therapeutic effects of Diospyros kaki L.f. leaves (DK) on transient focal cerebral ischemic injury and underlying mechanisms using a middle cerebral artery occlusion (MCAO) model of mice. The animals received the MCAO operation on day 0. The daily administrations of DK (50 and 100mg/kg, p.o) and edaravone (6mg/kg, i.v), a reference drug with radical scavenging activity, were started 7days before (pre-treatment) or immediately after the MCAO operation (post-treatment) and continued during the experimental period. Histochemical, biochemical, and neurological changes and cognitive performance were evaluated. MCAO caused cerebral infarction and neuronal cell loss in the cortex, striatum, and hippocampus in a manner accompanied by spatial cognitive deficits. These neurological and cognitive impairments caused by MCAO were significantly attenuated by pre- and post-ischemic treatments with DK and edaravone, suggesting that DK, like edaravone, has therapeutic potential for cerebral ischemia-induced brain damage. DK and edaravone suppressed MCAO-induced changes in biomarkers for apoptosis (TUNEL-positive cell number and cleaved caspase-3 protein expression) and oxidative stress (glutathione and malondialdehyde contents) in the brain. Interestingly, DK, but not edaravone, mitigated an increase in blood-brain permeability and down-regulation of vascular endothelial growth factor protein expression caused by MCAO. Although the exact chemical constituents implicated in the effects of DK remain to be clarified, the present results indicate that DK exerts neuroprotective and therapeutic activity against transient focal cerebral ischemia-induced injury probably by suppressing oxidative stress, apoptotic process, and mechanisms impairing blood-brain barrier integrity in the brain.

Structural and functional analysis of human pannexin 2 channel

The pannexin 2 channel (PANX2) participates in multiple physiological processes including skin homeostasis, neuronal development, and ischemia-induced brain injury. However, the molecular basis of PANX2 channel function remains largely unknown. Here, we present a cryo-electron microscopy structure of human PANX2, which reveals pore properties contrasting with those of the intensely studied paralog PANX1. The extracellular selectivity filter, defined by a ring of basic residues, more closely resembles that of the distantly related volume-regulated anion channel (VRAC) LRRC8A, rather than PANX1. Furthermore, we show that PANX2 displays a similar anion permeability sequence as VRAC, and that PANX2 channel activity is inhibited by a commonly used VRAC inhibitor, DCPIB. Thus, the shared channel properties between PANX2 and VRAC may complicate dissection of their cellular functions through pharmacological manipulation. Collectively, our structural and functional analysis provides a framework for development of PANX2-specific reagents that are needed for better understanding of channel physiology and pathophysiology.

Exosomes from bone marrow mesenchymal stem cells are a potential treatment for ischemic stroke.

Exosomes derived from human bone marrow mesenchymal stem cells (MSC-Exo) are characterized by easy expansion and storage, low risk of tumor formation, low immunogenicity, and anti-inflammatory effects. The therapeutic effects of MSC-Exo on ischemic stroke have been widely explored. However, the underlying mechanism remains unclear. In this study, we established a mouse model of ischemic brain injury induced by occlusion of the middle cerebral artery using the thread bolt method and injected MSC-Exo into the tail vein. We found that administration of MSC-Exo reduced the volume of cerebral infarction in the ischemic brain injury mouse model, increased the levels of interleukin-33 (IL-33) and suppression of tumorigenicity 2 receptor (ST2) in the penumbra of cerebral infarction, and improved neurological function. In vitro results showed that astrocyte-conditioned medium of cells deprived of both oxygen and glucose, to simulate ischemia conditions, combined with MSC-Exo increased the survival rate of primary cortical neurons. However, after transfection by IL-33 siRNA or ST2 siRNA, the survival rate of primary cortical neurons was markedly decreased. These results indicated that MSC-Exo inhibited neuronal death induced by oxygen and glucose deprivation through the IL-33/ST2 signaling pathway in astrocytes. These findings suggest that MSC-Exo may reduce ischemia-induced brain injury through regulating the IL-33/ST2 signaling pathway. Therefore, MSC-Exo may be a potential therapeutic method for ischemic stroke.

Inhibition of the LRRC8A channel promotes microglia/macrophage phagocytosis and improves outcomes after intracerebral hemorrhagic stroke.

Promoting microglial/macrophage (M/Mφ) phagocytosis accelerates hematoma clearance and improves the prognosis of intracerebral hemorrhagic stroke (ICH). Cation channels such as Piezo1 modulate bacterial clearance by regulating M/Mφ. Whether LRRC8A, an anion channel, affects M/Mφ erythrophagocytosis and functional recovery after ICH was investigated here. We found that LRRC8A is highly expressed on M/Mφ in the perihematomal region of ICH mice. Conditional knockout of Lrrc8a in M/Mφ or treatment with an LRRC8A channel blocker accelerated hematoma clearance, reduced neuronal death, and improved functional recovery after ICH. Mechanistically, the LRRC8A channel inhibition promoted M/Mφ phagocytosis by activating AMP-activated protein kinase (AMPK), thereby inducing nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2) and increasing Cd36 transcription. Our findings illuminate the regulation of M/Mφ phagocytosis by the LRRC8A channel via the AMPK-Nrf2-CD36 pathway after ICH, suggesting that LRRC8A is a potential target for hematoma clearance in ICH treatment.

Protective effect of an ethanol extract mixture of Aralia elata, Chaenomeles sinensis fruit, and Glycyrrhizae radix against cerebral ischemia-reperfusion injury in rats and excitotoxic and oxidative neuronal damage

Aralia elata, Chaenomeles sinensis fruit, and Glycyrrhizae radix have been widely used as oriental medicinal plants in Korea, China and Japan and found to possess anti-oxidative and anti-inflammatory activities. The current study was conducted to investigate the neuroprotective effect of an ethanol extract of a mixture of A. elata, C. sinensis fruit, and Glycyrrhizae radix (ACG) against ischemia-induced brain injury in rats and excitotoxic and oxidative neuronal death in primarily cultured rat cortical neurons. Transient focal cerebral ischemia was induced by 2 h middle cerebral artery occlusion followed by 24 h reperfusion (MCAO/R) in rats. Oral administration of ACG (10, 25, and 50 mg/kg) 30 min before MCAO, after 1 h of MCAO, and after 1 h of reperfusion reduced MCAO/R-induced brain infarct and edema formation. ACG also inhibited development of behavioral disabilities in MCAO/R-treated rats. Exposure of cultured cortical neurons to 500 μM glutamate for 12 h resulted in neuronal cell death. ACG (1, 10, and 50 μg/mL) inhibited glutamate-induced neuronal death. Furthermore, ACG inhibited 100 μM hydrogen peroxide (H2O2)- and hypoxia-induced neuronal death. These results suggest that the neuroprotective effect of ACG against ischemia-induced brain damage might be associated with its anti-excitotoxic and anti-oxidative activity and that ACG may have a therapeutic role for prevention of neurodegeneration in stroke.

Mitochondrial Proteins Unveil the Mechanism by Which Physical Exercise Ameliorates Memory, Learning and Motor Activity in Hypoxic Ischemic Encephalopathy Rat Model.

Background: Physical exercise has been shown to improve cognitive and motor functions, promoting neurogenesis and demonstrating therapeutic benefits in neurodegenerative disorders. Nonetheless, it is crucial to investigate the cellular and molecular mechanisms by which this occurs. The study aimed to investigate and evaluate the effect of swimming exercise on the changes of mitochondrial proteins in the brains of rats with hypoxic ischemic encephalopathy (HIE). Methods: the vertical pole and Morris water maze tests were used to assess the animals’ motor and cognitive functions, and western blot and immunofluorescence of brain tissue were used to assess the biomarkers of mitochondrial apoptosis and cristae stability in response to exercise training. Four groups of rats were used: (1) sham sedentary group (SHAM, NT), (2) sham exercise training group (SHAM, T) (3) hypoxic ischemic encephalopathy sedentary group (HIE, NT), and (4) hypoxic ischemic encephalopathy exercise training group (HIE, T). Results: animals with HIE showed motor and cognitive deficits, as well as increased apoptotic protein expression. Exercise, on the other hand, improved motor and cognitive functions while also suppressing the expression of apoptotic proteins. Conclusions: By stabilizing the mitochondrial cristae and suppressing the apoptotic cascade, physical exercise provided neuroprotection in hypoxic ischemia-induced brain injury.

JLX001 ameliorates cerebral ischemia injury by modulating microglial polarization and compromising NLRP3 inflammasome activation via the NF-κB signaling pathway

Ischemic stroke is a devastating disease with high morbidity and mortality rates, and the proinflammatory microglia-mediated inflammatory response directly affects stroke outcome. Previous studies have reported that JLX001, a novel compound with a structure similar to that of cyclovirobuxine D (CVB-D), exerts antiapoptotic, anti-inflammatory and antioxidative effects on ischemia-induced brain injury. However, the role of JLX001 in microglial polarization and nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome regulation after ischemic stroke has not been fully investigated. In this study, we used the middle cerebral artery occlusion (MCAO) method to establish a focal cerebral ischemia model and found that JLX001 attenuated the brain infarct size and improved cerebral damage. Moreover, the expression levels of proinflammatory cytokines (interleukin [IL]-1β and tumor necrosis factor [TNF]-α) were significantly reduced while those of the anti-inflammatory cytokine IL-10 were increased in the JLX001-treated group. Immunofluorescence staining and flow cytometry revealed an increased number of anti-inflammatory phenotypic microglia and a reduced number of proinflammatory phenotypic microglia in JLX001-treated MCAO mice. Western blotting analysis showed that JLX001 inhibited the expression of NLRP3 and proteins related to the NLRP3 inflammasome axis in vivo. Furthermore, JLX001 reduced the number of NLRP3/Iba1 cells in ischemic penumbra tissues. Finally, mechanistic analysis revealed that JLX001 significantly inhibited the expression of proteins related to the NF-κB signaling pathway. Additionally, pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, ameliorated cerebral ischemia–reperfusion injury by suppressing microglial polarization towards the proinflammatory phenotype and NLRP3 activation in vivo, further suggesting that these protective effects of JLX001 were mediated by inhibition of the NF-κB signaling pathway. These results suggest that JLX001 is a promising therapeutic approach for ischemic stroke.

Pioglitazone Ameliorates Lipopolysaccharide-Induced Behavioral Impairment, Brain Inflammation, White Matter Injury and Mitochondrial Dysfunction in Neonatal Rats.

Previous studies have demonstrated that pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, inhibits ischemia-induced brain injury. The present study was conducted to examine whether pioglitazone can reduce impairment of behavioral deficits mediated by inflammatory-induced brain white matter injury in neonatal rats. Intraperitoneal (i.p.) injection of lipopolysaccharide (LPS, 2 mg/kg) was administered to Sprague–Dawley rat pups on postnatal day 5 (P5), and i.p. administration of pioglitazone (20 mg/kg) or vehicle was performed 5 min after LPS injection. Sensorimotor behavioral tests were performed 24 h after LPS exposure, and changes in biochemistry of the brain was examined after these tests. The results show that systemic LPS exposure resulted in impaired sensorimotor behavioral performance, reduction of oligodendrocytes and mitochondrial activity, and increases in lipid peroxidation and brain inflammation, as indicated by the increment of interleukin-1β (IL-1β) levels and number of activated microglia in the neonatal rat brain. Pioglitazone treatment significantly improved LPS-induced neurobehavioral and physiological disturbances including the loss of body weight, hypothermia, righting reflex, wire-hanging maneuver, negative geotaxis, and hind-limb suspension in neonatal rats. The neuroprotective effect of pioglitazone against the loss of oligodendrocytes and mitochondrial activity was associated with attenuation of LPS-induced increment of thiobarbituric acid reactive substances (TBARS) content, IL-1β levels and number of activated microglia in neonatal rats. Our results show that pioglitazone prevents neurobehavioral disturbances induced by systemic LPS exposure in neonatal rats, and its neuroprotective effects are associated with its impact on microglial activation, IL-1β induction, lipid peroxidation, oligodendrocyte production and mitochondrial activity.

Abstract P747: Gpr68 Play a Protective Role in Ischemic Stroke in Mice

Introduction: Acidosis is one prevalent phenomenon in ischemic stroke. The literature has shown that protons directly gate acid-sensing ion channels (ASICs) and proton-activated chloride channel, both lead to neuronal injury However, it is unclear whether protons activate metabotropic pathways in brain neurons. There are four proton-sensitive G-protein coupled receptors (GPCRs): GPR4, GPR65, GPR68 and GPR132. It remains unknown whether any of these GPCRs mediate acid-induced signals in brain neurons or whether they contribute to ischemia-induced brain injury. Methods: Total RNA from human cortical tissue or mouse brain was isolated using TRIzol and an RNase Kit. Standard RT-PCR was performed to determine the expression of these GPCRs in the brain. An in vitro slice injury model was used for functional screening. To determine the effect of ischemia, WT and knockout male mice were subjected to MCAO. To study brain injury, brains were sectioned coronally at 1 mm intervals and stained by vital dye immersion: (2%) 2,3,5-triphenyltetrazolium hydrochloride (TTC). Locomotor analysis and corner test were used to assess behavior outcome. Adeno-associated virus (AAV) -mediated gene delivery was used to determine the outcome of GPR68 overexpression. Results: RT-PCR showed that brain tissue expressed GPR4, -65, and -68. The expression of GPR68 was evident at 30 cycles. In organotypic slices, compared to the WT, deleting GPR4 or GPR65 had no effect while deleting GPR68 significantly increased acidosis-induced neuronal injury. At both 24 hour and 72 hour after 45 minutes MCAO, GPR68 deletion increased brain injury (p=0.0020 for 24hour, p=0.0392 for 72hour, Mann-Whitney U test). WT and GPR68-/- mice did not differ in baseline locomotor activities or corner test. On the third day following MCAO, GPR68-/- exhibited significantly more left rotations (p=0.0287, Mann-Whitney U test) than WT animals. Lastly, mice receiving AAV-GPR68 exhibited an average infarct of 21.97 ± 12.4%, significantly (p = 0.0022, Mann-Whitney U test) smaller than those receiving AAV-GFP (37.2 ± 6.8%). Conclusion: These data showed that GPR68 functions as a neuroprotective proton receptor in the brain.

β-1, 3-galactosyltransferase 2 deficiency exacerbates brain injury after transient focal cerebral ischemia in mice

Glycosyltransferases are enzymes that catalyze the formation of a variety of glycoconjugates. Glycoconjugates play vital roles in the nervous system. β-1, 3-Galactosyltransferase 2 (B3galt2) is one of the major types of glycosyltransferases, which has not been reported in ischemia induced-brain injury. The purpose of this study was to explore the role of B3galt2 exerts and its underlying mechanism in cerebral ischemia in mice. Wild-type (WT) and heterozygous B3galt2 knockout (B3galt2−/+) mice were subjected to 90 min transient focal cerebral ischemia by middle cerebral artery occlusion (MCAO). The brain samples were analyzed at 24 h after reperfusion. The B3galt2 level in the peri-infarct penumbra was quantified. The cerebral infarct volume, neurological deficits, apoptosis and the levels of Reelin and Dab1 were assessed. Compared with control mice, B3galt2−/+ mice not only showed severe brain damage, neurologic functional deficits, but also showed severe neuronal apoptosis in the cortical penumbra after ischemia/reperfusion (I/R). The Caspase-3 activity was increased and the levels of Reelin and Dab1 were decreased in B3galt2−/+ mice. Recombinant human Reelin (rh-Reelin) administered intracerebroventricularly before MCAO significantly reduced infarct volume, and prevented neuronal loss in B3galt2−/+ mice after I/R. Our results suggest B3galt2 deficiency exacerbates ischemic brain damage in acute ischemic stroke in mice, and this was reversed by giving rh-Reelin. B3galt2 might play a beneficial role for neurons survival in the penumbra through modulation of Reelin pathway.

The protective effects of Pimavanserin against cerebral ischemia-induced brain injury

ABSTRACT The integrity of the blood-brain barrier (BBB) is mainly maintained by the brain vascular endothelial cells and the tight junctions amongst them. Pimavanserin is a novel agent approved for the treatment of Parkinson’s disease and exerts neuroprotective properties. The present study aims to explore the possibility that Pimavanserin might be an effective agent used for the treatment of cerebral ischemia stroke. Middle cerebral artery occlusion (MCAO) was established in mice, and oxygen-glucose deprivation/reoxygenation (OGD/R) was established in brain bEND.3 endothelial cells. Mice were randomly divided into four groups: (1) Sham operation group; (2). Pimavanserin (1 mg/kg); (3). MCAO; (4). Pimavanserin+ MCAO. We found that compared to the Sham group, the elevated neurological deficit score and brain water content increased production of inflammatory factors, increased BBB permeability, and downregulated Claudin 5 expression were observed in the MCAO group and were all dramatically reversed by the administration of Pimavanserin. Brain bEND.3 endothelial cells were treated with Pimavanserin before the exposure to OGD/R. The significantly increased lactate dehydrogenase (LDH) release, declined cell viability, increased endothelial permeability, downregulated Claudin 5 and Krüppel-like factors 6 (KLF6) were observed in the OGD/R group and were all reversed by the introduction of Pimavanserin. Lastly, the effects of Pimavanserin on the expression level of Claudin 5 and endothelial permeability in OGD/R-challenged endothelial cells were both abolished by the knockdown of KLF6. Taken together, our data revealed that Pimavanserin protected against cerebral ischemia injury by regulating the BBB integrity in a KLF6-dependent manner.

Subanesthetic isoflurane abates ROS-activated MAPK/NF-κB signaling to repress ischemia-induced microglia inflammation and brain injury.

Isoflurane (ISO) elicits protective effects on ischemia-induced brain injury. We investigated whether sub-anesthetic (0.7%) ISO post-conditioning attenuates the inflammation and apoptosis in oxygen-glucose deprivation (OGD)-insulted co-cultures (microglia and neurons) in vitro and the brain injury of the middle cerebral arterial occlusion (MCAO) rat. We demonstrated that ISO augmented the viability of OGD-treated microglia and neurons. ISO reduced the expression and activation of COX2 and iNOS in OGD-challenged microglia. ISO repressed the production of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-8, and monocyte chemoattractant protein-1 in OGD-exposed microglia. ISO also decreased nucleosomal fragmentation and caspase-3 activity but increased mitochondrial membrane potential in OGD-stimulated microglia and neurons. Mechanistically, ISO suppressed OGD-induced microglial inflammation by blocking ROS-regulated p38 MAPK/NF-κB signaling pathway and hampered OGD-triggered microglial apoptosis in a ROS- or NO-dependent fashion. In vivo results with MCAO rats were partly consistent with the in vitro observation. These findings indicate that sub-anesthetic ISO post-conditioning abates the inflammation and apoptosis in OGD-stimulated rat microglia and the apoptosis of OGD-exposed neurons and the brain injuries of MCAO rats, suggesting it as a potentially effective therapeutic approach for ischemic brain damages.

Kellerin from Ferula sinkiangensis exerts neuroprotective effects after focal cerebral ischemia in rats by inhibiting microglia-mediated inflammatory responses

Ethnopharmacological relevanceFerula sinkiangensis K. M. Shen is a traditional Chinese medicine that has a variety of pharmacological properties relevant to neurological disorders and inflammations. Kellerin, a novel compound extracted from Ferula sinkiangensis, exerts a strong anti-neuroinflammatory effect by inhibiting microglial activation. Microglial activation plays a vital role in ischemia-induced brain injury. However, the potential therapeutic effect of kellerin on focal cerebral ischemia is still unknown. Aim of the studyTo explore the effect of kellerin on cerebral ischemia and clarify its possible mechanisms, we applied the middle cerebral artery occlusion (MCAO) model and the LPS-activated microglia model in our study. Materials and methodsNeurological outcome was examined according to a 4-tiered grading system. Brain infarct size was measured using TTC staining. Brain edema was calculated using the wet weight minus dry weight method. Neuron damage and microglial activation were observed by immunofluorescence in MCAO model in rats. In in vitro studies, microglial activation was examined by flow cytometry and the viability of neuronal cells cultured in microglia-conditioned medium was measured using MTT assay. The levels of pro-inflammatory cytokines were measured by qRT-PCR and ELISA. The proteins involved in NF-κB signaling pathway were determined by western blot. Intracellular ROS was examined using DCFH-DA method and NADPH oxidase activity was measured using the NBT assay. ResultsWe found that kellerin improved neurological outcome, reduced brain infarct size and decreased brain edema in MCAO model in rats. Under the pathologic conditions of focal cerebral ischemia, kellerin alleviated neuron damage and inhibited microglial activation. Moreover, in in vitro studies of LPS-stimulated BV2 cells kellerin protected neuronal cells from being damaged by inhibiting microglial activation. Kellerin also reduced the levels of pro-inflammatory cytokines, suppressed the NF-κB signaling pathway, and decreased ROS generation and NADPH oxidase activity. ConclusionsOur discoveries reveal that the neuroprotective effects of kellerin may largely depend on its inhibitory effect on microglial activation. This suggests that kellerin could serve as a novel anti-inflammatory agent which may have therapeutic effects in ischemic stroke.

Neuroprotective and anti-inflammatory effects of myricetin 3-glucoside in a rat model of cerebral ischemia

Purpose: To investigate the effect of myricetin 3-glucoside (M3GLS) on middle cerebral artery occlusion (MCAO)-induced cerebral ischemia in a rat model, and the mechanism of action involved.Methods: A cerebral ischemia rat model was established using MCAO under 10 % chloral hydrate anesthesia. Neurological severity score was determined by analyzing reflex, motor and sensory functions, as well as balancing potential. Infarction volume was determined using triphenyl tetrazolium chloride dye, while counting of Nissl bodies was done after toluidine blue staining. The protein expression levels of Bax and Bcl-2 were assayed using western blotting, while cytokine levels were determined by enzyme-linked immunosorbent assay (ELISA).Results: Treatment of cerebral ischemia rats with M3GLS effectively reduced infarct volume, when compared to vehicle-treated group (p < 0.05). Moreover, M3GLS treatment significantly increased the population of Nissl bodies and effectively improved neurologic scores (p < 0.05). In M3GLS-pretreated rats, cerebral ischemia-induced elevation of protein expressions of TNF-α, IL-6 and IL-1β were significantly suppressed. M3GL treatment significantly reversed cerebral ischemia-mediated downregulation of Bcl-2 protein level, but markedly reduced cerebral ischemia-induced upregulation of Bax protein level (p < 0.05).Conclusion: M3GLS exerts protective effect against cerebral ischemia-induced brain injury in rats via downregulation of inflammatory cytokines. It reduces infarction volume in the brain of cerebral ischemia rats, and regulates Bcl-2/Bax protein ratio. Thus, M3GLS has a potential for use in the clinical management of cerebral ischemia.
 Keywords: Myricetin, Neuroprotection, Anti-inflammation, Cerebral ischemia, Cytokines, Infarction

Cerebrovascular effects of glibenclamide investigated using high-resolution magnetic resonance imaging in healthy volunteers

Glibenclamide inhibits sulfonylurea receptor (SUR), which regulates several ion channels including SUR1-transient receptor potential melastatin 4 (SUR1-TRPM4) channel and ATP-sensitive potassium (KATP) channel. Stroke upregulates SURl-TRPM4 channel, which causes a rapid edema formation and brain swelling. Glibenclamide may antagonize the formation of cerebral edema during stroke. Preclinical studies showed that glibenclamide inhibits KATP channel-induced vasodilation without altering the basal vascular tone. The in vivo human cerebrovascular effects of glibenclamide have not previously been investigated. In a randomized, double-blind, placebo-controlled, three-way cross-over study, we used advanced 3 T MRI methods to investigate the effects of glibenclamide and KATP channel opener levcromakalim on mean global cerebral blood flow (CBF) and intra- and extracranial artery circumferences in 15 healthy volunteers. Glibenclamide administration did not alter the mean global CBF and the basal vascular tone. Following levcromakalim infusion, we observed a 14% increase of the mean global CBF and an 8% increase of middle cerebral artery (MCA) circumference, and glibenclamide did not attenuate levcromakalim-induced vascular changes. Collectively, the findings demonstrate the vital role of KATP channels in cerebrovascular hemodynamic and indicate that glibenclamide does not inhibit the protective effects of KATP channel activation during hypoxia and ischemia-induced brain injury.