Focal Cerebral Ischemia Research Articles

Nitric oxide synthase inhibitor is an effective therapy for ischemia‐reperfusion injury in mice

BackgroundInhibition of nitric oxide synthase (NOS) has been shown to paradoxically protect against ischemic brain injury by an unknown mechanism. Recently, we found that NOS inhibitor, N (gamma)‐nitro‐L‐arginine methyl ester (L‐NAME), protects primary cultures of endothelial cells and neurons against oxygen‐glucose deprivation‐reoxygenation injury by preventing NOS uncoupling. Thus, the objective of the present study was to demonstrate the effectiveness of locally administered L‐NAME against ischemia‐reperfusion (I‐R) injury.MethodsTransient focal cerebral ischemia in male mice (C57Bl/6, 2‐4 months) was achieved by filament method of MCAO with a 60 min occlusion. We employed a novel approach to inject L‐NAME (1 mg/Kg dosages at a rate of 60 ug/minute) locally into the ischemic zone by threading a catheter up the middle cerebral artery. This was timed to the onset of reperfusion by rapidly followng the removal of the occlusion with the silicon suture. Following 24‐hour reperfusion, infarct size was determined by 2,3,5‐Triphenyltetrazolium chloride (TTC) staining and neurological function was assessed by employing a standard neurological score (0‐4 range) and Rotarod performance test.ResultsAdmnistration of FITC Dextran 40K and fluorescence microscopy of the brain slices 1 hour post‐ischemia confirmed the effective delivery of injected L‐NAME into the ischemic tissue. Localized injection of L‐NAME was protective against I‐R injury and promoted significant recovery of neurological and motor function (Figure).ConclusionLocal NOS inhibition could serve as an adjuvant for reperfusion therapies of ischemic stroke.

Efficacy of post‐injury anti‐miR‐181a and anti‐miR‐200c intravenous treatment in protection against experimental stroke in aged mice

IntroductionStroke remains the second leading cause of death worldwide and the primary cause of long‐term disability in the US. To date, the only pharmacological treatment for stroke remains tissue plasminogen activator. A major factor for translational failure is likely overwhelming use of young adult rodent models despite the fact that stroke occurs predominantly in aged populations. MicroRNAs (miRs) are non‐coding RNAs that regulate protein translation, and we have previously demonstrated in young adult mice neuroprotection against experimental stroke with anti‐miR‐181a and anti‐miR‐200c. In the present study, to extend translational impact, we tested efficacy of post‐injury intravenous anti‐miR‐181a and anti‐miR‐200c in clinically relevant aged mice.MethodsAll studies were conducted in accordance with National Institutes of Health guidelines for the use of experimental animals, with protocols approved by the Stanford Animal Care and Use Committee. Animal treatment groups were randomized by coin flip, and all analyses were performed by an observer blinded to conditions. Transient focal cerebral ischemia was induced by suture occlusion of the middle cerebral artery (MCAO) for 1h in 20 month‐old male C57BL/6 mice (n=8 per cohort and treatment group). Sham surgery consisted of anesthesia and internal carotid exposure without suture occlusion (n=4 per group). Intravenous infusion: animals were re‐anesthetized and 30 pmol/g anti‐miR‐181a, anti‐miR‐200c or control (MM) in sterile saline (100 μl) was infused over 1 minute into the internal jugular vein at 2h reperfusion. Neurological status was assessed by 4‐point neurologic deficit score at 48h after MCAO. Infarct volume was assessed after sacrifice by anesthesia overdose at 48h after MCAO with triphenyl tetrazolium chloride staining. Infarct volume was calculated as percentage of hemisphere corrected for edema.ResultsMCAO in all groups resulted in significant decreases in neurological score and increased infarct volume relative to sham treated animals. Both anti‐miR‐181a and anti‐miR‐200c treatment resulted in significantly (p<0.05) higher neurological scores and significantly lower infarct volumes relative to MM‐control treated animals. No differences in neurological score or in infarct volume were observed between anti‐miR‐181a and anti‐miR‐200c treatments.ConclusionsBoth anti‐miR‐181a and anti‐miR‐200c are effective non‐invasive post‐stroke interventions to reduce injury in clinically‐relevant aged mice. Future investigations should be extended to testing in aged female cohorts and identifying relevant downstream targets of miR‐181a and miR‐200c.

Sex‐differences and cell type‐specific alterations in brain Bcl2 expression after transient focal cerebral ischemia in aged mice

IntroductionStroke remains the second leading cause of death worldwide and remains the primary cause of long‐term disability in the US. To date, the only pharmacological treatment for stroke remains tissue plasminogen activator. A major factor for translational failure is likely overwhelming use of young male rodents despite the fact that stroke occurs predominantly in aged populations and is a sexually dimorphic disease with sex differences in incidence, prevalence, and outcomes. The Bcl2 family is known to play an important role in the evolution of injury following cerebral ischemia. Overexpression of pro‐survival Bcl2 protects against cerebral ischemia in vivo and in vitro. In the present study we explored whether Bcl2 could play a role in known age‐ and sex‐related differences in stroke.MethodsAll studies were conducted in accordance with National Institutes of Health guidelines for the use of experimental animals, with protocols approved by the Stanford Animal Care and Use Committee. Transient focal cerebral ischemia was induced by suture occlusion of the middle cerebral artery (MCAO) for 1h in 20 month‐old male and female C57BL/6 mice. Sham surgery consisted of anesthesia and internal carotid exposure without suture occlusion (n=6 per group). Histological assessment: Animals were sacrificed at post‐MCAO days 1, 3, and 30 (n=6 per group, per time point), and brains fixed with 4% phosphate‐buffered paraformaldehyde for stereological analysis. Fixed sections were stained for Bcl2, the astrocyte marker glial fibrillary acidic protein (GFAP), and the mature neuronal marker NeuN. An observer blinded to conditions quantified the cell‐type specific relative intensity of Bcl2 from maximum projection Z‐stack images.ResultsIn sham animals Bcl2 expression was significantly (p<0.05) higher in both female astrocytes and neurons relative to male. After MCAO male neuronal Bcl2 expression was significantly depressed from days 1 until 30, while female neuronal Bcl2 remained unchanged. Conversely in astrocytes, female Bcl2 expression decreased significantly at post‐injury days 1 and 3, but recovered by day 30, while male astrocyte Bcl2 decreased by day 3 and remained significantly repressed at day 30.ConclusionsBaseline differences in Bcl2 expression between aged males and females, and cell‐type specific differences in post‐injury Bcl2 expression may account for age‐ and sex‐related differences in injury outcome from stroke. Bcl2 may represent a therapeutic target for stroke for both sexes in clinically‐relevant aged populations.

An Optimal Animal Model of Ischemic Stroke Established by Digital Subtraction Angiography-Guided Autologous Thrombi in Cynomolgus Monkeys.

ObjectiveIschemic stroke seriously threatens human health, characterized by the high rates of incidence, disability, and death. Developing a reliable animal model that mimics most of the features of stroke is critical for pathological studies and clinical research. In this study, we aimed to establish and examine a model of middle cerebral artery occlusion (MCAO) guided by digital subtraction angiography (DSA) in cynomolgus monkeys.Materials and MethodsIn this study, 15 adult male cynomolgus monkeys were enrolled. Under the guidance of DSA, a MCAO model was established by injecting an autologous venous clot into the middle cerebral artery (MCA) via femoral artery catheter. Thrombolytic therapy with alteplase (rt-PA) was given to eight of these monkeys at 3 h after the occlusion. Blood test and imaging examination, such as computed tomography angiography (CTA), CT perfusion (CTP), brain magnetic resonance imaging (MRI), and brain magnetic resonance angiography (MRA), were performed after the operation to identify the post-infarction changes. The behavioral performance of cynomolgus monkeys was continuously observed for 7 days after operation. The animals were eunthanized on the 8th day after operation, and then the brain tissues of monkeys were taken for triphenyltetrazolium chloride (TTC) staining.ResultsAmong the 15 cynomolgus monkeys, 12 of them were successfully modeled, as confirmed by the imaging findings and staining assessment. One monkey died of brain hernia resulted from intracranial hemorrhage confirmed by necropsy. DSA, CTA, and MRA indicated the presence of an arterial occlusion. CTP and MRI showed acute focal cerebral ischemia. TTC staining revealed infarct lesions formed in the brain tissues.ConclusionOur study may provide an optimal non-human primate model for an in-depth study of the pathogenesis and treatment of focal cerebral ischemia.

Identification of optimal reference genes for gene expression studies in a focal cerebral ischaemia model-Spatiotemporal effects.

A proper reference gene (RG) is required to reliably measure mRNA levels in biological samples via quantitative reverse transcription PCR (RT‐qPCR). Various experimental paradigms require specific and stable RGs. In studies using rodent models of brain ischaemia, a variety of genes, such as β‐actin (Actb), hypoxanthine phosphoribosyltransferase 1 (Hprt1), peptidyl‐propyl isomerase A (Ppia) and glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh), are used as RGs. However, most of these genes have not been validated in specific experimental settings. The aim of this study was to evaluate the time‐ and brain region‐dependent expression of RG candidates in a rat model of transient middle cerebral artery occlusion (tMCAO). The following genes were selected: Actb, Hprt1, Ppia, Gapdh, tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein, zeta (Ywhaz) and beta‐2 microglobulin (B2m). Focal cerebral ischaemia was induced by 90 min of tMCAO in male Sprague‐Dawley rats. Expression was investigated at four time points (12 and 24 h; 3 and 7 days) and in three brain areas (the frontal cortex, hippocampus and dorsal striatum) within the ischaemic brain hemisphere. The RT‐qPCR results were analysed using variance analysis and the ΔCt, GeNorm, NormFinder and BestKeeper methods. Data from these algorithms were ranked using the geometric mean of ranks of each analysis. Ppia, Hprt1 and Ywhaz were the most stable genes across the analysed brain areas and time points. B2m and Actb exhibited the greatest fluctuations, and the results for Gapdh were ambiguous.

Neuroprotective efficacy of N-t-butylhydroxylamine (NtBHA) in transient focal ischemia in rats.

The pharmacological or toxicological activities of the degradation products of drug candidates have been unaddressed during the drug development process. Ischemic stroke accounts for 80% of all strokes and is responsible for considerable mortality and disability worldwide. Despite decades of research on neuroprotective agents, tissue plasminogen activators (t-PA), a thrombolytic agent, remains the only approved acute stroke pharmacological therapy. NXY-059, a free radical scavenger, exhibited striking neuroprotective properties in preclinical models and met all the criteria established by the Stroke Academic Industry Roundtable (STAIR) for a neuroprotective agent. In phase 3 clinical trials, NXY-059 exhibited significant neuroprotective effects in one trial (SAINT-I), but not in the second (SAINT-II). Some have hypothesized that N-t-butyl hydroxylamine (NtBHA), a breakdown product of NXY-059 was the actual neuroprotective agent in SAINT-I and that changes to the formulation of NXY-059 to prevent its breakdown to NtBHA in SAINT -II was the reason for the lack of efficacy. We evaluated the neuroprotective effect of NtBHA in N-methyl-D-aspartate (NMDA)-treated primary neurons and in rat focal cerebral ischemia. NtBHA significantly attenuated infarct volume in rat transient focal ischemia, and attenuated NMDA-induced cytotoxicity in primary cortical neurons. NtBHA also reduced free radical generation and exhibited mitochondrial protection.

Retraction Note to: Alleviation of glutamate-mediated neuronal insult by piroxicam in rodent model of focal cerebral ischemia: a possible mechanism of GABA agonism.

Retraction Note to: Alleviation of glutamate-mediated neuronal insult by piroxicam in rodent model of focal cerebral ischemia: a possible mechanism of GABA agonism.

Effects of electroacupuncture on angiogenesis and cortical VEGF and BDNF expression in rats with focal cerebral ischemia

Effects of electroacupuncture on angiogenesis and cortical VEGF and BDNF expression in rats with focal cerebral ischemia

COMPREHENSIVE ASSESSMENT OF NEUROLOGICAL DEFICIT AND ITS CORRECTION WITH PIR-10 COMPOUND IN EXPERIMENTAL FOCAL CEREBRAL ISCHEMIA OF RATS

Introduction: Correction of motor dysfunction resulting from focal cerebral ischemia is one of the significant problems of pharmacology and experimental therapy. Pyrimidine derivatives in the experiment on rats previously showed cerebroprotective activity, as a result of which it was decided to test the PIR-10 compound for the ability to reduce neurological deficit. Methods: A study was conducted to assess the effect of pyrimidine derivative (PIR-10 50 mg/kg) on neurological deficiency in the conditions of focal cerebral ischemia in rats. The study used mature male Wistar rats weighing 220-240 g (n=40). Focal cerebral ischemia was reproduced by coagulation of the left middle cerebral artery (anesthesia chloralhydrate 350 mg/kg). Neurological deficits were assessed using the McGraw, Combs and D'alecy, Garcia scales. Results: The studied compound PIR-10 contributes to the reduction of neurological deficits in all selected schools in comparison with the control group of animals (McGraw by 68% (p<0,05), Combs and D'alecy by 4,3 times (p<0,05), Garcia by 84% (p<0,05)). In addition, the results obtained after administration of PIR-10 to rats showed a statistically significantly difference from the group of rats receiving a reference drug cavinton. However, complete recovery of motor functions after modeling focal cerebral ischemia was not observed in any group of rats. Conclusion: The PIR10 compound is able to partially correct the neurological deficit that occurs in conditions of cerebral circulatory insufficiency, surpassing the activity of the reference drug cavinton.

Lithium upregulates growth-associated protein-43 (GAP-43) and postsynaptic density-95 (PSD-95) in cultured neurons exposed to oxygen-glucose deprivation and improves electrophysiological outcomes in rats subjected to transient focal cerebral ischemia following a long-term recovery period

ABSTRACT Objectives Lithium has numerous neuroplastic and neuroprotective effects in patients with stroke. Here, we evaluated whether delayed and short-term lithium treatment reduces brain infarction volume and improves electrophysiological and neurobehavioral outcomes following long-term recovery after cerebral ischemia and the possible contributions of lithium-mediated mechanisms of neuroplasticity. Methods Male Sprague Dawley rats were subjected to right middle cerebral artery occlusion for 90 min, followed by 28 days of recovery. Lithium chloride (1 mEq/kg) or vehicle was administered via intraperitoneal infusion once per day at 24 h after reperfusion onset. Neurobehavioral outcomes and somatosensory evoked potentials (SSEPs) were examined before and 28 days after ischemia-reperfusion. Brain infarction was assessed using Nissl staining. Primary cortical neuron cultures were exposed to oxygen-glucose deprivation (OGD) and treated with 2 or 20 μM lithium for 24 or 48 h; subsequent brain-derived neurotrophic factor (BDNF), growth-associated protein-43 (GAP-43), postsynaptic density-95 (PSD-95), and synaptosomal-associated protein-25 (SNAP-25) levels were analyzed using western blotting. Results Compared to controls, lithium significantly reduced infarction volume in the ischemic brain and improved electrophysiological and neurobehavioral outcomes at 28 days post-insult. In cultured cortical neurons, BDNF, GAP-43, and PSD-95 expression were enhanced by 24- and 48-h treatment with lithium after OGD. Conclusion Lithium upregulates BDNF, GAP-43, and PSD-95, which partly accounts for its improvement of neuroplasticity and provision of long-term neuroprotection in the ischemic brain. Abbreviations: BDNF: brain-derived neurotrophic factor; ECM: extracellular matrix; EDTA: ethylenediaminetetraacetic acid; GAP-43: growth-associated protein-43; GSK-3β: glycogen synthase kinase-3β; HBSS: Hank’s balanced salt solution; LCBF: local cortical blood perfusion; LDF: laser-Doppler flowmetry; MCAO: middle cerebral artery occlusion; MMP: matrix metalloproteinase; NMDA: N-methyl-D-aspartate; NMDAR: N-methyl-D-aspartate receptor; OCT: optimal cutting temperature compound; OGD: oxygen-glucose deprivation; PSD-95: postsynaptic density-95; SDS: sodium dodecyl sulfate; SNAP-25: synaptosomal-associated protein-25; SSEP: somatosensory evoked potential

Liraglutide Ameliorates Cerebral Ischemia in Mice via Antipyroptotic Pathways.

It was recently shown that pyroptosis, an inflammatory form of programmed cell death, is critically involved in the pathogenesis of ischemic stroke. Liraglutide (Lg) is a novel long-acting analog of glucagon-like peptide-1 that has potential protective effects against stroke. However, the relationship between Lg and pyroptosis in the brain is not well defined. In this study, we found that injection of Lg significantly improved the recovery of motor function, increased cerebral blood flow and ameliorated cerebral damage in a mouse model of focal cerebral cortical ischemia. Our results revealed that Lg treatment significantly reduced the levels of NLRP3, Caspase1, IL-1β and the pore-forming protein gasdermin D in microglial cells in vitro, suggesting that the neuroprotective effect of Lg may be achieved through the inhibition of pyroptosis. Furthermore, by using a specific inhibitor of NOD-like receptor protein 3 (NLRP3), we confirmed that the antipyroptotic mechanism of Lg may be mediated by NLRP3 in vivo. Our present study unveils a novel neuroprotective mechanism through which Lg alleviates ischemia by exerting NLRP3-dependent antipyroptotic effects.

Effects of Long-Term Vagus Nerve Electrical Stimulation Therapy on Acute Cerebral Infarction and Neurological Function Recovery in Post MCAO Mice.

Background Vagus nerve stimulation therapy is proven to produce neuroprotective effects against central nervous system diseases and reduce neurological injury, having a positive effect on the recovery of neurological functions in mouse model of stroke. Objective This study was aimed at exploring a wider time window for VNS treatment, investigating the long-term behavioral improvement of long-term VNS in mice after pMCAO, and exploring the antiapoptotic properties of VNS and its role in autophagy, all of which may be a permanent deficiency potential mechanism of neuroprotection in hemorrhagic stroke. Methods Permanent focal cerebral ischemia and implantation of vagus nerve stimulator were performed through intracavitary occlusion of the right middle cerebral artery (MCA). The vagus nerve stimulation group received five times vagus nerve stimulation from 6 h after surgery for 5 days. Adhesive removal test and NSS neurological score were used to evaluate the neurological deficit of mice. TTC staining of mouse brain tissue was performed one week after surgery in order to assess the area of cerebral infarction. Additionally, frozen sections were stained with Fluoro-Jade B to observe the apoptotic cells in the ischemic penumbra of brain tissue. Finally, Western blot was used to detect the changes in the levels of apoptosis-related proteins such as cleaved-caspase3 and Bcl-2 and autophagy-related proteins such as mTOR, Beclin-1, and LC3-II in brain tissue. Results VNS can effectively reduce the behavioral score of pMCAO mice; TTC results showed that VNS could effectively reduce the infarct area after pMCAO (P < 0.05). After VNS intervention of the pMCAO group compared with the pMCAO+VNI group, the FJB-positive cells in the VNS group were significantly decreased (P < 0.05); Western Blot analysis showed that the expression of cleaved-caspase3 in the brain tissue of mice increased after pMCAO (P < 0.05), and the expression of Bcl-2 decreased (P < 0.05). This change could be effectively reversed after VNS intervention (P < 0.05). Conclusion VNS could effectively improve the behavioral performance of mice after permanent stroke in addition to significantly reducing the infarct size of the brain tissue. The mechanism may be related to the effective reduction of cell apoptosis and excessive autophagy after pMCAO by VNS.

Loss of Kir6.1 facilitates peri-infarct depolarizations in focal cerebral ischemia

ABSTRACT Objective Peri-infarct depolarizations (PIDs) are spontaneous waves that propagate slowly across the penumbra region following stroke, contributing to secondary infarct growth and negatively affecting stroke outcomes. KATP channels are generally spread in the brain. Under conditions of ischemia and/or hypoxia, KATP channels play a cytoprotective role in neurons. However, it is still unknown whether KATP channels are involved in the initiation and propagation of PIDs. Methods The Kir6.1 knockout (Kir6.1−/−) mice, Kir6.2 knockout (Kir6.2−/−) mice, and wild-type C57Bl6 mice (n = 8) were used. The middle cerebral artery occlusion (MCAO) stroke model was made and PIDs were detected by an optical intrinsic signal (OIS) imaging system. Results Much more PIDs appeared in Kir6.1−/−mice than that in Kir6.2−/− and WT mice in both the first hour and 4 hours following MCAO (3.9 ± 0.7 vs. 1.5 ± 0.3, p < 0,05; 3.9 ± 0.7 vs. 1.9 ± 0.3, p < 0.05; 20.0 ± 2.5 vs. 10.4 ± 2.4, p < 0.05; 20.0 ± 2.5 vs. 11.3 ± 1.4, p < 0.05). Furthermore, the first PID occurred much earlier in Kir6.1−/− mice than that in Kir6.2−/− mice and WT mice (21.3 ± 2.1 min vs. 34.1 ± 4.8 min, p < 0.05; 21.3 ± 2.1 min vs. 38.8 ± 3.4 min, p < 0.01). No significant differences in other characteristics of PIDs including originating sites, duration time, propagation patterns, and velocity were detected. Additionally, the migration of originating sites was observed. Conclusion This study shows that loss of Kir6.1, not Kir6.2 facilitates the induction of PIDs in focal cerebral ischemia, indicating that Kir6.1-forming channels in the brain may provide protection against PIDs.

EVALUATION OF ABCB1-PROTEIN INHIBITION PROSPECTIVENESS IN HEMATOENCEPHALIC BARRIER AS METHOD FOR INCREASING EFFICIENCY OF PHARMACOTHERAPY IN CEREBRAL ISCHEMIA

The aim of the article is to evaluate the ABCB1 protein inhibition in the blood-brain barrier to increase the effectiveness of neuroprotective therapy for focal and global cerebral ischemia.Materials and methods. The work was performed on 103 male Wistar rats. In the 1st group (n=33), the neuroprotective activity of the ABCB1 protein substrate, nimodipine (0.4 mg/kg), was analyzed in terms of reducing the area of the brain necrosis after a 1-hour occlusion of the middle cerebral artery with a 24-hour reperfusion (focal ischemia). In the 2nd group (n=60), the effectiveness of nimodipine was analyzed by reducing the lethality of rats and the neurological deficit (ND) level against the background of the bilateral occlusion of the common carotid arteries (global ischemia). In both groups, nimodipine was used alone or in the combination with omeprazole, the ABCB1 protein inhibitor (17.6 mg/kg). The drugs were administered intravenously.Results. The nimodipine administration to the 1st group led to the reduction of the necrosis focus by 28% compared with the control series. Omeprazole did not cause a change in the area of the necrosis. The combination of drugs caused a decrease in the area of the necrosis in relation to the control by 29%; there were no differences in comparison with the nimodipine series. Nimodipine reduced the rats’ lethality in the 2nd group against the background of the pathology (a tendency). Omeprazole alone did not change the mortality. The drug combination reduced the mortality compared to the control and nimodipine series. The administration of omeprazole alone did not reduce the neurological deficit score relative to the control. In the nimodipine series, ND was 88% lower than the control, after 24 hours. With the administration of the drugs combination, this indicator decreased in relation to the control by 88%, 80%, 88%, 87% and 86% after 4, 12, 24, 48 and 72 hours, respectively, and in relation to the nimodipine series it decreased by 60% and 67% after 4 and 48 hours.Conclusion. The ABCB1 protein inhibition is promising for increasing the effectiveness of neuroprotective therapy for global ischemia, but not for focal cerebral ischemia.

Calcitriol Pretreatment Attenuates Glutamate Neurotoxicity by Regulating NMDAR and CYP46A1 Gene Expression in Rats Subjected to Transient Middle Cerebral Artery Occlusion.

Although the neuroprotective effects of calcitriol have been demonstrated in a variety of neurological diseases, such as stroke, the precise molecular mechanism has yet to be determined. This study aimed to investigate the possible role of calcitriol as a neuroprotective agent via CYP46A1 and glutamate receptors in a middle cerebral artery occlusion (MCAO) animal model. The MCAO technique was performed on adult male Wistar rats to induce focal cerebral ischemia for 1 hour followed by 23 hours of reperfusion. Calcitriol was given for 7 days prior to stroke induction. Sensorimotor functional tests were done 24 hours after ischemia/reperfusion, and infarct volume was estimated by tetrazolium chloride staining of brain sections. Gene expression of NR2A, NR2B, NR3B, and CYP46A1 was evaluated by RT-PCR followed by western blotting for NR3B protein. Our data revealed that calcitriol pretreatment reduced lesion volume and improved ischemic neurobehavioral parameters. Calcitriol therapy altered the expression of glutamate receptor and CYP46A1 genes. A possible molecular mechanism of calcitriol to reduce the severity and complications of ischemia may be through alterations of glutamate receptor and CYP46A1 gene expression.

Neurons Release Injured Mitochondria as "Help-Me" Signaling After Ischemic Stroke.

Mitochondrial dysfunction has been regarded as one of the major contributors of ischemic neuronal death after stroke. Recently, intercellular mitochondrial transfer between different cell types has been widely studied and suggested as a potential therapeutic approach. However, whether mitochondria are involved in the neuron-glia cross-talk following ischemic stroke and the underlying mechanisms have not been explored yet. In this study, we demonstrated that under physiological condition, neurons release few mitochondria into the extracellular space, and the mitochondrial release increased when subjected to the challenges of acidosis, hydrogen peroxide (H2O2), N-methyl-D-aspartate (NMDA), or glutamate. Acidosis reduced the mitochondrial basal respiration and lowered the membrane potential in primary-cultured mouse cortical neurons. These defective mitochondria were prone to be expelled to the extracellular space by the injured neurons, and were engulfed by adjacent astrocytes, leading to increased astrocytic expressions of mitochondrial Rho GTPase 1 (Miro 1) and mitochondrial transcription factor A (TFAM) at mRNA level. In mice subjected to transient focal cerebral ischemia, the number of defective mitochondria in the cerebrospinal fluid increased. Our results suggested that the neuron-derived mitochondria may serve as a “help-me” signaling and mediate the neuron-astrocyte cross-talk following ischemic stroke. Promoting the intercellular mitochondrial transfer by accelerating the neuronal releasing or astrocytic engulfing might be a potential and attractive therapeutic strategy for the treatment of ischemic stroke in the future.

MiR-204-5p is sponged by TUG1 to aggravate neuron damage induced by focal cerebral ischemia and reperfusion injury through upregulating COX2

Studies have reported that miR-204-5p is involved in multiple biological processes. However, little is known about the expression and mechanism of miR-204-5p in cerebral ischemia and reperfusion injury. This study found that miR-204-5p expression was significantly downregulated in the blood of patients with ischemic stroke, MCAO/R rat brains, and OGD/R neurons. Overexpression of miR-204-5p markedly reduced infarct volume and neurological impairment and alleviated the inflammatory response in vivo. miR-204-5p promoted neuronal viability and reduced apoptotic cells in vitro. Mechanically, miR-204-5p was negatively regulated by the expression lncRNA TUG1 upstream and down-regulated COX2 expression downstream. Therefore, the TUG1/miR-204-5p/COX2 axis was involved in ischemia and reperfusion-induced neuronal damage. This finding may provide a novel strategy for the treatment of cerebral ischemia and reperfusion injury.

Therapeutic Potential of a Combination of Electroacupuncture and Human iPSC-Derived Small Extracellular Vesicles for Ischemic Stroke.

This paper aimed to explore the roles of the combination of electroacupuncture (EA) and induced pluripotent stem cell-derived small extracellular vesicles (iPSC-EVs) on mice with ischemic stroke and the underlying mechanisms. A focal cerebral ischemia model was established in C57BL/6 mice through middle cerebral artery occlusion (MCAO). After 3 days, neurological impairment and motor function were examined by performing behavioral tests. The infarct volume and neuronal apoptosis were examined using TTC staining and TUNEL assays. Flow cytometry was performed to assess the proliferation of T lymphocytes. The changes in the interleukin (IL)-33/ST2 axis were evaluated by immunofluorescence and Western blotting. The combination of EA and iPSC-EVs treatment ameliorated neurological impairments and reduced the infarct volume and neuronal apoptosis in MCAO mice. EA plus iPSC-EVs suppressed T helper (Th1) and Th17 responses and promoted the regulatory T cell (Treg) response. In addition, EA plus iPSC-EVs exerted neuroprotective effects by regulating the IL-33/ST2 axis and inhibiting the microglia and astrocyte activation. Taken together, the study shows that EA and iPSC-EVs exerted a synergistic neuroprotective effect in MCAO mice, and this treatment may represent a novel potent therapy for ischemic stroke and damage to other tissues.

Effect and Mechanisms of Quercetin for Experimental Focal Cerebral Ischemia: A Systematic Review and Meta-Analysis.

Quercetin, a naturally occurring flavonoid, is mainly extracted from tea, onions, and apples. It has the underlying neuroprotective effect on experimental ischemic stroke. A systematic review and meta-analysis were used to assess quercetin's efficacy and possible mechanisms in treating focal cerebral ischemia. Compared with the control group, twelve studies reported a remarkable function of quercetin in improving the neurological function score (NFS) (P < 0.05), and twelve studies reported a significant effect on reducing infarct volume (P < 0.05). Moreover, two and three studies showed that quercetin could alleviate blood-brain barrier (BBB) permeability and brain water content, respectively. The mechanisms of quercetin against focal cerebral ischemia are diverse, involving antioxidation, antiapoptotic, anti-inflammation, and calcium overload reduction. On the whole, the present study suggested that quercetin can exert a protective effect on experimental ischemic stroke. Although the effect size may be overestimated because of the quality of studies and possible publication bias, these results indicated that quercetin might be a promising neuroprotective agent for human ischemic stroke. This study is registered with PROSPERO, number CRD 42021275656.

Effect of remote limb ischemic post-conditioning on the expression of miR-21-5p/PirB in the brain of rats with focal cerebral ischemia.

Cerebral ischemia causes great damage to ischemic brain regions. Remote limb post-conditioning (RLIPostC) has neuroprotective effects on cerebral ischemia. Paired immunoglobulin receptor B (PirB) has been confirmed to affect the cerebral ischemia injury. Therefore, this study investigated the mechanism of RLIPostC on cerebral ischemia injury. The middle cerebral artery occlusion (MCAO) model was established in rats, followed by RLIPostC (by blocking the blood flow in the distal limb for 10 min after MCAO, and restored for 10 min, three cycles), or in vivo lentivirus infection of miR-21-5p agomir, sh-PirB and oe-PirB. The locomotor abilities were assessed using the foot fault test and balance beam walking test at 48 h, 7 and 21 days after operation, and neurological function was measured on day 21. The cerebral infarct area, Nissl bodies and the pathology of brain tissues were examined using histological staining, followed by the assessment of miR-21-5p and PirB levels and the binding relationship between miR-21-5p and PirB. miR-21-5p was poorly expressed in MCAO rats. Both RLIPostC and overexpression of miR-21-5p reduced motor dysfunction, neurological function score, cerebral infarction area and brain edema; increased Nissl bodies in MCAO rats and thus alleviated the brain damage caused by cerebral ischemia. RLIPostC promoted miR-21-5p expression. PirB was highly expressed in cerebral ischemia, and miR-21-5p targeted PirB. Overall, RLIPostC promotes miR-21-5p expression and then inhibits PirB to alleviate cerebral ischemia injury, which provides theoretical basis for basic research on motor dysfunction and neuron injury after cerebral ischemia and the development of neuroscience.