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Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented γ-hydroxybutyrate (GHB) ligands as the first small molecules selectively targeting and stabilizing the CaMKIIα hub domain. Here, we report that the cyclic GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), improves sensorimotor function after experimental stroke in mice when administered at a clinically relevant time and in combination with alteplase. Further, we observed improved hippocampal neuronal activity and working memory after stroke. On the biochemical level, we observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated ischemia-specific expression of a constitutively active CaMKII kinase proteolytic fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. Similarly, HOCPCA's effects on dampening inflammatory changes require further investigation as an underlying protective mechanism. HOCPCA's selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy. [ABSTRACT FROM AUTHOR]

In experimental stroke, ovariectomized (OVX) adult rats have larger infarct volumes and greater sensory-motor impairment as compared to ovary-intact females and is usually interpreted to indicate that ovarian hormones are neuroprotective for stroke. Previous work from our lab shows that middle-aged, acyclic reproductively senescent (RS) females have worse stroke outcomes as compared to adult (normally cycling) females. We hypothesized that if loss of ovarian estrogen is the critical determinant of stroke outcomes, then ovary-intact middle-aged acyclic females, who have reduced levels of estradiol, should have similar stroke outcomes as age-matched OVX. Instead, the data demonstrated that OVX RS animals showed better sensory-motor function after stroke and reduced infarct volume as compared to ovary-intact females. Inflammatory cytokines were decreased in the aging ovary after stroke as compared to non-stroke shams, which led to the hypothesis that immune cells may be extravasated from the ovaries post-stroke. Flow cytometry indicated reduced overall T cell populations in the aging ovary after middle cerebral artery occlusion (MCAo), with a paradoxical increase in regulatory T cells (Tregs) and M2-like macrophages. Moreover, in the brain, OVX RS animals showed increased Tregs, increased M2-like macrophages, and increased MHC II + cells as compared to intact RS animals, which have all been shown to be correlated with better prognosis after stroke. Depletion of ovary-resident immune cells after stroke suggests that there may be an exaggerated response to ischemia and possible increased burden of the inflammatory response via extravasation of these cells into circulation. Increased anti-inflammatory cells in the brain of OVX RS animals further supports this hypothesis. These data suggest that stroke severity in aging females may be exacerbated by the aging ovary and underscore the need to assess immunological changes in this organ after stroke. [ABSTRACT FROM AUTHOR]

The α-tocotrienol (TCT) form of natural vitamin E is more potent than the better known α-tocopherol against stroke. Angiographic studies of canine stroke have revealed beneficial cerebrovascular effects of TCT. This work seeks to understand the molecular basis of such effect. In mice, TCT supplementation improved perfusion at the stroke-affected site by inducing miR-1224. miRNA profiling of a laser-capture-microdissected stroke-affected brain site identified miR-1224 as the only vascular miR induced. Lentiviral knockdown of miR-1224 significantly blunted the otherwise beneficial effects of TCT on stroke outcomes. Studies on primary brain microvascular endothelial cells revealed direct angiogenic properties of miR-1224. In mice not treated with TCT, advance stereotaxic delivery of an miR-1224 mimic to the stroke site markedly improved stroke outcomes. Mechanistic studies identified Serpine1 as a target of miR-1224. Downregulation of Serpine1 augmented the angiogenic response of the miR-1224 mimic in the brain endothelial cells. The inhibition of Serpine1, by dietary TCT and pharmacologically, increased cerebrovascular blood flow at the stroke-affected site and protected against stroke. This work assigns Serpine1, otherwise known to be of critical significance in stroke, a cerebrovascular function that worsens stroke outcomes. miR-1224-dependent inhibition of Serpine1 can be achieved by dietary TCT as well as by the small-molecule inhibitor TM5441.

Mitogen-activated protein kinase (MAPK) pathway is a major mechanism of acute brain damage in ischemic stroke. Pre-ischemic exercise is an effective method to reduce ischemic injury. However, the regulation by pre-ischemic exercise of MAPK pathway and associated mechanisms in animal models remains unclear.

Abstract Intraluminal monofilament model of middle cerebral artery occlusion (MCAO) is widely adopted for ischemic stroke; and Sprague–Dawley (SD) rats are commonly used rodents for preclinical research. Due to the paucity of information on the appropriate monofilament size for inducing MCAO in SD rats and the importance of including middle-aged models in ischemic stroke studies, we aimed to: (i). determine an appropriate Doccol® monofilament size for middle-aged male SD rats which weighed > 500 g following 24-h transient MCAO survival as well as (ii). demonstrate the optimal Doccol® filament size for middle-aged males (≤ 500 g) and females (273–300 g) while using young adult male SD rats (372–472 g) as control for severity of infarct volume following 7-days post-MCAO. All rats were subjected to 90-min transient MCAO. We show that 0.43 mm Doccol® monofilament size is more appropriate to induce large infarct lesion and optimal functional deficit when compared to 0.45 mm and 0.47 mm at 24 h post-MCAO. Our data on infarct volumes at 7 days post-MCAO as well as the observed weight loss and functional deficits at post-MCAO days 1, 3 and 7 demonstrate that 0.41 mm, 0.37 mm and 0.39 mm are optimal Doccol® filament sizes for middle-aged male (477.3 ± 39.61 g) and female (302.6 ± 26.28 g) as well as young-adult male (362.2 ± 28.38 g) SD rats, respectively.

Stroke known as a neurological disease has significant rates of disability and mortality. Middle cerebral artery occlusion (MCAO) models in rodents is crucial in stroke research to mimic human stroke. Building the mRNA and non-conding RNA network is essential for preventing MCAO-induced ischemic stroke occurrence. Herein, genome-wide mRNA, miRNA, and lncRNA expression profiles among the MCAO group at 3 h, 6 h, and 12 h after surgery and controls using high-throughput RNA sequencing. We detected differentially expressed mRNAs (DE-mRNAs), miRNAs (DE-miRNAs), and lncRNAs (DE-lncRNAs) between the MCAO and control groups. In addition, biological functional analyses were conducted, including GO/KEGG enrichment analysis, and protein-protein interaction analysis (PPI). GO analysis indicated that the DE-mRNAs were mainly enriched in several important biological processes as lipopolysaccharide, inflammatory response, and response to biotic stimulus. The PPI network analysis revealed that the 12 DE-mRNA target proteins showed more than 30° with other proteins, and the top three proteins with the highest node degree were Alb, IL-6, and TNF. In the DE-mRNAs, we found the mRNA of Gp6 and Elane interacting with two miRNAs (novel_miR_879 and novel_miR_528) and two lncRNAs (MSTRG.348134.3 and MSTRG.258402.19). As a result of this study, a new perspective can be gained into the molecular pathophysiology leading to the formation of MCAO. The mRNA-miRNA-lncRNA regulatory networks play an important role in MCAO-induced ischemic stroke pathogenesis and could be applied to the treatment and prevention of ischemic stroke in the future. [ABSTRACT FROM AUTHOR]

Simple Summary: There are no effective treatments for patients with cancers that induce airway narrowing via extrinsic pressure to the bronchus (i.e., extrinsic malignant central airway obstruction—MCAO). The effects of these cancerous tumors must be quickly alleviated to allow normal breathing and delay disease progression. Currently, stents are used to keep the airway open, but stents do not halt the progression of the cancerous tumor that can crush the stent. We have shown that interstitial photodynamic therapy (I-PDT) can be a safe and beneficial treatment option for patients with extrinsic MCAO. Image-based pre-treatment planning is critical for patient safety and tumor response in I-PDT. Herein, we present and validate novel image-based computer optimization methods for guiding light administration in I-PDT of extrinsic MCAO, based on a rate-based light dose metric. We demonstrate the benefit of our approach in data from representative patients with extrinsic MCAO who were treated with I-PDT. There are no effective treatments for patients with extrinsic malignant central airway obstruction (MCAO). In a recent clinical study, we demonstrated that interstitial photodynamic therapy (I-PDT) is a safe and potentially effective treatment for patients with extrinsic MCAO. In previous preclinical studies, we reported that a minimum light irradiance and fluence should be maintained within a significant volume of the target tumor to obtain an effective PDT response. In this paper, we present a computational approach to personalized treatment planning of light delivery in I-PDT that simultaneously optimizes the delivered irradiance and fluence using finite element method (FEM) solvers of either Comsol Multiphysics® or Dosie™ for light propagation. The FEM simulations were validated with light dosimetry measurements in a solid phantom with tissue-like optical properties. The agreement between the treatment plans generated by two FEMs was tested using typical imaging data from four patients with extrinsic MCAO treated with I-PDT. The concordance correlation coefficient (CCC) and its 95% confidence interval (95% CI) were used to test the agreement between the simulation results and measurements, and between the two FEMs treatment plans. Dosie with CCC = 0.994 (95% CI, 0.953–0.996) and Comsol with CCC = 0.999 (95% CI, 0.985–0.999) showed excellent agreement with light measurements in the phantom. The CCC analysis showed very good agreement between Comsol and Dosie treatment plans for irradiance (95% CI, CCC: 0.996–0.999) and fluence (95% CI, CCC: 0.916–0.987) in using patients' data. In previous preclinical work, we demonstrated that effective I-PDT is associated with a computed light dose of ≥45 J/cm2 when the irradiance is ≥8.6 mW/cm2 (i.e., the effective rate-based light dose). In this paper, we show how to use Comsol and Dosie packages to optimize rate-based light dose, and we present Dosie's newly developed domination sub-maps method to improve the planning of the delivery of the effective rate-based light dose. We conclude that image-based treatment planning using Comsol or Dosie FEM-solvers is a valid approach to guide the light dosimetry in I-PDT of patients with MCAO. [ABSTRACT FROM AUTHOR]

Background: The oxygen extraction fraction (OEF) indicates the brain's oxygen consumption and can be estimated by using the quantitative susceptibility mapping (QSM) MRI technique. Recent studies have suggested that OEF alteration following stroke is associated with the viability of at-risk tissue. In the present study, the temporal evolution of OEF in the monkey brain during acute stroke was investigated using QSM. Methods: Ischemic stroke was induced in adult rhesus monkeys (n = 8) with permanent middle cerebral artery occlusion (pMCAO) by using an interventional approach. Diffusion-, T2-, and T2*-weighted images were conducted on day 0, day 2, and day 4 post-stroke using a clinical 3T scanner. Progressive changes in magnetic susceptibility and OEF, along with their correlations with the transverse relaxation rates and diffusion indices, were examined. Results: The magnetic susceptibility and OEF in injured gray matter of the brain significantly increased during the hyperacute phase, and then decreased significantly on day 2 and day 4. Moreover, the temporal changes of OEF in gray matter were moderately correlated with mean diffusivity (MD) (r = 0.52; p = 0.046) from day 0 to day 4. Magnetic susceptibility in white matter progressively increased (from negative values to near zero) during acute stroke, and significant increases were seen on day 2 (p = 0.08) and day 4 (p = 0.003) when white matter was significantly degenerated. However, significant reduction of OEF in white matter was not seen until day 4 post-stroke. Conclusion: The preliminary results demonstrate that QSM-derived OEF is a robust approach to examine the progressive changes of gray matter in the ischemic brain from the hyperacute phase to the subacute phase of stroke. The changes of OEF in gray matter were more prominent than those in white matter following stroke insult. The findings suggest that QSM-derived OEF may provide complementary information for understanding the neuropathology of the brain tissue following stroke and predicting stroke outcomes. [ABSTRACT FROM AUTHOR]

Carnosol is a phytopolyphenol (diterpene) found and extracted from plants of Mediterranean diet, which has anti-tumor, anti-inflammatory and antioxidant effects. However, its role in ischemic stroke has not been elucidated. Primary neurons subjected to oxygen-glucose deprivation (OGD) was used to investigate the effect of carnosol in vitro. A mouse MCAO model was used to evaluate the effect of carnosol on ischemic stroke in vivo. The mRNA level of inflammatory and apoptosis-related genes was determined by RT-PCR. The protein level of total and phosphorylated AMPK was determined by WB. H&E and Immunofluorescent assay was used to investigate the necrosis, inflammation and apoptosis in brain tissue. Carnosol protected the activity of primary neurons subjected to oxygen-glucose deprivation (OGD) in vitro, as well as inhibited inflammation and apoptosis. Furthermore, carnosol could significantly reduce the infarct and edema volume and protect against neurological deficit in vivo, and had a significant inhibitory effect on brain neuroinflammation and apoptosis. Mechanically, carnosol could activate AMPK, and the effect of carnosol on cerebral ischemia-reperfusion injury cell model could be abolished by AMPK phosphorylation inhibitor. Carnosol has a protective effect on ischemic stroke, and this effect is achieved through AMPK activation. Our study demonstrates the protective effect of carnosol on cerebral ischemia-reperfusion injury and provides a new perspective for the clinical treatment of ischemic stroke. • A new function of carnosol in regulating cerebral ischemia reperfusion injury was found. • It was found that carnosol could inhibit OGD/R induced neurons injury, inflammatory response and apoptosis. • It was found that carnosol can promote phosphorylation of AMPK in cerebral ischemia reperfusion injury. [ABSTRACT FROM AUTHOR]

Objective: Neurological disorders is an increasing problem in the society. Our study aims to explore the secondary metabolites and evaluate the neuroprotective effect of leave extract of Dodonaea viscosa. Methods: The ethyl acetate extract was chromatographed and produced one new entity which was characterized by spectroscopy (IR, UV, NMR, MS) studies. This entity was named as (6S, 9R)-vomifoliol-9-β-D-glucopyranosyle (1 -3)- O-α-L-rhamnopyranoside (DVE-8) while the known entities were identified as viscosine (DVE-12), and catechin (DVE-18). The neuroprotective potential of D. viscosa leaves extract was evaluated using the transient middle cerebral artery occlusion (MCAO) method to induce focal cerebral ischemia–reperfusion injury in rats. Results: The levels of proinflammatory cytokines such as TNF-α, IL-6, and NF-κB gene expression along with the level of anti-apoptotic Bcl–2, BAX, and caspase–3 gene expression was evaluated. Neurological findings showed a significant reduction in the levels of pro-inflammatory cytokines (TNF-α and, IL-6; both P

Weitong Song,1 Lu Bai,1 Yuya Yang,1 Yongchao Wang,2 Pingxiang Xu,1,3 Yuming Zhao,1 Xuelin Zhou,1,3 Xiaorong Li,1,3 Ming Xue1,3 1Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing, People’s Republic of China; 2CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing, People’s Republic of China; 3Beijing Laboratory for Biomedical Detection Technology and Instrument, Beijing, People’s Republic of ChinaCorrespondence: Ming Xue, Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Youanmen, Beijing, 100069, People’s Republic of China, Tel +8610-83950416, Fax +8610-83911520, Email xuem@ccmu.edu.cnPurpose: We designed a novel isoliquiritigenin (ISL) loaded micelle prepared with DSPE-PEG2000 as the drug carrier modified with the brain-targeting polypeptide angiopep-2 to improve the poor water solubility and low bioavailability of ISL for the treatment of acute ischemic stroke.Methods: Thin film evaporation was used to synthesize the ISL micelles (ISL-M) modified with angiopep-2 as the brain targeted ligands. The morphology of the micelles was observed by the TEM. The particle size and zeta potential were measured via the nanometer particle size analyzer. The drug loading, encapsulation and in vitro release rates of micelles were detected by the HPLC. The UPLC-ESI-MS/MS methods were used to measure the ISL concentrations of ISL in plasma and main tissues after intravenous administration, and compared the pharmacokinetics and tissue distributions between ISL and ISL-M. In the MCAO mice model, the protective effects of ISL and ISL-M were confirmed via the behavioral and molecular biology experiments.Results: The results showed that the drug loading of ISL-M was 7.63 ± 2.62%, the encapsulation efficiency was 68.17 ± 6.23%, the particle size was 40.87 ± 4.82 nm, and the zeta potential was − 34.23 ± 3.35 mV. The in vitro release experiments showed that ISL-M had good sustained-release effect and pH sensitivity. Compared with ISL monomers, the ISL-M could significantly prolong the in vivo circulation time of ISL and enhance the accumulation in the brain tissues. The ISL-M could ameliorate the brain injury induced by the MCAO mice via inhibition of cellular autophagy and neuronal apoptosis. There were no the cellular structural damages and other adverse effects for ISL-M on the main tissues and organs.Conclusion: The ISL-M could serve as a promising and ideal drug candidate for the clinical application of ISL in the treatment of acute ischemic stroke.Keywords: isoliquiritigenin, micelle, brain distribution, pharmacokinetics, ischemic stroke, MCAO

Tannic acid (TA) is a polyphenolic compound that exerts protective effects under pathological conditions. The diverse mechanisms of TA can exert beneficial anti-oxidative, anti-inflammatory, and anti-cancer effects. Herein, we reported that TA affords robust neuroprotection in an animal model of stroke (transient middle cerebral artery occlusion; tMCAO) and exhibits Zn2+-chelating and anti-oxidative effects in primary cortical neurons. Following tMCAO induction, intravenous administration of TA (5 mg/kg) suppressed infarct formation by 32.9 ± 16.2% when compared with tMCAO control animals, improving neurological deficits and motor function. We compared the chelation activity under several ionic conditions and observed that TA showed better Zn2+ chelation than Cu2+. Furthermore, TA markedly decreased lactate dehydrogenase release following acute Zn2+ treatment and subsequently reduced the expression of p67 (a cytosolic component of NADPH oxidase), indicating the potential mechanism underlying TA-mediated Zn2+ chelation and anti-oxidative effects in primary cortical neurons. These findings suggest that anti-Zn2+ toxicity and anti-oxidative effects participate in the TA-mediated neuroprotective effects in the postischemic brain.

Moringa oleifera (MO), has been studied extensively, and has numerous medicinal and socioeconomic benefits. Emerging research has investigated the efficacy of MO extract and/or its phytochemical derivatives against ischemic stroke in-vivo. To date, no studies comprehensively reviewing the effects of MO extract and/or its phytochemical derivatives against ischemic stroke have been published. A systematic review and meta-analysis was conducted to assess the effects of MO extract and/or its phytochemical derivatives against focal ischemic stroke, modeled in-vivo. Compared with control groups, significant reduction in infarct volume and malondialdehyde levels, and signficant increase in antioxidant enzymes superoxide dismutase, glutathione peroxidase and catalase. The primary mechanism of action of MO extract and its phytochemical derivatives which confers neuroprotection is reduction in oxidative stress by increasing antioxidant enzymes. On the whole, the present systematic review critically assessed evidence which demonstrated that MO extract may confer protective effect on experimental ischemic stroke. Although effect size may have been overestimated due to the limited number of included studies, small sample sizes and possible publication bias, results generated in this meta-analysis dmeonstrate that MO extract may be a promising neuroprotective agent against human ischemic stroke.

The prognosis of ischemic stroke patients is highly associated with the collateral circulation. And the competing endogenous RNAs (ceRNAs) generated from different compensatory supply regions may also involve in the regulation of ischemic tissues prognosis. In this study, we found the apoptosis progress of ischemic neurons in posterior circulation-supplied regions (close to PCA, cortex2) was much slower than that in anterior circulation-supplied territory (close to ACA, cortex1) in MCAO-3-h mice. Using the RNA sequencing and functional enrichment analysis, we analyzed the difference between RNA expression profile in cortex1 and cortex2 and the related biological processes. The results indicated that the differential expressed ceRNAs in cortex1 were involved in cell process under acute injury, while the differential expressed ceRNAs in cortex2 was more likely to participate in long-term injury and repair process. Besides, by establishing the miRNA–ceRNA interaction network we further sorted out two specifically distributed miRNAs, namely mmu-miR446i-3p (in cortex1) and mmu-miR3473d (in cortex2). And the specifically increased mmu-miR3473d in cortex2 mainly involved the angiogenesis and cell proliferation after ischemic stroke, which may be the critical reason for the longer therapeutic time window in cortex2. In conclusion, the present study reported the specific changes of ceRNAs in distinct compensatory regions potentially involved in the evolution of cerebral ischemic tissues and the unbalance prognosis after stroke. It provided more evidence for the collateral compensatory effects on patients' prognosis and carried out the new targets for the ischemic stroke therapy. [ABSTRACT FROM AUTHOR]

Introduction: Despite all the research, no definitive treatment for stroke has been found yet. Cinnamon is a plant that has been shown to have health benefits effects. In this study, the effect of pretreatment of cinnamon on ischemia tolerance and the expression of Zonula occludens 1(ZO-1) gene in the brains of rats receiving a high-fat diet was investigated. Methods: In this study, 72 rats were divided into six groups: control, sham, model (stroke), vehicle, lovastatin, and cinnamon. All groups except the control group received a high-fat diet for 8 weeks. Then the last three groups received Carboxymethyl cellulose, lovastatin, and cinnamon 130 mg accordingly for 6 weeks. Stroke was induced by middle cerebral artery occlusion (MCAO). Twelve hours later, the animals were examined for the extent of serum lipids, brain edema, anti-oxidant capacity and gene expression of ZO-1. Results: Cinnamon was effective in reducing serum cholesterol and triglyceride. Cinnamon treatment significantly diminished brain edema. It also restored anti-oxidant capacity. ZO-1 gene expression was increased in the ischemic brains after cinnamon treatment (P< 0.05). Conclusion: Pretreatment with Cin130 had beneficial effects on the serum lipid profile, edema volume in ischemic brain and anti-oxidant capacity. It increased ZO-1 gene expression and so maintained cellular integrity and prevented the subsequent edema. [ABSTRACT FROM AUTHOR]

Background: Stroke is one of the major diseases that endangers human health. It is widely reported that enriched environment (EE) can improve the neurological function in different brain injury models. Recently, relevant researches have indicated that MAPK pathway is closely related to the inflammatory response in nervous system related diseases. However, whether pre exposure to EE (EE pretreatment) has a preventive effect, and its mechanism are not clear. Therefore, this study aimed to determine the possible benefits and related mechanisms of EE in preventing brain injury after acute ischemia-reperfusion. Methods: Adult Sprague Dawley rats were kept in enriched or standardized environments for 21 days. Then the middle cerebral artery of rats was occluded for one hour and 30 min, and then reperfusion was performed. Then their neurological deficit score was evaluated. Cerebral edema, along with ELISA and protein quantities of p38MAPK, JNK, ERK, IL-1β, TNF-α, and co-localization of Iba1 were assessed. Changes in neuroinflammation and apoptosis were also detected in the penumbra cortex. Results: Our research showed that EE pretreatment significantly alleviated acute cerebral ischemia-reperfusion injury in rats. Including the reduction of brain edema and apoptosis, and the improvement of neurological scores. In addition, the protein level of p38MAPK was significantly down regulated in EE pretreatment group, and the downstream protein STAT1 had the same trend. In addition, immunofluorescence results showed that Iba1 in EE pretreatment group decreased, the ELISA results showed that the classical proinflammatory cytokines increased significantly, while anti-inflammatory cytokines in EE pretreatment group increased, and the same results were obtained by Western blot analysis. Conclusion: On the whole, our research demonstrated that EE pretreatment can have a protective effect on the organism by inhibiting the p38 MAPK/STAT1 pathway. Thus, EE can be one of the most promising means of disease prevention. Secondly, p38MAPK/STAT1 pathway may be a latent target for the prevention of acute ischemic stroke. [ABSTRACT FROM AUTHOR]

[Display omitted] • OGD/R-induced astrocytes damaged and released PRDX6. • The release of PRDX6 aggravated the neuroapoptosis suffering OGD/R. • The mechanism underlying PRDX6 release could be linked to aiPLA2 activities. • PRDX6's pro-apoptotic role in neurons may be associated with RAGE and JNK signaling pathways. Peroxiredoxin-6 (PRDX6), a member of the peroxiredoxin family, has progressively emerged as a possible therapeutic target for a variety of brain diseases, particularly Alzheimer's disease and ischemic stroke. However, the role of PRDX6 in neurons under ischemic conditions has remained elusive. Here, we found that astrocytes could release PRDX6 extracellularly after OGD/R, and that PRDX6 release actually worsened neuroapoptosis under OGD/R. We discovered a unique PRDX6/RAGE/JNK signaling pathway that contributes to the effect of neuroapoptosis. We applied a specific inhibitor of the RAGE signaling pathway in a mouse MCAO model and observed significant alterations in animal behavior. Considered together, our findings show the crucial role of the astrocyte-released PRDX6 in the process of neuroapoptosis caused by OGD/R, and could provide novel insights for investigating the molecular mechanism of protecting brain function from ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

Ischemic lesions stimulate adult neurogenesis in the dentate gyrus, however, this is not associated with better cognitive function. Furthermore, increased neurogenesis is associated with the formation of aberrant neurons. In a previous study, we showed that a running task after a stroke not only increases neurogenesis but also the number of aberrant neurons without improving general performance. Here, we determined whether stimulation in an enriched environment after a lesion could increase neurogenesis and cognitive function without enhancing the number of aberrant neurons. After an ischemic stroke induced by MCAO, animals were transferred to an enriched environment containing a running wheel, tunnels and nest materials. A GFP-retroviral vector was delivered on day 3 post-stroke and a modified water maze test was performed 6 weeks after the lesion. We found that the enriched environment significantly increased the number of new neurons compared with the unstimulated stroke group but not the number of aberrant cells after a lesion. Increased neurogenesis after environmental enrichment was associated with improved cognitive function. Our study showed that early placement in an enriched environment after a stroke lesion markedly increased neurogenesis and flexible learning but not the formation of aberrant neurons, indicating that rehabilitative training, as a combination of running wheel training and enriched environment housing, improved functional and structural outcomes after a stroke. [ABSTRACT FROM AUTHOR]

MicroRNA-141-3p (miR-141-3p) has been found to be altered in the brain following a stroke. Herein, we investigate the impact of miR-141-3p on the apoptosis of neural stem cells (NSCs) in mice with middle cerebral artery occlusion (MCAO) and the potential mechanisms involved. Eight-week-old mice were injected intracerebroventricularly with miR-141-3p, antagomir-141-3p, or agomir negative control 2 h before MCAO, and animal behavior tests and infraction volume measurements were performed 24 h later. MCAO-mediated brain injury and NSCs apoptosis were observed by H&E, TTC, and TUNEL staining. The expression of cleaved caspase-3 and Ki67 was detected by western blotting. The luciferase reporter assay proved that miR-141-3p in combination with its target gene PBX homeobox 1 (PBX1). Exogenous miR-141-3p (agomir-141-3p) treatment increased infraction volume and brain edema and damaged neurological functions compared to control mice. Agomir-141-3p increased miR-141-3p expression in brain tissue of mice with MCAO and suppressed PBX1 expression. The effects of the agomir-141-3p-induced apoptosis in NSCs treated with oxygen–glucose deprivation (OGD)/reoxygenation (R) were abolished by PBX1 overexpression. The results from UCSC and JASPAR database showed that prokineticin 2 (PROK2) was a transcription factor of PBX1. The expression of PROK2 was transcriptionally regulated by PBX1 using RT-PCR and western blot assays. The effects of the apoptosis-promoting caused by PBX1 inhibition in NSCs treated with OGD/R were reversed by PROK2 inhibition. In conclusion, the miR-141-3p/PBX1/PROK2 axis might be a novel therapeutic target for the apoptosis of NSCs in MCAO. [ABSTRACT FROM AUTHOR]

[Display omitted] • Pyroptosis-related genes upregulate in brain cell clusters concurrently after MCAO. • MANF inhibits pyroptosis. • Manf -modified BMSCs play a strong therapeutic role in cerebral infarction. Cerebral infarction is a common disease characterized by high mortality, a narrow therapeutic window, and limited therapeutic options. Recently, cell therapy based on gene modification has brought a glimmer of hope to the treatment of cerebral infarction although the explicit underlying mechanism is beyond being well dissected. In the present study, we constructed an animal model of middle cerebral artery occlusion (MCAO), compared differentially expressed genes (DEGs) between the sham and MCAO groups by single-cell RNA sequencing (scRNA-seq) to explore the potential cell death-related pathways involved in cerebral infarction, and transfected Manf into BMSCs by lentivirus. Subsequently, we injected BMSCs (bone marrow-derived mesenchymal stem cells), Manf -modified BMSCs, or lentivirus encoding Manf into the brain. Their effects on MANF content, apoptosis, pyroptosis, infarct volume in the brain, and neurological function were evaluated after MCAO. We found that the DEGs upregulated in four major cell clusters after MCAO and were enriched with not only apoptosis, ferroptosis, and necroptosis but also with pyroptosis-related pathways. In addition, transfection of Manf into BMSCs significantly increased the expression and secretion of MANF in BMSCs; BMSCs, Manf -modified BMSCs, and Manf treatment all resulted in an increase in Manf content in the brain, a decrease in the expression of apoptosis- and pyroptosis-related molecules, a reduction in infarct volume, and an improvement in neurological function after MCAO. Moreover, Manf -modified BMSCs have the strongest therapeutic effect. Collectively, Manf -modified BMSCs ameliorate ischemic injury after cerebral infarction by repressing apoptosis- and pyroptosis-related molecules, which represents a new cell therapy strategy for cerebral infarction. [ABSTRACT FROM AUTHOR]

Systemic transplantation of mesenchymal stem cells (MSCs) is a promising approach for the treatment of ischemia-associated disorders, including stroke. However, exact mechanisms underlying its beneficial effects are still debated. In this respect, studies of the transplanted cells distribution and homing are indispensable. We proposed an MRI protocol which allowed us to estimate the dynamic distribution of single superparamagnetic iron oxide labeled MSCs in live ischemic rat brain during intravenous transplantation after the transient middle cerebral artery occlusion. Additionally, we evaluated therapeutic efficacy of cell therapy in this rat stroke model. According to the dynamic MRI data, limited numbers of MSCs accumulated diffusely in the brain vessels starting at the 7th minute from the onset of infusion, reached its maximum by 29 min, and gradually eliminated from cerebral circulation during 24 h. Despite low numbers of cells entering brain blood flow and their short-term engraftment, MSCs transplantation induced long lasting improvement of the neurological deficit, but without acceleration of the stroke volume reduction compared to the control animals during 14 post-transplantation days. Taken together, these findings indicate that MSCs convey their positive action by triggering certain paracrine mechanisms or cell–cell interactions or invoking direct long-lasting effects on brain vessels. [ABSTRACT FROM AUTHOR]

The mechanism of electroacupuncture (EA) pretreatment-induced neuroprotection remains unclear. In this study, we found that neuronal Triggering receptor expressed on myeloid cells 2 (TREM2) expression was increased and peaked at 48 h and 72 h after ischemia/reperfusion. After specific knockdown of TREM2 in excitatory neurons, neurological function was damaged, and the infarct volume was enlarged. Furthermore, the expression of LC3II/LC3I and Beclin1 was decreased, while the expression of p62 was increased. EA pretreatment enhanced TREM2, LC3II/LC3I and Beclin1 expression while reducing p62 in the ischemic penumbra area. The EA-induced neuroprotective effects and improvements in autophagic flux were abolished by specific knockdown of TREM2 in excitatory neurons. Taken together, our findings provide novel mechanistic insight into EA-induced ischemic tolerance and suggest a promising therapeutic strategy of targeting neuronal TREM2 to treat brain ischemia. • TREM2 expression on excitatory neurons play a role in cerebral ischemia/reperfusion. • Electroacupuncture pretreatment enhanced TREM2 expression and autophagic flux. • Neuronal TREM2 knockdown abolished EA-induced neuroprotective effects. [ABSTRACT FROM AUTHOR]

Purpose: The average saturation efficiency filter (ASEF) is a novel method of improving the specificity of CEST; however, there is a mismatch between the magnetization transfer (MT) effect under high‐duty cycle and low‐duty cycle pulse trains. We explore measures of mitigation and the sensitivity and potential of ASEF imaging in phantoms and stroke rats. Methods: Simulation and nicotinamide phantoms in denatured protein were used to investigate the effect of different average saturation powers and MT pool parameters on matching coefficients used for correction as well as the ASEF ratio signal and baseline. Then, in vivo studies were performed in stroke rodents to further investigate the sensitivity and fidelity of ASEF ratio spectra. Results: Simulation and studies of nicotinamide phantoms show that the matching coefficient needed to correct the baseline MT mismatch is strongly dependent on the average saturation power. In vivo studies in stroke rodents show that the matching coefficient required to correct the baseline MT mismatch is different for normal versus ischemic tissue. Thus, a baseline correction was performed to further suppress the residue MT mismatch. After correction of the mismatch, ASEF ratio achieved comparable contrast at 3.6 ppm between normal and ischemic tissue when compared to the apparent amide proton transfer (APT*) approach. Moreover, contrasts for 2.0 and 2.6 ppm were also ascertainable from the same spectra. Conclusion: ASEF can improve the CEST signal specificity of slow exchange labile protons such as amide and guanidyl, with small loss to sensitivity. It has strong potential in the CEST imaging of various diseases. [ABSTRACT FROM AUTHOR]

To establish pathological features of skeletal muscle post-stroke and to provide a background for promising interventions. Adult male SD rats were selected and randomly divided into a control group, a sham group, and a middle cerebral artery occlusion (MCAO) group. The tolerance and capability of exercise were separately collected on days 1, 3, 5, and 7 after the MCAO operation. The neurological deficits, brain infarct volume, soleus histopathology, mRNA-seq analysis, flow cytometry, immunofluorescence, and protein expression analysis were performed on the seventh day. Rats in the MCAO group showed that soleus tissue weight, pulling force, exercise capacity, endurance, and muscle structure were significantly decreased. Moreover, the RNA sequencing array revealed that mitochondrial-mediated autophagy was the critical pathological process, and the result of transcriptomic findings was confirmed at the translational level. The mitochondrial membrane potential and the mfn2 and p62 protein expression were decreased, and the Beclin-1, ATG5, Parkin, PINK1, LC3B, and Drp1 expression were upregulated; these results were consistent with immunohistochemistry. This is the first report on the pathological features of limbic symptoms on day 7 after MCAO surgery in rats. In addition, we further confirmed that autophagy is one of the main causative mechanisms of reduced muscle function after stroke. [ABSTRACT FROM AUTHOR]

Objective: This study explored the underlying therapeutic mechanism of human umbilical cord mesenchymal stem cells (hUCMSCs) for ischemic stroke (IS), and determined the optimal administration time windows and dose-effect relationship. Methods: The levels of SDF-1α, IL-10, IL-6, TNF-α, BDNF, IL-1β, and VEGF-A factors in serum and brain tissue lysate were measured by ELISA. The pathological status of brain tissues was evaluated by Hematoxylin-Eosin (HE) staining, and apoptosis of nerve cells was detected by tunel. The protein expression of CXCR-4, NeuN, and Nestin in the brain tissues was assessed through immunofluorescence. The balance beam, forelimb muscle strength, and limb placement were tested on MCAO rats at different time points and doses. The infarct area of the rat brain tissues was measured at the end of the experiment. Results: The hUCMSC treatment during the acute phase of MCAO significantly reduced the secretion of IL-6, TNF-α, IL-1β but increased IL-10 in serum, and the levels of SDF-α and BDNF in serum and brain tissues lysate were also increased. The pathological results showed that there were more neurons in the treatment group compared to the model group. Immunofluorescence assays showed that the expression of CXCR4、Nestin、NeuN was relatively higher than that in the model group. The d4 and d7 treatment significantly improves the motor function, promotes the recovery of forelimb muscle strength, increases the forelimb placement rate and reduces the scope of cerebral infarction, but the d14 treatment group has less therapeutic effect compared to the d4 and d7 treatment. The 2×107/kg treatment showed the best therapeutic effect, followed by the 1×107/kg treatment, and the worst is 0.5×107/kg treatment from the test of balance beam, forelimb muscle strength, limb placement and the infarct area of the rat brain tissues. Conclusion: The hUCMSCs can inhibit the infiltration of inflammatory cells in the brain tissue, and promote the repair of brain tissue structure and function. Early intervention by injecting high-dose of hUCMSCs can significantly improve the recovery of neurological/motor function and reduce the size of cerebral infarction in rats. [ABSTRACT FROM AUTHOR]

Background Previous data have indicated the regulation of circular RNA (circRNA) toward cerebral ischemia. This study aims to reveal the effects of circNUFIP2 on cerebral ischemia and the underlying mechanism. Methods Oxygen-glucose deprivation (OGD) hippocampal neuron (HT22) cell model and middle cerebral artery occlusion (MCAO) mouse model were used for this study. The expression of circRNA nuclear FMR1 interacting protein 2 (circNUFIP2), microRNA-1224-5p (miR-1224-5p) and growth differentiation factor 11 (GDF11) was detected by quantitative real-time polymerase-chain reaction. Protein expression was checked by Western blotting. The binding relationships among circNUFIP2, miR-1224-5p and GDF11 were identified by dual-luciferase reporter assay, RNA pull-down assay, and RNA immunoprecipitation assay. Cell proliferation and apoptosis were investigated by 5-Ethynyl-29-deoxyuridine and flow cytometry analysis, respectively. Results CircNUFIP2 and GDF11 expression were decreased, but miR-1224-5p was increased in OGD-treated HT22 cells when compared with their expression in control groups. OGD treatment inhibited HT22 cell proliferation but induced cell apoptosis and oxidative stress; however, these effects were attenuated after circNUFIP2 overexpression. Also, circNUFIP2 upregulation assuaged the cerebral infarction of MCAO mice. Besides, circNUFIP2 bound to miR-1224-5p and mediated OGD-induced HT22 cell damage through miR-1224-5p. Meanwhile, knockdown of GDF11, a target gene of miR-1224-5p, relieved miR-1224-5p depletion-caused effects in OGD-treated HT22 cells. Furthermore, circNUFIP2 regulated GDF11 expression by interacting with miR-1224-5p. Conclusion CircNUFIP2 overexpression protected neuron cells against cerebral ischemia-induced damage, at least in part, by the miR-1224-5p/GDF11 pathway, providing a possible target for the therapy of cerebral ischemic stroke [ABSTRACT FROM AUTHOR]

Due to the complex pathogenesis of acute ischemic stroke (AIS), further investigation into its underlying mechanisms is necessary. Presently, existing literature indicates a close association between ferroptosis and AIS injury; however, the precise mechanism and molecular target of ferroptosis in AIS injury remain elusive. By RNA sequencing, we found a significant increase in LCN2 expression in the ischemic cortex. In order to investigate the potential role of LCN2 in modulating AIS injury through the regulation of ferroptosis, we utilized RNA interference (RNAi) knockdown and gene overexpression experiments. The findings from experiments conducted both in vitro and in vivo revealed a marked increase in ferroptosis levels within the AIS model group. Suppression of the LCN2 gene resulted in a significant reduction in ferroptosis levels in OGD/R cells. Conversely, upregulation of LCN2 exacerbated ferroptosis levels in OGD/R cells. The results suggest that elevated levels of ferroptosis may result from heightened expression of LCN2, thereby exacerbating ischemia/reperfusion injury. This study indicates the involvement of ferroptosis in the pathogenesis of AIS and highlights LCN2 as a regulator of ferroptosis in AIS-induced injury, suggesting a potential therapeutic target for ischemic stroke. • The expression of LCN2 significantly increased in the MCAO/R model and OGD/R PC12 cells. • Silencing LCN2 reduced the level of ferroptosis in OGD/R-injured cells. • Overexpression of LCN2 exacerbated the level of ferroptosis in OGD/R-injured cells. [ABSTRACT FROM AUTHOR]

Stroke is a major public health concern, with limited clinically approved interventions available to enhance sensorimotor recovery beyond reperfusion. Remarkably, spontaneous recovery is observed in certain stroke patients, suggesting the existence of a brain self-repair mechanism not yet fully understood. In a rat model of permanent cerebral ischemia, we described an increase in oligodendrocytes expressing 3RTau in damaged area. Considering that restoration of myelin integrity ameliorates symptoms in many neurodegenerative diseases, here we hypothesize that this cellular response could trigger remyelination. Our results revealed after ischemia an early recruitment of OPCs to damaged area, followed by their differentiation into 3RTau+ pre-myelinating cells and subsequent into remyelinating oligodendrocytes. Using rat brain slices and mouse primary culture we confirmed the presence of 3RTau in pre-myelinating and a subset of mature oligodendrocytes. The myelin status analysis confirmed long-term remyelination in the damaged area. Postmortem samples from stroke subjects showed a reduction in oligodendrocytes, 3RTau+ cells, and myelin complexity in subcortical white matter. In conclusion, the dynamics of oligodendrocyte populations after ischemia reveals a spontaneous brain self-repair mechanism which restores the functionality of neuronal circuits long-term by remyelination of damaged area. This is evidenced by the improvement of sensorimotor functions in ischemic rats. A deep understanding of this mechanism could be valuable in the search for alternative oligodendrocyte-based, therapeutic interventions to reduce the effects of stroke. [Display omitted] • In the long term, spontaneous sensorimotor improvement occurs in ischemic rats. • Changes in oligodendrocyte populations after ischemia results in remyelination. • OLGs colonizing damaged area do not originate from cell division after ischemia. • Premyelinating oligodendrocytes and a subset of matures express 3RTau. • Samples from stroke donors showed a reduction in 3RTau+ cells and myelin complexity. [ABSTRACT FROM AUTHOR]

Background: Somatosensory deficits are frequently seen in acute stroke patients and may recover over time and affect functional outcome. However, the underlying mechanism of function recovery remains poorly understood. In the present study, progressive function alteration of the secondary somatosensory cortex (S2) and its relationship with regional perfusion and neurological outcome were examined using a monkey model of stroke. Methods and materials: Rhesus monkeys (n = 4) were induced with permanent middle cerebral artery occlusion (pMCAo). Resting-state functional MRI, dynamic susceptibility contrast perfusion MRI, diffusion-weighted, T1 and T2 weighted images were collected before surgery and at 4–6, 48, and 96 h post stroke on a 3T scanner. Progressive changes of relative functional connectivity (FC), cerebral blood flow (CBF), and CBF/Tmax (Time to Maximum) of affected S2 regions were evaluated. Neurological deficits were assessed using the Spetzler approach. Results: Ischemic lesion was evidently seen in the MCA territory including S2 in each monkey. Relative FC of injured S2 regions decreased substantially following stroke. Spetzler scores dropped substantially at 24 h post stroke but slightly recovered from Day 2 to Day 4. Relative FC progressively increased from 6 to 48 and 96 h post stroke and correlated significantly with relative CBFand CBF/Tmax changes. Conclusion: The present study revealed the progressive alteration of function connectivity in S2 during acute stroke. The preliminary results suggested the function recovery might start couple days post occlusion and collateral circulation might play a key role in the recovery of somatosensory function after stroke insult. The relative function connectivity in S2 may provide additional information for prediction of functional outcome in stroke patients.

Stroke is a devastating neurovascular disorder that results in damage to neurons and white matter tracts. It has been previously demonstrated that neuregulin-1 (NRG-1) protects neurons from ischemic injury following stroke. Here, diffusion tensor imaging (DTI) was utilized to characterize the effects of NRG-1 treatment on cererbral infarction and integrity of white matter after ischemic insult using a permanent middle celebral artery occlusion (pMCAo) rat model. In the present study, sixteen Sprague-Dawley rats underwent pMCAo surgery and received either a single intra-arterial bolus (20 μg/kg) dose of NRG-1 or saline immediately prior to pMCAo. MRI including T2-weighted imaging and DTI was performed in the first 3 h post stroke, and repeated 48 h later. It is found that the stroke infarction was significantly reduced in the NRG-1 treated group. Also, NRG-1 prevented the reduction of fractional anisotropy (FA) in white matter tracts of fornix and corpus callosum (CC), indicating its protection of CC and fornix white matter bundles from ischemia insult. As a conclusion, the present DTI results demonstrate that NRG-1 has significantly neuroprotective effects in both cerebral cortex and white matter including corpus callosum and fornix during acute stroke. In particular, NRG-1 is more effective on stroke lesion with mild ischemia. As CC and fornix white matter bundles play critical roles in transcallosal connectivity and hippocampal projections respectively in the central nervous system, the findings could provide complementary information for better understanding the biological mechanism of NRG-1's neuroprotection in ischemic tissues and neurobehavioral effects.

Strokes are one of the leading causes of mortality and chronic morbidity in the world, yet with only limited successful interventions available at present. Our previous studies revealed the potential role of the glucocorticoid receptor (GR) in the pathogenesis of neonatal hypoxic-ischemic encephalopathy (HIE). In the present study, we investigate the effect of GR knockdown on acute ischemic brain injuries in a model of focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in adult male CD1 mice. GR siRNAs and the negative control were administered via intracerebroventricular (i.c.v.) injection 48 h prior to MCAO. The cerebral infarction volume and neurobehavioral deficits were determined 48 h after MCAO. RT-qPCR was employed to assess the inflammation-related gene expression profiles in the brain before and after MCAO. Western Blotting was used to evaluate the expression levels of GR, the mineralocorticoid receptor (MR) and the brain-derived neurotrophic factor/tropomyosin receptor kinase B (BDNF/TrkB) signaling. The siRNAs treatment decreased GR, but not MR, protein expression, and significantly enhanced expression levels of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) in the brain. Of interest, GR knockdown suppressed BDNF/TrkB signaling in adult mice brains. Importantly, GR siRNA pretreatment significantly increased the infarction size and exacerbated the neurobehavioral deficits induced by MCAO in comparison to the control group. Thus, the present study demonstrates the important role of GR in the regulation of the inflammatory responses and neurotrophic BDNF/TrkB signaling pathway in acute ischemic brain injuries in adult mice, revealing a new insight into the pathogenesis and therapeutic potential in acute ischemic strokes.

Buzhong Yiqi decoction (BZYQD) has been developed for preventing or reducing the recurrence of ischemic stroke for a long time in China. However, the mechanism of action of the BZYQD is not completely understood. Our research aims to determine whether the mechanism of action of BZYQD is by regulating gut microbiota using 16SR RNA and fecal microbiota transplantation. In a cerebral ischemia mouse model, the results showed that prophylactic administration of BZYQD could reduce brain infarct volume and improve neurological function and behavior. The prophylactic administration of BZYQD could regulate intestinal microbiota and increase the abundance of butyrate-producing Prevotellaceae_NK3B31_group and probiotic Akkermansia in mice 72 h after surgery. Transplanting BZYQD-administered bacterial flora into antibiotic-depleted mice could reproduce the therapeutic effects of BZYQD. Overall, our study provided molecular insights into the mechanism and impact of BZYQD in the prevention of cerebral ischemic damage and highlighted the potential of regulation of intestinal microbiota as a therapeutic approach for ischemic stroke. [ABSTRACT FROM AUTHOR]

Intraluminal monofilament model of middle cerebral artery occlusion (MCAO) is widely adopted for ischemic stroke; and Sprague–Dawley (SD) rats are commonly used rodents for preclinical research. Due to the paucity of information on the appropriate monofilament size for inducing MCAO in SD rats and the importance of including middle-aged models in ischemic stroke studies, we aimed to: (i). determine an appropriate Doccol® monofilament size for middle-aged male SD rats which weighed > 500 g following 24-h transient MCAO survival as well as (ii). demonstrate the optimal Doccol® filament size for middle-aged males (≤ 500 g) and females (273–300 g) while using young adult male SD rats (372–472 g) as control for severity of infarct volume following 7-days post-MCAO. All rats were subjected to 90-min transient MCAO. We show that 0.43 mm Doccol® monofilament size is more appropriate to induce large infarct lesion and optimal functional deficit when compared to 0.45 mm and 0.47 mm at 24 h post-MCAO. Our data on infarct volumes at 7 days post-MCAO as well as the observed weight loss and functional deficits at post-MCAO days 1, 3 and 7 demonstrate that 0.41 mm, 0.37 mm and 0.39 mm are optimal Doccol® filament sizes for middle-aged male (477.3 ± 39.61 g) and female (302.6 ± 26.28 g) as well as young-adult male (362.2 ± 28.38 g) SD rats, respectively. [ABSTRACT FROM AUTHOR]

Background: Cardiovascular diseases like stroke cause changes to sphingolipid mediators like sphingosine 1-phosphate (S1P) or its ceramide analogs, which bear the potential to either alleviate or exacerbate the neurological damage. Therefore, the precise identification of alterations within the sphingolipidome during ischemic stroke (IS) and hemorrhagic transformation (HT) harbors a putative therapeutic potential to orchestrate local and systemic immunomodulatory processes. Due to the scarcity of research in this field, we aimed to characterize the sphingolipidome in IS and HT. Methods: C57BL/6 mice underwent middle cerebral artery occlusion (MCAO) and specimens of the peri-infarct tissue were taken for sphingolipid profiling. Results: Ischemic stroke resulted in reduced S1P whilst ceramides were elevated six hours post ischemia onset. However, these differences were nearly revoked at 24 hours post ischemia onset. Moreover, the topmost S1P and ceramide levels were linked to the presence of HT after MCAO. In this study we show the characterization of the sphingolipidomic landscape of the peri-infarct tissue after ischemic stroke and HT. Especially, highest values of S1P, C18 lactosylceramide, C18 glucosylceramide, and C24:1 ceramide were nearly entirely expressed by mice with HT. Conclusions: Our results warrant further investigations into the immunomodulatory consequences of altered sphingolipid species for the development of HT after IS. [ABSTRACT FROM AUTHOR]

Objective: This study sought to evaluate the feasibility of multifunctional gastrodin (GAS)‐containing nano‐drug carrier system against cerebral ischemia–reperfusion injury (CIRI). Methods: The drug‐loaded nanocomposite (Au‐G5.NHAc‐PS/GAS) with certain encapsulation efficiency (EE) was prepared by physical adsorption method using different proportions of GAS and drug‐carrying system (Au‐G5.NHAc‐PS). High‐performance liquid chromatography was used to determine the drug loading and EE. Cultured rat astrocytes and hypothalamic neurons were assigned into four groups: PBS, Au‐G5.NHAc‐PS, Au‐G5.NHAc‐PS/GAS, and GAS. CCK‐8 assay, flow cytometry, and quantitative real‐time PCR were performed to examine the cell viability, apoptosis, and the expression of tumor necrosis factor‐α (TNF‐α), IL‐1β, and IL‐6 in the astrocytes and hypothalamic neurons, respectively. Cellular uptake of GAS and Au‐G5.NHAc‐PS/GAS was analyzed by using Hoechst 33342 staining. The animal model with focal cerebral ischemia was generated by middle cerebral artery occlusion (MCAO) in healthy male Sprague Dawley (SD) rats, and pathological changes of brain tissue and major organs in the rats were identified by hematoxylin and eosin (HE) staining. Apoptosis in rat astrocytes and hypothalamic neurons was detected by TUNEL staining and flow cytometry. Results: Au‐G5.NHAc‐PS had a spherical shape with a uniform size of 157.3 nm. Among the nanoparticles, Au‐G5.NHAc‐PS/GAS with an EE of 70.3% displayed the best release delay effect. Moreover, we observed that in vitro cytotoxicity and cellular uptake of Au‐G5.NHAc‐PS/GAS were higher than those of GAS, whereas the expression of TNF‐α, IL‐1β, and IL‐6 was significantly downregulated in Au‐G5.NHAc‐PS/GAS group as compared to G5.NHAc‐PS group. Notably, HE staining revealed that although Au‐G5.NHAc‐PS/GAS had no toxic and side effects on the main organs of rats, it alleviated the damage of brain tissue in the MCAO rats. Besides, Au‐G5.NHAc/GAS markedly reduced MCAO‐induced apoptosis. Conclusion: Au‐G5.NHAc‐PS showed favorable surface morphology, sustained drug release ability, no measurable toxicity, and good biocompatibility, indicating that GAS exerts anti‐inflammatory and antiapoptotic effects on CIRI. [ABSTRACT FROM AUTHOR]

Objective: Brain ischemia leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen and consequently, ER stress. To help cells restore ER function, a series of adaptive stress response pathways, collectively termed the unfolded protein response (UPR), are activated. We have previously demonstrated that the UPR pathway initiated by ATF6 is pro-survival in transient ischemic stroke. However, the effect of ATF6 activation on the outcome after permanent ischemic stroke remains unknown. Here, we addressed this knowledge gap. Method: sATF6-KI mice with functional short-form ATF6 (sATF6) predominantly expressed in forebrain neurons were subjected to two ischemic stroke models: photothrombotic stroke and permanent middle cerebral artery occlusion (pMCAO). Both short-term and long-term functional outcomes were evaluated. Changes in neuroinflammation and cerebrovascular density after pMCAO were also assessed. Results: Compared to littermate controls, sATF6-KI mice performed significantly better in open field, cylinder, and foot fault tests on day 1 or 3 after photothrombotic stroke. However, on days 7 and 14 after stroke, the performance of these functional tests was not significantly different between groups, which is likely related to mild brain damage associated with this stroke model. Thus, to evaluate the long-term effects of ATF6 activation in permanent stroke, we turned to our pMCAO model. We first found that on day 4 after pMCAO, functional outcome was better, and infarct volumes were smaller in sATF6-KI mice vs controls. Next, the 15-day stroke outcome study indicated that compared to control mice, sATF6-KI mice consistently exhibited improved performance in neurologic scoring, tight rope test, and tape removal test, after pMCAO. Moreover, sATF6-KI mice showed higher vascular density and lower activation of both astrocytes and microglia around stroke regions on day 16 after pMCAO. Conclusions: Here, we presented the first evidence that activation of the ATF6 UPR branch is protective in permanent ischemic stroke, which further supports the therapeutic potential of targeting the ATF6 pathway in stroke. [ABSTRACT FROM AUTHOR]

Cerebrovascular disease is the leading cause of disability and death, and ischemic stroke accounts for most stroke cases. However, few effective drugs are available for the treatment of ischemic stroke; thus, there is an urgent need to develop effective drugs to treat ischemic stroke. DL-3-n-butylphthalide (NBP) is clinically approved as an anti-ischemic drug in China, but its potential hepatotoxicity limits its use. G-3702 (a structural analogue of NBP) is synthesized with the boron hydroxyl group replacing carbonyl group. G-3702 significantly enhanced the survival of middle cerebral artery occlusion (MCAO) rats, decreased neurobehavioral deficit scores and cerebral infarct volume, comparable with NBP, which was also supported by tissue damage assessment, immunohistochemistry staining, biochemical parameters and ELISA assay. G-3702 showed better anti-stroke activity than NBP according to 1H NMR spectroscopy-based metabolomics analysis, demonstrating the feasibility of metabolomics approach to assess drug efficacy. G-3702 markedly ameliorated energy metabolism, attenuated oxidative and inflammatory stress during ischemia/reperfusion (I/R). G-3702 exhibited good neuroprotective effects against I/R induced injury and favorable little possibility of hepatotoxicity, which made it a promising anti-stroke drug and better NBP alternative. [ABSTRACT FROM AUTHOR]

Background: In the analysis of brain fluorodeoxyglucose positron emission tomography (FDG-PET) images, intensity normalization is a necessary step to reduce inter-subject variability. However, the choice of the most appropriate normalization method in stroke studies remains unclear, as demonstrated by inconsistent findings in the literature., Materials and Methods: Here, we propose a regression- and single-subject-based model for analyzing FDG-PET images without intensity normalization. Two independent data sets were collected before and after middle cerebral artery occlusion (MCAO), with one comprising 120 rats and the other 96 rats. After data preprocessing, voxel intensities in the same region and hemisphere were paired before and after the MCAO scan. A linear regression model was applied to the paired data, and the coefficient of determination R 2 was calculated to measure the linearity. The R 2 values between the ipsilateral and contralateral hemispheres were compared, and significant regions were defined as those with reduced linearity. Our method was compared with voxel-wise analysis under different intensity normalization methods and validated using the triphenyl tetrazolium chloride (TTC) staining data., Results: The significant regions identified by the proposed method showed a large degree of overlap with the infarcted regions identified by TTC data, as measured by the Dice similarity coefficient (DSC). The average DSC of the proposed method was 59.7%, whereas the DSCs of the existing approaches ranged from 41.4%∼51.3%. Additional validation using receiver operating characteristic (ROC) demonstrated that the area under the curve (AUC) of the average ROC curves reached 0.84 using the proposed method, whereas existing methods achieved AUCs ranging from 0.77∼0.79. The identified regions were consistent across the two independent data sets, and some findings were corroborated by other publications., Conclusions: The proposed model presents a novel quantitative approach for identifying injury regions post-stroke using FDG-PET images. The calculation does not require intensity normalization and can be applied to individual subjects. The method yields more sensitive results compared to existing identification methods., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Glycine Transporter Type 1 (GlyT1) inhibition confers neuroprotection against different forms of cerebral damage. This effect occurs through the elevation of synaptic glycine concentrations, which enhances N-methyl-d-aspartate receptor (NMDAR) activation by glutamate. To investigate the neuroprotective mechanism of GlyT1 inhibition, we used the Middle Cerebral Artery Occlusion (MCAO) model in male C57BL/6 mice, aged 10-12 weeks. We administered N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl] sarcosine (NFPS), a GlyT1 inhibitor, 24 h prior to ischemia induction. NFPS pretreatment provided significant neuroprotection in the MCAO model, associated with modulation of pathways related to long-term potentiation. Specifically, GluN2A subunit expression was upregulated, while GluN2B subunit expression was downregulated in cortical areas, correlating with enhanced phosphorylation of CaMKIV and CREB proteins. Coadministration with the GluN2B antagonist Eliprodil or the CREB inhibitor C646 did not affect the neuroprotective effects of NFPS pretreatment, but TCN-201, a specific GluN2A antagonist, disrupted these effects. These findings suggest that GlyT1 inhibition mediates neuroprotection through activation of GluN2A-containing NMDARs and the GluN2A/CaMKIV/CREB signaling cascade, thereby modulating the balance between GluN2A and GluN2B subunits., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sleep disturbances, including insomnia and excessive daytime sleepiness, are highly prevalent in patients with ischemic stroke (IS), which severely impacts recovery and rehabilitation efforts. However, how IS induces sleep disturbances is unclear. Three experiments were performed on middle-aged C57BL/6J mice, instrumented with sleep recording electrodes and/or subjected to 1 h of middle cerebral artery (MCAO; Stroke group) or sham (Sham group) occlusion to induce IS. After 48 h of reperfusion (a) experiment 1 verified sensorimotor deficit (using Garcia scale) and infarction (using TTC staining) in this mouse model; (b) experiment 2 examined the effects of IS on the quality (sleep latency and NREM delta power) and quantity (duration) of sleep; and (c) experiment 3 determined the effects of IS on sleep homeostasis using sleep deprivation (SD) and recovery sleep (RS) paradigm. Stroke mice display (a) a significant correlation between sensorimotor deficit and cerebral infarction; (b) insomnia-like symptoms (increased sleep latency, reduced NREM duration and delta power) during the light (inactive) period and daytime sleepiness-like symptoms during the dark (active) period mimicking sleep in IS patients; and (c) impairments in the markers of sleep pressure (during SD) and sleep dissipation (during RS). Our results suggest that IS disrupts sleep homeostasis to cause sleep disturbances. [ABSTRACT FROM AUTHOR]

Although tissue-type plasminogen activator was approved by the FDA for early reperfusion of occluded vessels, there is a need for an effective neuroprotective drug for stroke patients. In this study, we established tumor susceptibility gene (TSG)101-overexpressing human neural stem cells (F3.TSG) and investigated whether they showed enhanced secretion of exosomes and whether treatment with exosomes during reperfusion alleviated ischemia-reperfusion-mediated brain damage. F3.TSG cells secreted higher amounts of exosomes than the parental F3 cells. In N2A cells subjected to oxygen–glucose deprivation (OGD), treatment with exosomes or coculture with F3.TSG cells significantly attenuated lactate dehydrogenase release, the mRNA expression of proinflammatory factors, and the protein expression of DNA-damage-related proteins. In a middle cerebral artery occlusion (MCAO) rat model, treatment with exosomes, F3 cells, or F3.TSG cells after 2 h of occlusion followed by reperfusion reduced the infarction volume and suppressed inflammatory cytokines, DNA-damage-related proteins, and glial fibrillary acidic protein, and upregulated several neurotrophic factors. Thus, TSG101-overexpressing neural stem cells showed enhanced exosome secretion; exosome treatment protected against MCAO-induced brain damage via anti-inflammatory activities, DNA damage pathway inhibition, and growth/trophic factor induction. Therefore, exosomes and F3.TSG cells can affect neuroprotection and functional recovery in acute stroke patients. [ABSTRACT FROM AUTHOR]

Tannic acid (TA) is a polyphenolic compound that exerts protective effects under pathological conditions. The diverse mechanisms of TA can exert beneficial anti-oxidative, anti-inflammatory, and anti-cancer effects. Herein, we reported that TA affords robust neuroprotection in an animal model of stroke (transient middle cerebral artery occlusion; tMCAO) and exhibits Zn2+-chelating and anti-oxidative effects in primary cortical neurons. Following tMCAO induction, intravenous administration of TA (5 mg/kg) suppressed infarct formation by 32.9 ± 16.2% when compared with tMCAO control animals, improving neurological deficits and motor function. We compared the chelation activity under several ionic conditions and observed that TA showed better Zn2+ chelation than Cu2+. Furthermore, TA markedly decreased lactate dehydrogenase release following acute Zn2+ treatment and subsequently reduced the expression of p67 (a cytosolic component of NADPH oxidase), indicating the potential mechanism underlying TA-mediated Zn2+ chelation and anti-oxidative effects in primary cortical neurons. These findings suggest that anti-Zn2+ toxicity and anti-oxidative effects participate in the TA-mediated neuroprotective effects in the postischemic brain. [ABSTRACT FROM AUTHOR]

Ischemic stroke leads to hypoxia-induced neuronal death and behavioral abnormity, and is a major cause of death in the modern society. However, the treatments of this disease are limited. Brilliant Blue FCF (BBF) is an edible pigment used in the food industry that with multiple aromatic rings and sulfonic acid groups in its structure. BBF and its derivatives were proved to cross the blood-brain barrier and have advantages on the therapy of neuropsychiatric diseases. In this study, BBF, but not its derivatives, significantly ameliorated chemical hypoxia-induced cell death in HT22 hippocampal neuronal cell line. Moreover, protective effects of BBF were attributed to the inhibition of the extracellular regulated protein kinase (ERK) and glycogen synthase kinase-3β (GSK3β) pathways as evidenced by Western blotting analysis and specific inhibitors. Furthermore, BBF significantly reduced neurological and behavioral abnormity, and decreased brain infarct volume and cerebral edema induced by middle cerebral artery occlusion/reperfusion (MCAO) in rats. MCAO-induced increase of p-ERK in ischemic penumbra was reduced by BBF in rats. These results suggested that BBF prevented chemical hypoxia-induced otoxicity and MCAO-induced behavioral abnormity via the inhibition of the ERK and GSK3β pathways, indicating the potential use of BBF for treating ischemic stroke [ABSTRACT FROM AUTHOR]

Tannic acid (TA) is a polyphenolic compound that exerts protective effects under pathological conditions. The diverse mechanisms of TA can exert beneficial anti-oxidative, anti-inflammatory, and anti-cancer effects. Herein, we reported that TA affords robust neuroprotection in an animal model of stroke (transient middle cerebral artery occlusion; tMCAO) and exhibits Zn2+-chelating and anti-oxidative effects in primary cortical neurons. Following tMCAO induction, intravenous administration of TA (5 mg/kg) suppressed infarct formation by 32.9 ± 16.2% when compared with tMCAO control animals, improving neurological deficits and motor function. We compared the chelation activity under several ionic conditions and observed that TA showed better Zn2+ chelation than Cu2+. Furthermore, TA markedly decreased lactate dehydrogenase release following acute Zn2+ treatment and subsequently reduced the expression of p67 (a cytosolic component of NADPH oxidase), indicating the potential mechanism underlying TA-mediated Zn2+ chelation and anti-oxidative effects in primary cortical neurons. These findings suggest that anti-Zn2+ toxicity and anti-oxidative effects participate in the TA-mediated neuroprotective effects in the postischemic brain. [ABSTRACT FROM AUTHOR]

Background: Exosomes can activate microglia to modulate neural activity and synaptic plasticity by phagocytosis of neural spines or synapses. Our previous research found that an early 4-week exercise intervention in middle cerebral artery occlusion (MCAO) rats can promote the release of exosomes and protect the brain. This study intended to further explore the intrinsic mechanism of neuroprotection by exosome release after exercise. Methods: Rats were randomly divided into four groups: the sham operation (SHAM), middle cerebral artery occlusion (MCAO) with sedentary intervention (SED-MCAO), MCAO with exercise intervention (EX-MCAO), and MCAO with exercise intervention and exosome injection (EX-MCAO-EXO). Modified neurological severity score (mNSS), cerebral infarction volume ratio, microglial activation, dendritic complexity, and expression of synaptophysin (Syn) and postsynaptic density protein 95 (PSD-95) were detected after 28 days of intervention. Results: (1) The exercise improved body weight and mNSS score, and the survival state of the rats after exosome infusion was better. (2) Compared with the SED-MCAO group, the EX-MCAO (P = 0.039) and EX-MCAO-EXO groups (P = 0.002) had significantly lower cerebral infarct volume ratios (P < 0.05), among which the EX-MCAO-EXO group had the lowest (P = 0.031). (3) Compared with the SED-MCAO group, the EX-MCAO and EX-MCAO-EXO groups had a significantly decreased number of microglia (P < 0.001) and significantly increased process length/cell (P < 0.01) and end point/cell (P < 0.01) values, with the EX-MCAO-EXO group having the lowest number of microglia (P = 0.036) and most significantly increased end point/cell value (P = 0.027). (4) Compared with the SED-MCAO group, the total number of intersections and branches of the apical and basal dendrites in the EX-MCAO and EX-MCAO-EXO groups was increased significantly (P < 0.05), and the increase was more significant in the EX-MCAO-EXO group (P < 0.05). (5) The expression levels of Syn and PSD-95 in the EX-MCAO (PSyn = 0.043, PPSD-95 = 0.047) and EX-MCAO-EXO groups were significantly higher than those in the SED-MCAO group (P<0.05), and the expression levels in the EX-MCAO-EXO group were significantly higher than those in the EX-MCAO group (P < 0.05). Conclusion: Early exercise intervention after stroke can inhibit the excessive activation of microglia and regulate synaptic plasticity by exosome release. [ABSTRACT FROM AUTHOR]

Background: The blood-brain barrier (BBB) maintains the balance of the internal environment of the brain and strictly controls substance exchange between the brain and blood dynamically but stably. Transient increases in the permeability of the BBB plays an important role in helping macromolecular drugs enter the brain to exert their pharmacological effects. Previous research has revealed that electronic acupuncture (EA) stimulation connecting Baihui (GV20) and Shuigou (GV26) at a specific frequency can enhance the permeability of the BBB at 8 minutes after the intervention and induce the entry of 20 kDa fluorescein isothiocyanate-dextran (FITC-dextran) into the cerebral cortex, but whether it can also allow drugs to pass the BBB remains unknown. We hypothesized that EA at a specific frequency could open the BBB and induce the entry of nerve growth factor (NGF) into the brain to exert its therapeutic effect. Methods: First, the middle cerebral artery occlusion (MCAO) model is adopted and changes in the permeability and structure of the BBB are assessed by measuring both the intensity of Evans blue (EB) staining and the cerebral infarction volume, and by evaluating the ultrastructure of the BBB. Then, a laser spectrometer and immunofluorescence are used to observe entry of NGF into the brain. Finally, the learning and memory ability of rats are assessed and the DeadEndTM Fluorometric TUNEL System is applied to assess apoptosis in the hippocampus. Results: Our results showed that, in the first, the BBB was essentially repaired three weeks after MCAO operation. Secondly, Electronic Acupuncture (EA) stimulation at a specific frequency can enhance BBB permeability in the prefrontal cortex and induce NGF uptake by prefrontal neurons. Finally, in the presence of EA stimulation, entry of NGF into the brain promoted learning and memory in rats and inhibited the apoptosis of neurons in the hippocampus. Conclusions: In this study, the timing of BBB repair in the MCAO model was determined under pathological conditions and the EA stimulation can induce the entry of NGF into the brain to exert its therapeutic effect. EA could serve as a new strategy for delivering therapeutics to the central nervous system (CNS), given that EA stimulation at a specific frequency was shown to increase the permeability of the BBB. Further study of the mechanism underlying the opening of the BBB and its timing is needed. [ABSTRACT FROM AUTHOR]