Mitophagy influences the progression and prognosis of ischemic stroke (IS). However, whether DNA methylation in the brain is associated with altered mitophagy in hypoxia-injured neurons remains unclear. Here, miR-138-5p was found to be highly expressed in exosomes secreted by astrocytes stimulated with oxygen and glucose deprivation/re-oxygenation (OGD/R), which could influence the recovery of OGD/R-injured neurons through autophagy. Mechanistically, miR-138-5p promotes the stable expression of Ras homolog enriched in brain like 1(Rhebl1) through DNA-methyltransferase-3a (DNMT3A), thereby enhancing ubiquitin-dependent mitophagy to maintain mitochondrial homeostasis. Furthermore, we employed glycosylation engineering and bioorthogonal click reactions to load mirna onto the surface of microglia and deliver them to injured region utilising the inflammatory chemotactic properties of microglia to achieve drug-targeted delivery to the central nervous system (CNS). Our findings demonstrate miR-138-5p improves mitochondrial function in neurons through the miR-138-5p/DNMT3A/Rhebl1 axis. Additionally, our engineered cell vector-targeted delivery system could be promising for treating IS. STATEMENT OF SIGNIFICANCE: : In this study, we demonstrated that miR-138-5p in exosomes secreted by astrocytes under hypoxia plays a critical role in the treatment of hypoxia-injured neurons. And we find a new target of miR-138-5p, DNMT3A, which affects neuronal mitophagy and thus exerts a protective effect by regulating the methylation of Rbebl1. Furthermore, we have developed a carrier delivery system by combining miR-138-5p with the cell membrane of microglia and utilized the inflammatory chemotactic properties of microglia to deliver this system to the brain via intravenous injection. This groundbreaking study not only provides a novel therapeutic approach for ischemia-reperfusion treatment but also establishes a solid theoretical foundation for further research on targeted drug delivery for central nervous system diseases with promising clinical applications.

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.

BACKGROUND: Buyang Huanwu Decoction (BHD) is widely used in Chinese clinical practice for the treatment and prevention of ischemic cerebral vascular diseases. This study was designed to investigate the effects of BHD on ischemic stroke (IS) and its underlying mechanism.
METHODS: The middle cerebral artery occlusion (MCAO) rat model and oxygen-glucose deprivation and reoxygenation (OGD/R) rat brain microvascular endothelial cell (RBMVEC) models were established. Brain infarction size and neurological score were calculated following MCAO surgery. Evans blue was used to measure blood brain barrier (BBB) permeability. Cell counting kit-8 (CCK-8) and TUNEL assays were performed to evaluate the cell viability and apoptosis of RBMVECs. Dual-luciferase reporter assay was used to analyze the transcriptional activities of apoptosis-related genes.
RESULTS: Results showed that higher infarction volume, neurological scores, and BBB permeability in the MCAO group rats were reduced after BHD treatment. Drug serum (DS) treatment had no impact on the normal RBMVECs\' cell viability and cell apoptosis. Besides, DS treatment decreased the lactate production, glucose uptake, and extracellular acidification rate in normal and OGD/R-induced RBMVECs. DS treatment downregulated the protein levels of pan-lysine lactylation (kla), histone H3 lysine 18 lactylation (H3K18la), and the transcriptional of apoptotic protease activating factor-1 (Apaf-1) in OGD/R-treated RBMVECs. In addition, Apaf-1 overexpression decreased cell viability and increased apoptosis and glycolysis activity of OGD/R-treated RBMVECs.
CONCLUSIONS: In summary, BHD inhibited glycolysis and apoptosis via suppressing the pan-kla and H3K18la protein levels and the Apaf-1 transcriptional activity, thus restraining the progression of IS.

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.

BACKGROUND: Notch1 signaling inhibiton with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester] (DAPT) treatment could promote brain recovery and the intervention effect is different between striatum (STR) and cortex (CTX), which might be accounted for different changes of glial activities, but the in-depth mechanism is still unknown. The purpose of this study was to identify whether DAPT could modulate microglial subtype shifts and astroglial-endfeet aquaporin-4 (AQP4) mediated waste solute drainage.
METHODS: Sprague-Dawley rats (n=10) were subjected to 90min of middle cerebral artery occlusion (MCAO) and were treated with DAPT (n=5) or act as control with no treatment (n=5). Two groups of rats underwent MRI scans at 24h and 4 week, and sacrificed at 4 week after stroke for immunofluorescence (IF).
RESULTS: Compared with control rats, MRI data showed structural recovery in ipsilateral STR but not CTX. And IF showed decreased pro-inflammatory M1 microglia and increased anti-inflammatory M2 microglia in striatal lesion core and peri-lesions of STR, CTX. Meanwhile, IF showed decreased AQP4 polarity in ischemic brain tissue, however, AQP4 polarity in striatal peri-lesions of DAPT treated rats was higher than that in control rats but shows no difference in cortical peri-lesions between control and treated rats.
CONCLUSIONS: The present study indicated that DAPT could promote protective microglia subtype shift and striatal astrocyte mediated waste solute drainage, that the later might be the major contributor of waste solute metabolism and one of the accounts for discrepant recovery of STR and CTX.

Ischemic stroke remains a major cause of disability and death. Kaempferol (Kae) is a neuroprotective flavonoid compound. Thus, this study aimed to explore the impact of Kae on cerebral infarction. We generated the middle cerebral artery occlusion (MCAO) mouse model to study the effects of Kae on infarction volume and neurological function. The oxygen and glucose deprivation (OGD)/reoxygenation (R) model of neural stem cells (NSCs) was established to study the effects of Kae on cell viability, migration, and apoptosis. Cell processes were assessed by cell counting kit-8, Transwell assay, flow cytometry, and TUNEL analysis. The molecular mechanism was assessed using the Western blot. The results indicated that Kae attenuated MCAO-induced cerebral infarction and neurological injury. Besides, Kae promoted cell viability and migration and inhibited apoptosis of OGD/R-treated NSCs. Moreover, OGD/R suppressed total O-GlcNAcylation level and O-GlcNAcylation of β-catenin, thereby suppressing the Wnt/β-catenin pathway, whereas Kae reversed the suppression. Inactivation of the Wnt/β-catenin pathway abrogated the biological functions of NSCs mediated by Kae. In conclusion, Kae suppressed cerebral infarction by facilitating NSC viability, migration, and inhibiting apoptosis. Mechanically, Kae promoted O-GlcNAcylation of β-catenin to activate the Wnt/β-catenin pathway. Kae may have a lessening effect on ischemic stroke.

Ischemic stroke is a sudden and acute disease characterized by neuronal death, increment of reactive gliosis (reactive microglia and astrocytes), and a severe inflammatory process. Neuroinflammation is an early event after cerebral ischemia, with microglia playing a leading role. Reactive microglia involve functional and morphological changes that drive a wide variety of phenotypes. In this context, deciphering the molecular mechanisms underlying such reactive microglial is essential to devise strategies to protect neurons and maintain certain brain functions affected by early neuroinflammation after ischemia. Here, we studied the role of mammalian target of rapamycin (mTOR) activity in the microglial response using a murine model of cerebral ischemia in the acute phase. We also determined the therapeutic relevance of the pharmacological administration of rapamycin, a mTOR inhibitor, before and after ischemic injury. Our data show that rapamycin, administered before or after brain ischemia induction, reduced the volume of brain damage and neuronal loss by attenuating the microglial response. Therefore, our findings indicate that the pharmacological inhibition of mTORC1 in the acute phase of ischemia may provide an alternative strategy to reduce neuronal damage through attenuation of the associated neuroinflammation.

Metabolic disorders are risk factors for stroke exacerbating subsequent complications. Rapidly after brain injury, a glial scar forms, preventing excessive inflammation and limiting axonal regeneration. Despite the growing interest in wound healing following brain injury, the formation of a glial scar in the context of metabolic disorders is poorly documented. In this study, we used db/db mice to investigate the impact of metabolic perturbations on brain repair mechanisms, with a focus on glial scarring. First, we confirmed the development of obesity, poor glucose regulation, hyperglycaemia and liver steatosis in these mice. Then, we observed that 3 days after a 30-min middle cerebral artery occlusion (MCAO), db/db mice had larger infarct area compared with their control counterparts. We next investigated reactive gliosis and glial scar formation in db/+ and db/db mice. We demonstrated that astrogliosis and microgliosis were exacerbated 3 days after stroke in db/db mice. Furthermore, we also showed that the synthesis of extracellular matrix (ECM) proteins (i.e., chondroitin sulphate proteoglycan, collagen IV and tenascin C) was increased in db/db mice. Consequently, we demonstrated for the first time that metabolic disorders impair reactive gliosis post-stroke and increase ECM deposition. Given that the damage size is known to influence glial scar, this study now raises the question of the direct impact of hyperglycaemia/obesity on reactive gliosis and glia scar. It paves the way to promote the development of new therapies targeting glial scar formation to improve functional recovery after stroke in the context of metabolic disorders.

OBJECTIVE: To distinguish between the genuine cellular impact of the ischemic cascade by leukocytes and unspecific effects of edema and humoral components, two knock-in mouse lines were utilized. Mouse lines Y731F and Y685F possess point mutations in VE-cadherin, which lead to a selective inhibition of transendothelial leukocyte migration or impaired vascular permeability.
METHODS: Ischemic stroke was induced by a model of middle cerebral artery occlusion. Analysis contained structural outcomes (infarct volume and extent of brain edema), functional outcomes (survival analysis, rotarod test, and neuroscore), and the extent and spatial distribution of leukocyte migration (heatmaps and fluorescence-activated cell sorting (FACS) analysis).
RESULTS: Inhibition of transendothelial leukocyte migration as in Y731F mice leads to smaller infarct volumes (52.33 ± 4719 vs. 70.43 ± 6483 mm3 , p = .0252) and improved motor skills (rotarod test: 85.52 ± 13.24 s vs. 43.06 ± 15.32 s, p = .0285). An impaired vascular permeability as in Y685F mice showed no effect on structural or functional outcomes. Both VE-cadherin mutations did not influence the total immune cell count or spatial distribution in ischemic brain parenchyma.
CONCLUSIONS: Selective inhibition of transendothelial leukocyte migration by VE-cadherin mutation after ischemic stroke in a mouse model leads to smaller infarct volumes and improved motor skills.

OBJECTIVE: Ischemic stroke accounts for 87% of all strokes, and its death and disability bring a huge burden to society. Brain injury caused by ischemia-reperfusion (I/R) is also a major difficulty in clinical treatment and prognosis. Sophoricoside (SOP) is an isoflavone glycoside isolated from the seed of medical herb Sophora japonica L. Previously, SOP was found to be effective in anti-inflammation and glucose-lipid metabolism-related diseases. In order to investigate whether SOP has a regulatory effect on cerebral I/R injury, we conducted this study.
METHODS: Here, by application of SOP into MCAO (transient middle cerebral artery occlusion)-induced mice and OGD/R (oxygen glucose deprivation/reperfusion)-induced primary neurons, the regulation effects of SOP was analyzed by detecting neurological score of post-stroke mice, phenotypes of brains and brain sections, cell viabilities, and apoptosis- and inflammation-regulation. RNA sequencing and molecular biology experiments were performed to explore the mechanism of SOP regulating cerebral I/R injury.
RESULTS: SOP administration decreased the infarct size, neurological deficit score, neuronal cell injury, inflammation and apoptosis. Mechanistically, SOP exerted its protective effect by activating the AMP-activated protein kinase (AMPK) signaling pathway.
CONCLUSIONS: SOP inhibits cerebral I/R injury by promoting the phosphorylation of AMPK.