stroke-related-animal-models-methodologies-applications-translational

Stroke-Related Animal Models: Methodologies, Applications, and Translational Perspectives

Stroke remains a leading cause of death and disability worldwide. Despite hundreds of neuroprotective agents showing promise in preclinical studies, ...

The Second Affiliated Hospital of Kunming Medical University

This study investigated the role of integrin-linked kinase (ILK) in neuronal apoptosis induced by cerebral ischemia‒reperfusion injury (CIRI) and its interaction with a circRNA (0000964) and miR-758-3p. Using in vivo and in vitro rat models, we clarified how ILK regulates neuronal apoptosis during CIRI. Our findings revealed that ILK expression is upregulated in response to CIRI and is modulated by the circRNA (0000964)/miR-758-3p axis. This study provides new insights into the molecular mechanisms of CIRI and suggests potential therapeutic targets to reduce neuronal apoptosis. A CIRI rat model was created through middle cerebral artery occlusion (MCAO). After miR-758-3p overexpression, neurological deficits, CIRI volume, and the expression levels of circRNAs (0000964) and ILK were evaluated. Neurons were subjected to oxygen‒glucose deprivation (OGD) to simulate in vitro CIRI, and the same molecules were analyzed. MCAO-induced CIRI downregulated a circRNA (0000964) and upregulated ILK and miR-758-3p. Similarly, in vitro OGD-induced apoptosis downregulated a circRNA (0000964) and upregulated ILK and miR-758-3p. Further analysis confirmed that a circRNA (0000964) negatively regulates miR-758-3p, which in turn negatively regulates ILK. This axis controls ILK and Caspase-3 expression, influencing neuronal apoptosis. ILK has been identified as a key regulator of neuronal apoptosis in CIRI. The circRNA (0000964)/miR-758-3p axis modulates ILK, impacting neuronal survival. This molecular network offers new insights into CIRI pathophysiology and highlights possible therapeutic approaches.

miR-758-3p Interferes with Neuronal Apoptosis in Cerebral Ischemia-Reperfusion by Inhibiting ILK.

Ischemic stroke (IS) is a severe disease. The altered activation states of microglia play important roles in IS. In present study, a total of 125 C57BL/6 mice was used (N = 6 per group). Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD) were performed for in vivo and in vitro model construction. The infarct size was detected using TTC staining. The nerve injury was evaluated by a neurological deficit score. OGD-treated brain microvascular endothelial cells (BMECs) were co-cultured with BV2 cells. Cell viability was determined by CCK-8 assay, and the apoptosis rate was identified by flow cytometry analysis. Transendothelial electronic resistance (TEER) of the cells was measured by TEER measurement. Molecular interactions were analyzed using dual-luciferase reporter gene, ChIP, and Co-IP assays. All in vitro experiments were conducted with three replicates, and each experiment was performed in triplicate. We found that Src Homology 2B Adaptor Protein 3 (SH2B3) was overexpressed in the cerebral cortex tissues of MCAO treated mice (P < 0.01), and BMECs co-cultured with BV-2 cells under OGD conditions (P < 0.01). SH2B3 knockdown or Myocyte Enhancer Factor 2 A (MEF2A) overexpression reduced infarct size and improved neurological function in MCAO mice. SH2B3 knockdown enhanced OGD-treated cell viability (P < 0.05), inhibited cell apoptosis (P < 0.05) in BMECs, and ameliorated BBB (P < 0.01). Moreover, SH2B3 knockdown changed the activation status of microglia. MEF2A promoted the transcriptional activation of WW Domain Containing E3 Ubiquitin Protein Ligase 2 (WWP2) and WWP2 promoted the ubiquitination and degradation of SH2B3. SH2B3 overexpression reversed the effects of MEF2A overexpression on microglia states, BMECs injury and BBB function. In summary, MEF2A promoted the ubiquitination-mediated degradation of SH2B3 via transcription up-regulating WWP2, then changed the activation status of microglia, thus ameliorating BMEC injury, and finally ameliorating IS injury.

The Ubiquitination and Degradation of SH2B3 Mediated by MEF2A/WWP2 Axis Restores Microglial Homeostasis to Alleviate Cerebral Microvascular Endothelial Cell Injury in Ischemic Stroke.

Microglia/macrophage polarization is a crucial factor in inflammatory processes following ischemic stroke (IS). This study explores the molecular mechanisms through which lysine-specific histone demethylase 4 (KDM4A) regulates microglial polarization postischemic stroke. IS models are established  via transient middle cerebral artery occlusion (MCAO) surgery and  via oxygen-glucose deprivation (OGD) treatment. 2,3,5-Triphenyl tetrazolium chloride staining is conducted to determine the infarct size. RT-qPCR is used to determine mRNA expression. Immunofluorescence assay is used to detect the expressions of KDM4A and biomarkers of microglia. Western blot analysis is used to determine the expressions of KDM4A and serine peptidase inhibitor Kazal type 5 (SPINK5). The enrichment of H3K9me3 on the promoter of  is determined via chromatin immunoprecipitation assay. Neuronal apoptosis is detected via TUNEL assay. We find that KDM4A is upregulated in IS models. Downregulation of KDM4A mitigates neurological dysfunction, enhances motor capacity, and reduces inflammatory infiltration  while suppressing microglial activation and promoting M2 polarization. Mechanistically, KDM4A reduces the enrichment of H3K9me3 on the  promoter, thereby increasing SPINK5 expression. Moreover, overexpression of SPINK5 inhibits M2 microglial polarization and neuronal apoptosis. Overall, KDM4A exacerbates ischemic stroke-induced brain injury by promoting proinflammatory microglial polarization via SPINK5 signaling.

Article	Year
miR-758-3p Interferes with Neuronal Apoptosis in Cerebral Ischemia-Reperfusion by Inhibiting ILK. Molecular neurobiology\| PubMed ID: 39937418	2025
The Ubiquitination and Degradation of SH2B3 Mediated by MEF2A/WWP2 Axis Restores Microglial Homeostasis to Alleviate Cerebral Microvascular Endothelial Cell Injury in Ischemic Stroke. Neurochemical research\| PubMed ID: 40407948	2025
KDM4A regulates microglial polarization after ischemic stroke by regulating SPINK5 signaling. Acta biochimica et biophysica Sinica\| PubMed ID: 40999902	2025

Lei Xian

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