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Updated: Jan 12, 2026
Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke
Published on: January 3, 2025
The role of ROCK1/MLC/NMMHC IIA-actin signaling in ischemic stroke-induced blood-brain barrier disruption:
Liangying Bao1, Yuanhao Xu1, Yuchuan Ren1
1State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Pharmacology of Chinese Materia Medica, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People's Republic of China.
Background:
Disruption of the blood-brain barrier (BBB) is a key event in the onset of ischemic stroke (IS), primarily driven by endothelial cytoskeletal rearrangement. The interaction between non-muscle myosin heavy chain IIA (NMMHC IIA) and actin, along with the ROCK/MLC pathway, is central to this cytoskeletal reorganization. While our previous studies have shown that the Caspase-3/ROCK1/MLC/NMMHC IIA-actin positive feedback loop mediates H2O2-induced neuronal apoptosis, its role in cerebral ischemia-reperfusion (I/R) injury and BBB disruption remains unclear.
Methods:
In vivo, we used endothelial-specific NMMHC IIA conditional knockdown mice, NMMHC IIA-inducible endothelial conditional knock-in mice and C57BL/6J to establish a middle cerebral artery occlusion/reperfusion model. In vitro, we employed brain microvascular endothelial cells in an oxygen-glucose deprivation/reoxygenation model. The effects of the NMMHC IIA inhibitor blebbistatin, the ROCK1 inhibitor Y-27632, and the actin depolymerizer cytochalasin D were assessed for their impact on I/R-induced activation of the ROCK/MLC/NMMHC IIA-actin pathway, tight junction proteins (TJs) degradation, and brain damage.
Results:
Inhibition of NMMHC IIA expression and stress fiber depolymerization significantly reduced NMMHC IIA-actin interactions, suppressed the ROCK/MLC pathway, decreased TJs degradation, and alleviated cerebral I/R injury. Conversely, overexpression of NMMHC IIA further exacerbated cerebral I/R injury and BBB disruption and amplified activation of the ROCK1/MLC pathway. Y-27632 inhibited the ROCK/MLC/NMMHC IIA-actin pathway, mitigating I/R-induced BBB disruption.
Conclusions:
This study reveals that the ROCK1/MLC/NMMHC IIA-actin pathway is implicated in I/R-induced BBB disruption and operates as a positive feedback loop. These findings offer a promising therapeutic strategy for the treatment of IS and BBB damage.
