Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia‐enhanced hemorrhagic transformation after stroke

Abstract

Aims Pre‐existing hyperglycemia (HG) aggravates the breakdown of blood–brain barrier (BBB) and increases the risk of hemorrhagic transformation (HT) after acute ischemic stroke in both animal models and patients. To date, HG‐induced ultrastructural changes of brain microvascular endothelial cells (BMECs) and the mechanisms underlying HG‐enhanced HT after ischemic stroke are poorly understood. Methods We used a mouse model of mild brain ischemia/reperfusion to investigate HG‐induced ultrastructural changes of BMECs that contribute to the impairment of BBB integrity after stroke. Adult male mice received systemic glucose administration 15 min before middle cerebral artery occlusion (MCAO) for 20 min. Ultrastructural characteristics of BMECs were evaluated using two‐dimensional and three‐dimensional electron microscopy and quantitatively analyzed. Results Mice with acute HG had exacerbated BBB disruption and larger brain infarcts compared to mice with normoglycemia (NG) after MCAO and 4 h of reperfusion, as assessed by brain extravasation of the Evans blue dye and microtubule‐associated protein 2 immunostaining. Electron microscopy further revealed that HG mice had more endothelial vesicles in the striatal neurovascular unit than NG mice, which may account for their deterioration of BBB impairment. In contrast with enhanced endothelial transcytosis, paracellular tight junction ultrastructure was not disrupted after this mild ischemia/reperfusion insult or altered upon HG. Consistent with the observed increase of endothelial vesicles, transcytosis‐related proteins caveolin‐1, clathrin, and hypoxia‐inducible factor (HIF)‐1α were upregulated by HG after MCAO and reperfusion. Conclusion Our study provides solid structural evidence to understand the role of endothelial transcytosis in HG‐elicited BBB hyperpermeability. Enhanced transcytosis occurs prior to the physical breakdown of BMECs and is a promising therapeutic target to preserve BBB integrity.

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“…Mice were anesthetized with 5% isoflurane (R510-22-8, RWD) using a face mask and continuously inhaled with a pipeline maintenance respirator (BD21 4LZ, Medical supplies & Services) during the surgery. tMCAO was induced as previously described ( Xu et al., 2021 ). A 6-0 monofilament with a silicon-coated tip (6023PK5Re, Doccol Corporation) was introduced to the right external carotid artery and advanced along the internal carotid artery to occlude the MCA.…”

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“…Mice were anesthetized with iso urane. Transient MCAO was performed as described previously [32]. A midline ventral cervical skin incision was made to expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA).…”