Abstract: Reperfusion therapy is the preferred treatment for ischemic stroke, but is hindered by its short treatment window, especially in patients with diabetes whose reperfusion after prolonged ischemia is often accompanied by exacerbated hemorrhage. The mechanisms underlying exacerbated hemorrhage are not fully understood. This study aimed to identify this mechanism by inducing prolonged 2-hour transient intraluminal middle cerebral artery occlusion in diabetic Ins2Akita/+ mice to mimic patients with diabetes undergoing delayed mechanical thrombectomy. The results showed that at as early as 2 hours after reperfusion, Ins2Akita/+ mice exhibited rapid development of neurological deficits, increased infarct and hemorrhagic transformation, together with exacerbated down-regulation of tight-junction protein ZO-1 and up-regulation of blood-brain barrier-disrupting matrix metallopeptidase 2 and matrix metallopeptidase 9 when compared with normoglycemic Ins2+/+ mice. This indicated that diabetes led to the rapid compromise of vessel integrity immediately after reperfusion, and consequently earlier death and further aggravation of hemorrhagic transformation 22 hours after reperfusion. This observation was associated with earlier and stronger up-regulation of pro-angiogenic vascular endothelial growth factor (VEGF) and its downstream phospho-Erk1/2 at 2 hours after reperfusion, which was suggestive of premature angiogenesis induced by early VEGF up-regulation, resulting in rapid vessel disintegration in diabetic stroke. Endoplasmic reticulum stress-related pro-apoptotic C/EBP homologous protein was overexpressed in challenged Ins2Akita/+ mice, which suggests that the exacerbated VEGF up-regulation may be caused by overwhelming endoplasmic reticulum stress under diabetic conditions. In conclusion, the results mimicked complications in patients with diabetes undergoing delayed mechanical thrombectomy, and diabetes-induced accelerated VEGF up-regulation is likely to underlie exacerbated hemorrhagic transformation. Thus, suppression of the VEGF pathway could be a potential approach to allow reperfusion therapy in patients with diabetic stroke beyond the current treatment window. Experiments were approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong [CULATR 3834-15 (approval date January 5, 2016); 3977-16 (approval date April 13, 2016); and 4666-18 (approval date March 29, 2018)].

Key words: blood-brain barrier, brain injury, diabetes mellitus, hemorrhagic transformation, infarct, ischemia/reperfusion injury, middle cerebral artery occlusion, mouse model, stroke, vascular endothelial growth factor