Abstract Body: Background
Acute hyperglycemia is common post ischemic stroke, and is a robust predictor of poor stroke outcome. Even transient glucose elevation substantially increases the risk of mortality and long-term disability. Tight glucose control with insulin is effective in correcting hyperglycemia, but lacks efficacy in improving patient outcome, suggesting the presence of early hyperglycemic injuries that are resistant to later glucose control. Our recent clinical research revealed that abnormal red blood cells (RBCs) could be a novel mediator of the adverse effect of acute hyperglycemia on stroke outcome. Here, using in vitro and ex vivo models, we aim the explore the underlying mechanism.

Method
RBCs were isolated from healthy C57BL6 mice (10~12 wk) and exposed to in vitro normal glucose (NG: 5 mM) or high glucose (HG: 15 mM) for 24 hr. HG-exposed RBCs were also treated with ROS scavenger TEMPOL (HG+TEMPOL). The treated RBCs were then incubated with mouse brain blood vessels for additional 24 hr. Changes in vascular gene expression were profiled via RNA sequencing.

Result
HG exposure increased reactive oxygen species (ROS) production within RBCs, which was mitigated by TEMPOL (Figure 1A). Importantly, HG-challenged RBCs (HG-RBCs) led to a significant shift in brain vascular gene expression and function (Figure 1B-D). Specifically, the expression of endothelial nitric oxide synthase (eNOS), the enzyme responsible nitric oxide (NO) production, was decreased by HG-RBCs treatment, potentially reducing NO availability and compromising cerebral blood flow (Figure 2A). These changes were further validated by immunohistochemistry (Figure 2B, 2C). Moreover, RBCs affected vascular pathways linked to Alzheimer’s disease (Figure 1D). HG-RBCs suppressed Adam10 (α-secretase)-mediated non-amyloidogenic process and activated Bace1 (β-secretase)-mediated amyloidogenic process, potentially contributing to β-amyloid accumulation (Figure 2D, 2E). Most of these detrimental effects could be reversed by TEMPOL pretreatment (Figure 2A-E), and the overall vascular gene expression pattern was closer to those treated by NG-RBCs (Figure 1B, 1C), suggesting that hyperglycemia-induced RBC oxidative stress play a causal role in brain vascular dysfunction.

Conclusion
RBCs, beyond their oxygen transport role, are crucial regulators of vascular function and could be a previously overlooked contributor to vascular dysfunction in acute hyperglycemia post stroke (Figure 3).