Super-resolution localization microscopy visualizes ischemia-related cytoskeletal and vascular alterations at the...

Ischemic stroke results in acute and long-lasting brain damage with consecutive neuronal death. Advancements in visualizing ischemia-related structural alterations at the cellular level are crucial for understanding pathophysiological processes in more detail. Considering the already established concept of the neurovascular unit, both neuronal components and the vasculature are of particular interest. This study combined conventional fluorescence and super-resolution localization microscopy, specifically direct stochastic optical reconstruction microscopy (dSTORM), to visualize cell-stabilizing neuronal and vascular elements in the setting of stroke. For this approach, neurofilament light chain (NFL), microtubule-associated protein 2 (MAP2), and collagen IV (CollIV) were detected in non-altered and ischemic brain regions of mice following 24 h of focal cerebral ischemia. Super-resolution localization microscopy enabled the visualization of the cytoskeletal elements NFL and MAP2 as filamentous structures with varying diameters and CollIV associated with the vessel wall. Due to ischemia, gradually enlarged filament diameters and higher molecular densities were observed for NFL, while the number of localizations was largely reduced for MAP2 and gradually increased for CollIV. Although these observations do not allow for conclusions regarding the functional characteristics of targeted structures, ischemia-related structural alterations at the nanoscale, i.e., the molecular level, became visible by dSTORM. Super-resolution localization microscopy thus appears to be a valuable method for investigating the ischemic consequences on cytoskeletal elements and the vasculature. The emerging insights could help elucidate the mechanisms underlying stroke-related tissue damage in more detail and identify novel neuroprotective targets.