USING A SPATIAL RNA-PROTEIN CO-DETECTION ASSAY TO INVESTIGATE POST-MORTEM MICROVASCULAR CHANGES IN FORMER AMERICAN FOOTBALL PLAYERS

Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy associated with repetitive head impacts (RHI), most commonly observed in contact sport athletes. It is characterized by neurofibrillary tangle accumulation in neurons and glia surrounding small blood vessels at the depths of neocortical sulci, with pathology that may extend to deeper brain regions over time. Because CTE can only be definitively diagnosed postmortem, improved understanding of cellular and molecular changes in affected brain tissue is critical for biomarker discovery and therapeutic development.
Here, we apply single-cell spatial RNAscope technology to characterize trauma-related molecular and cellular changes in human postmortem dorsolateral frontal cortex. Using a novel RNA–protein co-detection assay, we simultaneously visualized gene and protein expression to investigate microvascular alterations associated with RHI. Hypoxic cells were identified using an RNA probe targeting HIF1A, while vascular basement membrane components were assessed using a COL4A1 RNA probe. Immunohistochemical markers were used to identify microglia (IBA1), astrocytes (GFAP), endothelial cells (CD31), and pathological tau (AT8).
Quantitative image analysis was performed using HALO software. Co-expression analysis of hypoxia markers with cell-type-specific markers in RHI-exposed and unexposed samples revealed trauma-associated alterations in brain microvasculature, including cell-specific hypoxic responses and basement membrane changes.
These results demonstrate that single-cell spatial RNAscope combined with RNA–protein co-detection provides a powerful multimodal approach for interrogating complex neuropathological tissue and identifying potential prognostic and therapeutic biomarkers in CTE.