Abstract Body: Introduction: Peripheral blood-based biomarker development for neurological diseases such as ischemic stroke has traditionally focused on measuring alterations in leukocyte number and composition. Deeper immunophenotyping of cells using fluorescent probes that detect alterations among organelles and structures can provide a better understanding of the biological function of leukocytes after ischemic stroke.
Methods: We evaluated the utility of BODIPY 493/503 and LysoTracker as fluorogenic probes to assess cellular changes in lipid content and lysosomal burden. Flow cytometric assessments were performed on peripheral mononuclear blood cells from healthy volunteers (HV, N=31), and from ischemic stroke patients (N=43) at 3d and 7d to capture the temporal profile of these biomarkers.
Results: We found decreased frequency of CD3+ T cells 3d after stroke, which was also seen at 7d after stroke. We found CD4:CD8 ratios were significantly and persistently increased in stroke patients compared to HV controls. Within the CD4 subset, we found a persistent increase in the frequency of effector CD4 T cells after stroke, this finding is consistent with previous literature. Interestingly, we noted a delayed, but profound increase in lipid content of all cell types at 7d after stroke compared controls. This difference was more pronounced among females. Lysosomal content was differentially altered by cell type, with increased levels in CD4 naïve T cells, but decreased levels in CD4 central memory, CD8 naïve, CD8 effector, CD8 central and effector memory T cells after stroke. While we did not see an association with increase in severity at admission, high lipid content at the acute phase was significantly associated with increased disability at 3 months after hospitalization.
Conclusion: Taken together, our findings provide greater context to the previously documented compositional changes in the blood leukocyte compartment after stroke. Ischemic stroke causes a pervasive increase in cellular lipidosis. Lysosomal content has also been significantly positively correlated to functional disability among stroke patients. Using cell-based functional assays with flow cytometric immunophenotyping may provide meaningful insights into biological health and function of circulating leukocytes after brain injury. Understanding the effects of these compositional changes seen in the acute phase in respect to functional outcomes should be a priority for researchers moving forward.