Introduction Cerebral thrombi, which cause acute ischemic stroke, can arise in various environments such as the rapid flow of atherosclerotic conditions, venous stasis, and the turbulent flow caused by atrial fibrillation. In clots retrieved through thrombectomy, a significant number of immune cells such as macrophages and neutrophils are observed. Both the pathology of the clot obtained from patients and the etiology of clot formation are complex. The evaluation of clot pathology by Martius, Scarlet, and Blue (MSB) staining does not necessarily correlate with the cause, and it doesn’t allow for a detailed view of the components that are classified as white blood cells. In this study, we investigated how thrombi formed in various flow environments (FE) affect the immune cells within them, using single-cell RNA-seq, and flow cytometry.

Methods Clots were generated in vitro via a modified Chandler-Loop design at 37°C at static condition, at 10rotations per minute (RPM, low FE) or at 40RPM (high FE) for 1 hour. Histological analysis was performed using martius scarlet blue (MSB) staining, and cellular quantification was conducted via Orbit Image Software. Single-cell RNA-seq using the 10X Genomics platform was performed to determine detailed cellular content, with a focus on WBC identity of clots generated at variable FEs. Top 3000 highly variable features were used for principal component analysis (PCA). Cells were clustered based on their PCA scores, utilizing the first 30 PCs.

Results MSB stained samples demonstrate increasing fibrin content with higher FE clots (Static 1%, 10RPM 8%, 40RPM 25%). Orbit image quantification shows higher erythrocyte content in static vs. high FE clots (95% vs. 56%), but leukocytes (WBCs) and platelets (PTLs) are more enriched in high flow clots than static clots (4% vs. 0.1% WBC, and 16% vs. 5% PLTs, respectively). Uniform Manifold Approximation and Projection (UMAP) plots were applied to single-cell RNA-seq of variable FE clots to visualize 20 distinct clusters, and expression patterns were annotated using curated data from PanglaoDB, and the Human Protein Atlas to identify cluster cellular identities. T cells are higher in clots formed at static or low FEs, by gene expression cell identification, however, the number of neutrophils, monocytes, and APCs increase in clots formed at high FEs. This observation with enrichment of myeloid cells in the high flow vs. static clots was partially validated via multiplex flow (77% vs 96%), and further studies are underway. Furthermore, our differential expression analysis of myeloid cells generated under variable FEs, has revealed unique cellular, and potentially targetable, differentially regulated signaling pathways at various FEs.

Conclusions Our studies demonstrate that FEs drive variable cellular composition of clots and alter cellular gene expression. Understanding these differences can identify novel therapeutic strategies in stroke associated with specific stroke risk conditions. Our future directions include further validation of RNA-Seq findings, via repeat multiplex flow cytometry, extrapolation of our findings via flow cytometry and/or immunofluorescence staining of fresh human clots, and functional evaluation of clot burden by targeting cell types and/or differentially expressed signaling pathways most upregulated at various FEs.

Disclosures Y. Ghochani: None. R. Kawaguchi: None. M. Ghovvati: None. T. Imahori: None. K. Sakuta: None. J. Hinman: None. N. Kaneko: None.