Abstract Body: Introduction:
The optimal therapeutic window for evacuation of intracerebral hemorrhage (ICH) is controversial, balancing risk of prolonged exposure to blood-induced neurotoxicity against risk of post-operative rebleeding. Pharmacologic mitigators of neurotoxicity have the potential to lengthen this therapeutic window, extending the opportunity for favorable surgical outcomes. Here, we identify a blood-induced transcriptomic signature in an organoid model of ICH and perform an in silico screen to identify reversers of blood-induced toxicity.

Methods:
Single-cell RNA sequencing was performed on 96,725 cells across 18 human neural organoids comprising multiple cell types, treated with 5% blood for either 6 or 24 hours. Alignment, filtering, and analysis was performed using cellranger, SoupX, Scrublet, and Seurat. Gene associations to blood exposure were conducted using mixed-effect linear modeling. Significant expression signatures were determined using Benjamini-Hochberg multiple testing correction (FDR <5%). To identify compounds that may reverse the blood-exposure signature, we applied the Broad Institute Connectivity Map query tool to calculate a weighted enrichment score between the blood-induced signature and curated expression signatures of known therapeutic compounds. Compounds with a significant enrichment score FDR<0.05 were considered “reversers”.

Results:
Single-cell sequencing identified distinct transcriptomic impacts of 24-hours of blood exposure, including astrocyte reactivity (TGFb response, p=2.41x10^-3), endothelial vasculogenesis (p=8.73x10^-9), and neurotoxicity (glutamate receptor signaling, p= 1.12x10^-3). To identify therapeutics with potential to reverse this signature, we performed an in silico drug screen, identifying 345 compounds targeting 119 unique mechanisms of action. Ten classes of drugs reversed blood-induced signatures in all three cell types, including adrenergic receptor antagonists, dopamine receptor antagonists, histamine receptor antagonists, acetylcholinesterase inhibitors, calcium channel blockers, phosphodiesterase inhibitors, HDAC inhibitors, MAPK inhibitors, and antibiotics.

Conclusion:
The neurotoxic effects of blood may be mitigated by a number of compounds targeting convergent mechanisms of toxicity across cell types. These candidates may represent novel therapeutic compounds to widen the window for safe and effective ICH evacuation and are ideal candidates for screening in scalable in vitro organoid models of ICH.