Secondary brain injury occurs four to 14 days after a subarachnoid hemorrhage (SAH). This delayed onset potentially allows for disease modulation and outcome improvement in patients. To enhance our understanding of the pathophysiology of secondary brain injury, we characterized the cellular immune response by longitudinal RNA-single-cell sequencing. We mapped cellular phenotypes to clinical endpoints.
Patients who were 18 years or older at the time of hospital admission and who had a confirmed aneurysmal subarachnoid hemorrhage and a cerebrospinal fluid (CSF) diversion were included in the study. CSF samples of patients were collected at three different time points and subjected to a leukocyte isolation protocol. Timepoint one was defined as day three to five post-SAH, timepoint two as day six to eight post-SAH, and timepoint three as day nine to eleven post-SAH. The obtained cells were processed using the 10x Genomics Chromium Single Cell 3" v3.1 Reagent Kit instruction guide (10x Genomics). Further, the 3ʹ Gene Expression libraries for each sample were pooled at an equimolar amount and sequenced on an Illumina NovaSeq 6000 system with a sequencing depth of 50000 reads per cell. (workflow depicted in Fig.1) The computational analysis was performed with R (R version 4.2.1) using the Seurat package (version 4.3.0).
To our knowledge, we have created the biggest RNA-single-cell data set on CSF-derived leukocytes.
We identified all major leukocyte subsets like T-cells, B cells, myeloid dendritic cells, monocytes, and neutrophils within the CSF of our aSAH cohort (celltypes depicted in Fig.2).
Outcome variables stratified patients according to the presence/absence of secondary brain injury and the presence/absence of a ventriculoperitoneal shunt three months after the initial bleeding. We identified significantly differentially expressed genes between either of the groups using a negative binomial model.
Characterizing the cellular immune response after aneurysmal subarachnoid hemorrhage is critical to understanding the pathophysiological course of the disease. Cell composition and cellular phenotypes within the CSF allow us to conclude about potentially modifiable adaptive processes. Further, this work enhances our knowledge about protective immune responses and those promoting SAH-SBI and shunt dependency.