Abstract Body: Background
Cardiac arrest (CA) is a life-threatening medical emergency, with most victims being elderly. Despite considerable advances in resuscitation, the rates of survival-to-discharge remain discouraging. Moreover, out-of-hospital CA has one of the highest rates of disability-adjusted life years. The high incidence of post-CA mortality and morbidity has been attributed primarily to brain injury. However, our understanding of the pathophysiology of post-CA brain injury is limited. One critical, yet poorly investigated, event following CA is the immune response in the brain. This study aims to dissect the immune cell populations and their potential roles in the post-CA brain using both young and aged mice.

Methods
Young (3-6 months old) and aged (19-22 months old) mice underwent sham or KCl-induced CA followed by cardiopulmonary resuscitation. On day 3 post-CA, CD45⁺ immune cells were isolated from the brain by fluorescence-activated cell sorting (FACS) and subjected to single-cell RNA sequencing (scRNA-seq) analysis. Flow cytometry, quantitative PCR, and cytokine array were used to further analyze the immune response in the brain after CA.

Results
Our unbiased scRNA-seq analysis of CD45⁺ cells revealed 15 transcriptionally distinct clusters (Figure). As expected, most clusters comprised microglia, which were further subdivided into 9 different subsets. Additionally, we identified T cells, B cells, monocytes/macrophages, neutrophils, and border-associated macrophages. Consistent with previous studies, aged brains in the sham group exhibited an inflammatory state, evidenced by a substantial presence of lymphocytes, elevated cytokine/chemokine levels, and a higher number of activated microglia. Interestingly, after CA, infiltrating immune cells were less abundant in the aged brain, and aged microglia showed an attenuated transcriptomic response to CA, suggesting impaired immune function with aging. Notably, there were fewer proliferative microglia but more pro-inflammatory microglia in the aged vs young brain after CA.

Conclusions
This is the first scRNA-seq study of immune cells in the post-CA brain using both young and aged mice. Our findings provide novel insights into the immune landscape in the post-CA brain, highlighting age-related differences in immune responses. A better understanding of these differences may inform the development of new post-CA immunomodulatory therapies to improve outcomes in young and elderly patients.