Abstract 3867: Robust Infiltration of Inflammatory Monocytes 12 Hours After Experimental Intracerebral Hemorrhage
Background and Purpose: Inflammation is a key component of injury in the initial hours following intracerebral hemorrhage (ICH). Neutrophils are thought to be the earliest cells infiltrating into brain, but traditional methods to identify leukocytes are unable to differentiate between infiltrating monocyte populations and resident microglia. We used flow cytometry on brains 12 hours after ICH to quantify the specific infiltrating leukocyte populations. This is an early timepoint when neurobehavioral deficit is maximal and treatment could realistically be initiated.
Methods: ICH was modeled by injecting 15μ l of blood from a C57BL/6 mouse (carrying leukocyte marker CD45.2) into the right striatum of a B6Ly5.2 mouse (which expresses CD45.1). The different CD45 isoforms allowed infiltrating leukocytes from the recipient mouse to be distinguished from the blood that was injected to model the ICH. Brains were harvested 12 hours later, dissociated mechanically and enzymatically, and cells collected from the interphase of a 30%/70% percoll gradient. Cells were then stained for CD45.1, CD45.2, CD3, CD11b, CD11c, Ly6G, and GR-1 and analyzed on a BD LSRII flow cytometer. Blood-derived leukocytes were identified as CD45.1hi, neutrophils as CD45.1hiCD11b+Ly6G+, inflammatory monocytes as CD45.1hiCD11b+Ly6G-CD11c-Gr1+, monocytes as CD45.1hiCD11b+Ly6G-CD11c-Gr1-, and dendritic cells as CD45.1hiCD11b+Ly6G-CD11c+. Mean cell counts in the ipsilateral hemisphere were compared to the contralateral hemisphere by t-test.
Results: At 12 hours after ICH, we observed a significant increase in blood-derived leukocytes within the ipsilateral hemisphere (p<0.01, see figure). Less than 4% of total leukocytes were CD45.2. Surprisingly, we noticed a robust inflammatory monocyte population in the ipsilateral hemisphere (p<0.05, figure). Significant changes were not observed in monocyte, dendritic, or T cell populations, although there was a trend towards an increase in neutrophils (p=.055, figure).
Conclusions: Using a CD45.1/CD45.2 blood transfer model, we determined that the cells injected into the striatum as part of the modeled hemorrhage are no longer detectable 12 hours later. Infiltrating inflammatory monocytes outnumbered neutrophils at 12 hours. This challenges conventional thought regarding the time course of invading immune cell populations following ICH, and suggests that future studies are needed to determine the contribution of inflammatory monocytes to early injury after ICH.
- © 2012 by American Heart Association, Inc.