Abstract 3822: Neutrophils and Toll-like receptor 4 Contribute to Injury after Intracerebral Hemorhage

Abstract

Background: Inflammation contributes to injury after intracerebral hemorrhage (ICH). We have previously demonstrated that activation of Toll-like receptor 4 (TLR4) on cells within the hemorrhage is a key initiator of the inflammatory response. TLR4-deficient mice had reduced infiltrating neutrophils, monocytes, inflammatory monocytes, and microglia. TLR4-deficient mice given an ICH comprised of blood from a WT donor had increased recruitment of these cell populations. Our current objective was to determine whether intrahematomal neutrophils contribute to the TLR4-dependent inflammatory response. Methods: WT (C3H/HeOuJ) mice were depleted using a neutrophil-selective antibody (anti-Ly6G, clone 1A8, 5 mg/kg) or control rat IgG the day prior to being used as blood donors for ICH in TLR4-deficient (C3H/HeJ) mice. ICH was created by stereotaxic injection of 15 μ L blood from a neutrophil-depleted or control donor into the right striatum. Mice were tested on the cylinder test for 3 days and then sacrificed for analysis of leukocyte populations in the brain by flow cytometry. Brains were mechanically and enzymatically digested and then cells harvested from a 30%-60% Percoll gradient. Cells were then stained with fluorophore-conjugated antibodies to CD45, CD3, CD11b, CD11c, Ly6G, and Gr1 and counted on a LSRII cytometer. Cells were identified as microglia (CD45lowCD11b+), leukocytes (CD45hi), neutrophils (CD45hiCD11b+Ly6G+), monocytes (CD45hiCD11b+Ly6G-CD11b-Gr1-), inflammatory monocytes (CD45hiCD11b+Ly6G-CD11b-Gr1+) or dendritic cells (CD45hiCD11b+CD11c+Ly6G-). Results: TLR4-deficient mice with a WT, neutrophil-depleted ICH showed reduced numbers of leukocytes (p<0.05), dendritic cells (p<0.01), and microglia (p<0.05) compared to the TLR4-deficient mice with a WT, control ICH. There was no difference in neutrophils, monocytes, or inflammatory monocytes. The TLR4-deficient mice with a neutrophil-depleted ICH had better functional outcomes throughout the first 3 days after ICH (p<0.05 each day). Conclusions: Neutrophils within the ICH contribute to increases in microglia and dendritic cells as well as the neurological deficit in the TLR4-deficient host. However, the specific populations of leukocytes reduced after neutrophil depletion did not mirror the findings in TLR4-deficient mice given an ICH using autologous blood, which had reduced neutrophils, monocytes, and inflammatory monocytes. This suggests that intrahematomal neutrophils have a role in initiating TLR4 signaling but other cells contribute to the recruitment of monocyte and neutrophil populations.