Intracerebral hemorrhage leads to infiltration of several leukocyte populations with concomitant pathophysiological changes

Abstract

Intracerebral hemorrhage (ICH) is a stroke subtype with high rates of mortality and morbidity. The immune system, particularly complement and cytokine signaling, has been implicated in brain injury after ICH. However, the cellular immunology associated with ICH has been understudied. In this report, we use flow cytometry to quantitatively profile immune cell populations that infiltrate the brain 1 and 4 days post-ICH. At 1 day CD45(hi) GR-1(+) cells were increased 2.0-fold compared with saline controls (P<or=0.05); however, we did not observe changes in any other cell populations analyzed. At 4 days ICH mice presented with a 2.4-fold increase in CD45(hi) cells, a 1.9-fold increase in CD45(hi) GR-1(-) cells, a 3.4-fold increase in CD45(hi) GR-1(+) cells, and most notably, a 1.7-fold increase in CD4(+) cells (P<or=0.05 for all groups), compared with control mice. We did not observe changes in the numbers of CD8(+) cells or CD45(lo) GR-1(-) cells (P=0.43 and 0.49, respectively). Thus, we have shown the first use of flow cytometry to analyze leukocyte infiltration in response to ICH. Our finding of a CD4 T-cell infiltrate is novel and suggests a role for the adaptive immune system in the response to ICH.

Figures

Figure 1

Physiologic and behavior changes associated with intracerebral hemorrhage (ICH). (A) ICH produced a mild to moderate hematoma in the anterolateral portion of the brain near the striatum. (B) ICH led to increased edema at 4 days when compared with saline-treated mice (n = 4 to 6 per group, P≤0.05). Edema was measured as total brain water content. (C) ICH showed poorer motor function at 1 and 4 days compared with saline controls (baseline versus 1 or 4 days; n = 6 to 7 per group, P≤0.05). Bars are mean ± s.e.m.; *P≤0.05 compared with saline of the same time point.

Figure 2

Representative scatter plots showing analysis of CD45hi cells at 4 days in a mouse receiving intracerebral hemorrhage (ICH) and a saline control. An overall increase in CD45hi inflammatory cell infiltrate was observed at 4 days post-ICH, compared with saline-treated mice (P≤0.05), but not at 1 day (P = 0.11). Cells were considered to be CD45hi if they expressed CD45 at 103.2 channels or higher. These cells were then gated and counted for statistical analysis. Gate P2 is considered to be CD45hi GR-1−, gate P3 is considered to be CD45hi GR-1 +, and gate P4 is considered to be CD45lo GR-1−.

Figure 3

Assessment of blood-derived inflammatory cells in the CNS 1 and 4 days after intracerebral hemorrhage (ICH). Brain infiltrating inflammatory cells were stained with anti-CD45, a pan marker for blood-derived cells, and anti-GR-1 antibodies. GR-1 is a pan surface marker for granulocytes. (A) At 4 days there were an increased number of blood-derived leukocytes in ICH brains compared with saline-treated brains (P≤0.05); this was not observed at 1 day (P = 0.11). (B) We did not observe a difference in CD45lo GR-1− cells (microglia) between ICH and saline mice at 1 or 4 days (P = 0.46 and P = 0.49, respectively). Conversely, we found an increased CD45hi GR-1 + cell population (neutrophils) at 1 and 4 days (C)(P≤0.05) and an increased CD45hi GR-1− cell population (cells of the lymphoid lineage and macrophages) at 4 days, but not 1 day (D) (P≤0.05 and P = 0.39). N = 3 to 4 per group; bars are mean±s.e.m.; *P≤0.05 compared with saline of the same time point.

Figure 4

CD4 + and CD8 + cells. (A) Representative scatter plots showing flow cytometric analysis of CD4 + cells in intracerebral hemorrhage (ICH) and saline brains at 4 days. (B) We observed increased numbers of CD4 + cells in ICH mice compared with saline controls (P≤0.05) at 4 days but not at 1 day (P = 0.13; C) There was not a difference in CD8 + cells between ICH and saline mice at 1- or 4-day post-ICH (P = 0.32 and P = 0.43, respectively). N = 3 to 4 per group; bars are mean±s.e.m.; *P≤0.05 compared with saline of the same time point.