Alternative activation-skewed microglia/macrophages promote hematoma resolution in experimental intracerebral hemorrhage

Abstract

Microglia/macrophages (MMΦ) are highly plastic phagocytes that can promote both injury and repair in diseased brain through the distinct function of classically activated and alternatively activated subsets. The role of MMΦ polarization in intracerebral hemorrhage (ICH) is unknown. Herein, we comprehensively characterized MMΦ dynamics after ICH in mice and evaluated the relevance of MMΦ polarity to hematoma resolution. MMΦ accumulated within the hematoma territory until at least 14days after ICH induction. Microglia rapidly reacted to the hemorrhagic insult as early as 1-1.5h after ICH and specifically presented a "protective" alternatively activated phenotype. Substantial numbers of activated microglia and newly recruited monocytes also assumed an early alternatively activated phenotype, but the phenotype gradually shifted to a mixed spectrum over time. Ultimately, markers of MMΦ classic activation dominated at the chronic stage of ICH. We enhanced MMΦ alternative activation by administering intraperitoneal injections of rosiglitazone, and subsequently observed elevations in CD206 expression on brain-isolated CD11b⁺ cells and increases in IL-10 levels in serum and perihematomal tissue. Enhancement of MMΦ alternative activation correlated with hematoma volume reduction and improvement in neurologic deficits. Intraventricular injection of alternative activation signature cytokine IL-10 accelerated hematoma resolution, whereas microglial phagocytic ability was abolished by IL-10 receptor neutralization. Our results suggest that MMΦ respond dynamically to brain hemorrhage by exhibiting diverse phenotypic changes at different stages of ICH. Alternative activation-skewed MMΦ aid in hematoma resolution, and IL-10 signaling might contribute to regulation of MMΦ phagocytosis and hematoma clearance in ICH.

Keywords: Hematoma resolution; Interleukin-10; Intracerebral hemorrhage; Microglia/macrophage polarization; Phagocytosis.

Figure 1

Microglia/macrophages (MMΦ) accumulate within the hematoma territory until 14 days after ICH. (A) Representative coronal brain sections show the temporal changes in hematoma resolution after ICH. The boxed area indicates the location of representative fluorescence images in the hemorrhagic brain. Scale bar: 1 cm. n = 3 mice per time point. (B) Identification of Iba1- and GFAP-positive cells in brain sections at 1, 3, 7, and 14 days post-ICH. Immunoreactivity of Iba1 (ionized calcium binding adaptor molecule 1; MMΦ marker) and GFAP (glial fibrillary acidic protein; astrocyte marker) are shown in green and red, respectively. The inset column represents higher magnification of the boxed area in the corresponding merged images. Nuclei were stained with DAPI (blue). Scale bars: 200 μm (merged column); 20 μm (inset column). n = 3 mice per time point.

Figure 2

Identification of Iba1- and GFAP-positive cells in brain sections at 1, 3, 7, and 14 days after sham operation. Immunoreactivity of Iba1 (ionized calcium binding adaptor molecule 1; MMΦ marker) and GFAP (glial fibrillary acidic protein; astrocyte marker) are shown in green and red, respectively. Nuclei were stained with DAPI (blue). Scale bars: 100 μm.

Figure 3

Markers of classically activated microglia/macrophages (MMΦ) increase over time after ICH. (A) Temporal profile of mRNA expression for each marker of classically activated MMΦ after ICH. n = 4 to 6 per time point. (B) Representative double-immunofluorescence staining of Iba1 (ionized calcium binding adaptor molecule 1; MMΦ marker; red) and CD16/32 (classic activation marker; green) on hemorrhagic brain sections at 1, 3, 7, and 14 days post-ICH. Dotted lines mark the hematoma boundary in the Iba1-fluorescent images. Arrows indicate the cells and area of colocalization. The inset images represent higher magnification of the boxed area in the corresponding merged images. Nuclei were stained with DAPI (blue). Scale bars: 100 μm (merged row), 10 μm (inset row). (C) Bar graph shows the degree of MMΦ and CD16/32 colocalization in gray pixel intensity at 1, 3, 7, and 14 days post-ICH. n = 4 mice at 1 and 7 days; n = 7 mice at 3 and 14 days. Values are mean ± SD; *P < 0.05 vs. sham or contralateral side; #P < 0.05 vs. previous time point. S, sham; Contra, contralateral; Ipsi, ipsilateral.

Figure 4

Markers of classically activated microglia/macrophages (MMΦ) over time after sham operation. Representative double-immunofluorescence staining of Iba1 (ionized calcium binding adaptor molecule 1; MMΦ marker; red) and CD16/32 (classic activation marker; green) on sham-operated brain sections at 1, 3, 7, and 14 days post-surgery. Dotted lines mark the needle track. Nuclei were stained with DAPI (blue). Scale bars: 100 μm (low-magnification image); 10 μm (high-magnification image).

Figure 5

Markers of alternatively activated microglia/macrophages (MMΦ) increase rapidly after ICH and subside with time. (A) Gene expression for markers of alternatively activated MMΦ at various time points after ICH determined by qPCR. n = 4 to 6 mice per time point. (B) Representative double-labeling of Iba1 (ionized calcium binding adaptor molecule 1; MMΦ marker; red) and CD206 (mannose receptor; alternative activation marker; green) on hemorrhagic brain sections at 1, 3, 7, and 14 days post-ICH. Dotted lines mark the hematoma boundary in the representative Iba1 images. Arrows indicate the cells and area of colocalization. The inset images represent higher magnification of the boxed area in the corresponding merged images. Nuclei were stained with DAPI (blue). Scale bars: 100 μm (merged row); 10 μm (inset row). (C) Bar graph shows the degree of MMΦ and CD16/32 colocalization in gray pixel intensity at 1, 3, 7, and 14 days post-ICH. n = 4 mice at 1 and 7 days; n = 7 mice at 3 and 14 days. (D) Bar graph shows IL-10 protein concentration in the sham (S) group and the contralateral (Contra) and ipsilateral (Ipsi) hemispheres of the ICH group at 1–1.5 h after ICH. n = 4 mice per group. (E) Top: Representative immunoblot of HO-1 protein expression in CD11b-positive cells isolated from the sham mice and contralateral and ipsilateral hemispheres of the ICH mice. Bottom: Densitometric analysis shows significantly higher HO-1 protein expression in CD11b-positive cells isolated from the ipsilateral hemisphere of ICH mice than in those isolated from the contralateral hemisphere at 1–1.5 h after ICH. n = 3 mice per group. Values are mean ± SD; *P < 0.05 vs. sham or contralateral group; #P < 0.05 vs. previous time point.

Figure 6

Markers of alternatively activated microglia/macrophages (MMΦ) over time after sham operation. Representative double-immunofluorescence staining of Iba1 (ionized calcium binding adaptor molecule 1; MMΦ marker; red) and CD206 (alternative activation marker; green) on sham-operated brain sections at 1, 3, 7, and 14 days post-surgery. Dotted lines mark the needle track. Nuclei were stained with DAPI (blue). Scale bars: 100 μm (low-magnification image); 10 μm (high-magnification image).

Figure 7

Administration of rosiglitazone accelerates hematoma resolution and improves neurologic function in 12-month-old mice subjected to ICH. (A) Representative serial coronal sections show the hematoma territory at 4 days (top) and 7 days (bottom) post-ICH in mice administered rosiglitazone (Rosi) or vehicle (Veh). (B) Quantification of the hematoma volume in rosiglitazone- and vehicle-treated groups at 4 and 7 days post-ICH. n = 8 mice per group. (C) Changes in body weight of rosiglitazone- and vehicle-treated mice. n = 9 to 14 mice per group. (D) Neurologic function of ICH mice was assessed by the neurologic deficit score (top left), corner turn test (top right), hindlimb placing test (bottom left), and forelimb placing test (bottom right). n = 9 to 14 mice per group. Values are mean ± SD; *P < 0.05 vs. vehicle group.

Figure 8

Administration of rosiglitazone promotes microglia/macrophage (MMΦ) alternative activation in 12-month-old mice subjected to ICH. (A) Representative dot-plot scatter analysis of CD11b-positive cells isolated from the brains of control and ICH animals after vehicle (Veh) or rosiglitazone (Rosi) treatment. A gate was drawn to exclude cellular debris from dead cells for further analysis. (B) Gated cells from (A) were analyzed for the expression of CD11b and CD45 to identify CD11b⁺CD45^high (macrophage) and CD11b⁺CD45^low (microglia) populations in control (left dot plots) and ICH (right dot plots) groups treated with or without rosiglitazone. CD45^low cells and CD45^high cells were classified as CD11b⁺ microglia and macrophages, respectively. (C) Representative dot plots showing expression of CD206-positive subsets of CD11b⁺CD45^high and CD11b⁺CD45^low brain macrophages and microglia in control and ICH groups at 3 days post-ICH. (D) Representative histograms show that CD206 staining of CD11b⁺CD45^high (bottom left) and CD11b⁺CD45^low (top left) cells was brighter in brains of rosiglitazone-treated mice (blue trace) than in brains of vehicle-treated mice (yellow trace). Isotype background staining is shown as a red trace. Quantification shows that rosiglitazone treatment increased the percentage of CD11b⁺CD45^highCD206⁺ and CD11b⁺CD45^lowCD206⁺ cells in the ICH brains (right). Cells were pooled from three mice per group for a total of three separate experiments. (E) Bar graphs represent mRNA expression of alternative activation markers YM1/2 and Arg1 in brains of vehicle-, rosiglitazone-, GW9662-, and rosiglitazone+GW9662-treated ICH mice. n = 3 to 5 mice per group. (F) Bar graphs show IL-10 mRNA expression in rosiglitazone-, vehicle-, GW9662-, and rosiglitazone+GW9662-treated mice at 3 days after ICH. Compared with vehicle treatment, rosiglitazone significantly increased IL-10 mRNA expression at 3 days in the ipsilateral hemisphere. IL-10 mRNA expression significantly decreased in mice that cotreatment of GW9662 with rosiglitazone, compared with rosiglitazone-treated group (top). n = 3 to 5 mice per group. Serum IL-10 protein levels were significantly higher in the rosiglitazone-treated group than in the vehicle-treated group at 2 and 3 days post-ICH (bottom). n = 4 to 6 mice per group. (G) Representative coronal brain sections show hematoma in vehicle- and IL-10-injected ICH brains at 3 days post-ICH (top). Hematoma volume of IL-10-injected ICH brains was significantly smaller than that of vehicle-injected ICH brains on day 3 post-ICH (bottom). Scale bar: 1 cm. Values are mean ± SD; *P < 0.05 vs. vehicle or control group; ^#P < 0.05 vs. rosiglitazone group; ^&P < 0.05 vs. GW9662 group. Con, contralateral; Ipsi, ipsilateral; n.d., not detectable; Veh, vehicle; Rosi, rosiglitazone; GW, GW9662.

Figure 9

Effect of repeated rosiglitazone treatment on CD206 expression in the ICH brain. Representative low-magnification images show CD206 immunoreactivity (red) within and around the hematoma on brain sections from vehicle (Veh)- and rosiglitazone (Rosi)-treated Cx3cr1^GFP/+ mice at 4 days post-ICH. Immunofluorescent signal of CD206 was barely detectable in the contralateral hemisphere (right). I, II, III, IV, and V indicate the images from five independent animals, and the numbers represent raw fluorescence intensity for each image. Scale bars: 100 μm. Quantification analysis of fluorescence intensity indicates that rosiglitazone administration significantly increased CD206 immunoreactivity in the ICH brain at 4 days post-ICH (left). n = 5 mice per group. Values are mean ± SD; *P < 0.05 vs. vehicle group. Contra, contralateral.

Figure 10

IL-10 receptor blockade inhibits phagocytosis by Cx3cr1^GFP/+ microglia in organotypic hippocampal slice cultures (OHSCs). (A) Stimulation of OHSCs with hemoglobin induced changes in mRNA expression of classic and alternative activation markers. RT-PCR was carried out with total RNA extracted from hemoglobin-treated or untreated OHSCs at 3 h, 6 h, 12 h, 1 day, and 3 days after stimulation. n = 3 independent experiments. (B) Representative images show Cx3cr1^GFP/+ microglia ingesting 1 μm-diameter fluorescent latex beads in unstimulated OHSCs (Con), OHSCs stimulated with hemoglobin (Hb), and OHSCs stimulated with rosiglitazone (Rosi). These groups were treated with either PBS (top row) or 20 μg/mL IL-10 receptor alpha antibody (RA; bottom row). The inset column represents higher magnification of the boxed area in each image. Scale bars: 10 μm. (C) Quantification of ingested fluorescent beads/cell in OHSCs after 24 h of treatment. n = 3 independent experiments. Values are mean ± SD; *P < 0.05 vs. control group; ^#P < 0.05 vs. PBS group. Con, control.

Figure 11

Effect of repeated rosiglitazone treatment on neuronal survival in the ICH brain. Representative images show NeuN immunoreactivity (red) in the perihematoma of brain sections from vehicle- and rosiglitazone-treated Cx3cr1^GFP/+ mice at 4 days post-ICH (top). Quantification analysis of NeuN-positive cells indicates that rosiglitazone administration significantly increased the number of surviving neurons in the ICH brain at 4 days post-ICH (bottom). Nuclei were stained with DAPI (blue). Scale bars: 50 μm. n = 5 mice per group. Values are mean ± SD; *P < 0.05 vs. vehicle group. Veh, vehicle; Rosi, rosiglitazone.