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. 2016 Jul 21;17(7):1181.
doi: 10.3390/ijms17071181.

High Morphologic Plasticity of Microglia/Macrophages Following Experimental Intracerebral Hemorrhage in Rats

Shu-Sheng Yang  1   2 , Li Lin  3   4 , Yue Liu  5   6 , Jie Wang  7 , Jiang Chu  8 , Teng Zhang  9 , Lin-Na Ning  10 , Yan Shi  11 , Ying-Yan Fang  12 , Peng Zeng  13 , Jian-Zhi Wang  14 , Ming-Yi Qiu  15 , Qing Tian  16
Affiliations

High Morphologic Plasticity of Microglia/Macrophages Following Experimental Intracerebral Hemorrhage in Rats

Shu-Sheng Yang et al. Int J Mol Sci. .

Abstract

As current efforts have limited effects on the clinical outcome of intracerebral hemorrhage (ICH), the mechanisms including microglia/macrophages that involved inflammation need further investigation. Here, 0.4 units of collagenase VII were injected into the left caudate putamen (CPu) to duplicate ICH rat models. In the brains of ICH rats, microglia/macrophages, the nearest cells to the hemorrhagic center, were observed as ameboid and Prussian-blue positive. Furthermore, the ameboid microglia/macrophages were differentiation (CD) 68 and interleukin-1β (IL-1β) positive, and neither CD206 nor chitinase3-like 3 (Ym1) positive, suggesting their strong abilities of phagocytosis and secretion of IL-1β. According to the distance to the hemorrhagic center, we selected four areas-I, II, III, and IV-to analyze the morphology of microglia/macrophages. The processes decreased successively from region I to region IV. Microglia/macrophages in region IV had no processes. The processes in region I were radially distributed, however, they showed obvious directivity towards the hemorrhagic center in regions II and III. Region III had the largest density of compactly arrayed microglia/macrophages. All these in vivo results present the high morphologic plasticity of microglia/macrophages and their functions in the pathogenesis of ICHs.

Keywords: interleukin-10; interleukin-1β; intracerebral hemorrhage; microglia.

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Figures

Figure 1
Figure 1
Increased and accumulated microglia/macrophages and astrocytes in the hemorrhagic caudate putamen (CPu) of rats. 0.4 U of collagenase VII (2 μL) was injected into the left CPu of three-month-old male Sprague–Dawley (SD) rats (A, ICH). Sham rats (Sham) received 2 μL 0.9% NaCl injection. Beam balance test (B, n = 5/group), elevated body swing test (C, n = 5/group), and brain magnetic resonance imaging (MRI) test (D, n = 4/group) were performed to evaluate the model. By immunohistochemistry staining on brain slices (20 μm), Iba-1 (marker of microglia/macrophage) positive cells and GFAP (marker of astrocyte) positive cells were shown in the collagenase VII injected CPu (E, Bar = 50 μm). By double-label immunofluorescence staining, microglia/macrophages (green) were found much closer to the hemorrhagic center (HC) than astrocytes (red) (F, Bar = 50 μm). The data were expressed as means ± standard error of the mean (SEM).* p < 0.05, ** p < 0.01, *** p < 0.001, vs. Sham.
Figure 2
Figure 2
Ameboid microglia/macrophages in the hemorrhagic center presented Prussian blue positive. Brain slices (20 μm) of intracerebral hemorrhage (ICH) rats were immunohistochemically stained by Iba-1 (A, n = 5) or further double stained with Prussian blue (B,C, n = 5). The optical densities of Prussian-blue staining were quantitatively analyzed in D (n = 5). Microglia/macrophages in the brain slices taken at 7, 14 and 28 days had stronger blue staining than those in three-day brain slices (C,D). Bar = 25 μm. Data were expressed as means ± SEM. * p < 0.05, ** p < 0.01, vs. ICH 3 d. Δ p < 0.05, vs. ICH 7 d.
Figure 3
Figure 3
Ameboid microglia/macrophages were mainly differentiation (CD)68 positive in the hemorrhagic center. Brain slices (20 μm) were taken from ICH rats at three days after collagenase VII injection. By double-label immunofluorescence staining, ameboid Iba1 positive (red, A) or CD11b positive (green, B,C) microglia/macrophages in the hemorrhagic center were CD68 positive (green, A, n = 3), neither CD206 nor chitinase 3-like 3 (Ym1) positive (red, B,C) (B and C, n = 3). Bar = 30 μm.
Figure 4
Figure 4
Increased IL-1β in the hemorrhagic CPu was detected at three and seven days after collagenase VII injection. IL-1β in the CPu was studied by immunohistochemistry (A,B, Bar = 100 μm) on brain slices (20 μm) at one, three, and seven days after collagenase VII injection (ICH 1 d, 3 d and 7 d, n = 3). Control CPu (Sham) was from 2 μL 0.9% NaCl injected rats (n = 3). Data were expressed as means ± SEM. *** p < 0.001, vs. Sham. ΔΔΔ p < 0.001 vs. ICH 1 d. To study the cytokine expressions of activated microglia in the hemorrhagic center, brain slices were double-stained with antibodies specific to pro-inflammatory cytokines interleukin (IL)-1β (C, green) ortumor necrosis factor-α (TNF-α) (D, green) and CD68 (red in C,D, n = 3). Bar = 50 μm.
Figure 5
Figure 5
Microglia/macrophages showed high morphologic plasticity. Brain slices (20 μm) were taken three days after operation from Sham rats (A) and ICH rats (B,C). By immunohistochemistry, Iba-1 marked microglia/macrophages in the left CPu were observed (n = 3, Bar = 200 μm). According to the distance to the hemorrhagic center (HC), we selected four areas (B,C) to analyze the morphologic features of activated microglia/macrophages, and the left CPu of sham rats was used as control (Sham, A,D,E). The process numbers (D) and the densities (E) of microglia/macrophages were studied. To evaluate the process directivity of a microglia/macrophage, the cell body was set as the origin, and the y-axis was set pointing to the hemorrhagic center (F). The directivity was calculated as the process number in quadrants I and IV deducted the number in quadrants II and III, and the difference was divided by the total process number (G). The morphologic characteristics of activated microglia/macrophages after ICH was summarized in (H). Data were expressed as means ± SEM. * p < 0.05, *** p < 0.001, vs. Sham. ΔΔ p < 0.01, ΔΔΔ p < 0.001, vs. region I. ## p < 0.01, ### p < 0.001, vs. region II. ▲▲▲ p < 0.001, vs. region III.
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