Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain

Abstract

Here we report in vivo evidence of a neuroprotective role of proliferating microglial cells in cerebral ischemia. Using transgenic mice expressing a mutant thymidine kinase form of herpes simplex virus driven by myeloid-specific CD11b promoter and ganciclovir treatment as a tool, we selectively ablated proliferating (Mac-2 positive) microglia after transient middle cerebral artery occlusion. The series of experiments using green fluorescent protein-chimeric mice demonstrated that within the first 72 h after ischemic injury, the Mac-2 marker [unlike Iba1 (ionized calcium-binding adapter molecule 1)] was preferentially expressed by the resident microglia. Selective ablation of proliferating resident microglia was associated with a marked alteration in the temporal dynamics of proinflammatory cytokine expression, a significant increase in the size of infarction associated with a 2.7-fold increase in the number of apoptotic cells, predominantly neurons, and a 1.8-fold decrease in the levels of IGF-1. A double-immunofluorescence analysis revealed a approximately 100% colocalization between IGF-1 positive cells and Mac-2, a marker of activated/proliferating resident microglia. Conversely, stimulation of microglial proliferation after cerebral ischemia by M-CSF (macrophage colony stimulating factor) resulted in a 1.9-fold increase in IGF-1 levels and a significant increase of Mac2+ cells. Our findings suggest that a postischemic proliferation of the resident microglial cells may serve as an important modulator of a brain inflammatory response. More importantly, our results revealed a marked neuroprotective potential of proliferating microglia serving as an endogenous pool of neurotrophic molecules such as IGF-1, which may open new therapeutic avenues in the treatment of stroke and other neurological disorders.

Figure 1.

Conditional ablation of early microglia/macrophage proliferation in CD11b-TK^mt30 transgenic mice. A–F, Representative double-immunofluorescence images of Iba1 (green) and BrdU (red) in WT (A–C) and transgenic mice (D–F) treated with GCV 72 h after transient MCAO. No colocalization was detected for BrdU and Iba1 staining in CD11b-TK^mt30-GCV-treated mice. Scale bar, 50 μm.

Figure 2.

Microglia/macrophages in CD11b-TK^mt30-GCV-treated mice. A, Schematic representation of the brain section used for the immunostaining experiment. Box represents the cortex area magnified in B–I. B–E, Photomicrographs of immunoreactivities for Iba1 (B, C) and Mac-2 (D, E) in WT and transgenic mice treated with GCV 24 h after transient MCAO. F–I, Photomicrographs of immunoreactivities for Iba1 (F, G) and Mac-2 (H, I) in WT and transgenic mice treated with GCV 72 h after transient MCAO. J, No significant change was detected in the total number of microglia/macrophages (Iba1 staining) and in the number of activated microglia/macrophages (Mac-2 staining) in CD11b-TK^mt30-GCV-treated mice compared with the WT-GCV control group (Iba1, n = 4, p = 0.2507; Mac-2, n = 4, p = 0.0756). A decrease of ∼40% was detected in the total number of microglia/macrophages (Iba1 staining) in CD11b-TK^mt30-GCV-treated mice compared with the WT-GCV controls group (n = 4–5; **p = 0.0042). A decrease of ∼65% was observed in the number of activated microglia/macrophages (Mac-2 staining) in transgenic mice treated with GCV compared with the WT-GCV. Data are expressed as mean ± SEM (n = 4; **p = 0.0019). Scale bars, 50 μm.

Figure 3.

Mac-2⁺ cells do not colocalize with GFP bone marrow-derived microglia/macrophages in chimeric mice. A–C, No colocalization between Mac-2 and GFP could be found 72 h after transient MCAO in chimeric mice by immunofluorescence. D–F, Majority of infiltrating bone marrow GFP cells colocalize with the marker of microglia/macrophages Iba1 by double immunofluorescence in chimeric mice (white arrows). Scale bars: A, 500 μm; A, inset, D, 50 μm.

Figure 4.

Alteration in the level of IκBα mRNA 72 h after transient MCAO. A, Schematic representation of the brain section used for the in situ hybridization experiments. Boxes represent the cortex area magnified in B–E and in Figures 5 and 6. B–E, Photomicrographs of in situ hybridization of IκBα mRNA in WT and transgenic mice treated with GCV 24 h (B, C) and 72 h (D, E) postinjury. F, Quantification of positive cells in the ipsilateral hemisphere did not reveal any significant changes in transgenic mice compared with the WT mice treated with GCV. Values indicate mean ± SEM (n = 4; p = 0.5862). At 72 h, an increase in the number of IκBα⁺ cells in transgenic mice treated with GCV was found compared with the WT-GCV group (n = 4; ***p = 0.0003). Scale bars: B, 500 μm; B, inset, 50 μm.

Figure 5.

Expression of inflammatory markers 24 h after transient MCAO. A–F, Photomicrographs of in situ hybridization for IL-1β (A, B), IL-6 (C, D), and TNF-α (E, F) mRNA in WT and transgenic mice treated with GCV 24 h postinjury. G, No significant change in the number of Il-1 β⁺ and TNF-α⁺ cells in transgenic mice treated with GCV compared with the WT-GCV treated mice was observed. Data are expressed as mean ± SEM (Il-1 β, n = 4, p = 0.0545; TNF-α, n = 3–5, p = 0.2498). An increase was detected in the number of IL-6⁺ cells in CD11b-TK^mt30-GCV compared with the WT-GCV (n = 7; **p = 0.0065). Scale bars: A, 500 μm; A, inset, 50 μm.

Figure 6.

Increase in proinflammatory cytokines 72 h after transient MCAO. A–F, Photomicrographs of in situ hybridization for IL-1β (A, B), IL-6 (C, D), and TNF-α (E, F) mRNA in WT and transgenic mice treated with GCV 72 h postinjury. G, Increase in protein levels of proinflammatory cytokines 72 h after transient MCAO. Brain lysates were generated and the presence of key inflammatory cytokine was determined using a cytokines array (RayBiotech). An increase in the protein amount of IL-6 and TNF-α was detected in CD11b-TK^mt30 mice compared with the WT GCV-treated littermates. No significant change was observed in IL-1β protein expression in transgenic mice treated with ganciclovir compared with the WT GCV-treated group (IL-1β, n = 4, p = 0.8759; IL-6, n = 4, **p = 0.0018; TNF-α, n = 4, ***p = 0.0010). H, Significant increases of IL-1β⁺, IL-6⁺, and TNF-α⁺ cells were detected in transgenic GCV treated mice compared with the WT-GCV treated mice (IL-1β, n = 4, **p = 0.0064; IL-6, n = 5, **p = 0.0019; TNF-α, n = 4, ***p = 0.0005). Data are expressed as mean ± SEM. Scale bars: A, 500 μm; A, inset, 50 μm.

Figure 7.

Increase of infarct area and apoptosis in CD11b-TK^mt30-GCV mice 72 h after cerebral ischemia. A, B, Representative brain sections of WT and transgenic brain treated with GCV stained with TTC. The size of ischemic lesion is expressed as a percentage of the control; the equivalent area of contralateral nonischemic hemisphere (100%) was measured 72 h after transient MCAO. A significant increase of ∼13% was observed in the size of ischemic lesion in transgenic mice treated with GCV. Each value represents a percentage of the mean value ± SEM (n = 7–9; *p = 0.05). C, D, Photomicrographs of immunoreactivities of cleaved caspase-3 in WT and transgenic mice treated with GCV 72 h after transient MCAO. Quantification of labeled cells showed an increase of cleaved caspase-3⁺ cells in transgenic mice treated with GCV compared with the WT-GCV. Values indicate mean ± SEM (n = 4; *p = 0.0422). E–P, Representative photomicrographs of double immunostaining for cleaved caspase-3 (red) and NeuN (green) (E–J) or Mac-2 (green) (K–P) in WT and transgenic mice by double immunofluorescence. E–J, Photomicrographs show colocalization for cleaved caspase-3 and NeuN in both groups (white arrows). G–J, In transgenic mice, more NeuN⁺ cells colocalize with cleaved caspase-3 compared with WT-GCV mice (white arrows) (H–J). K–M, In WT-GCV mice, no colocalization was observed for Mac-2 and cleaved caspase-3, but in transgenic mice (N–P) some Mac-2⁺ cells are cleaved caspase-3⁺. Scale bars: C, (in E) E–P, 25 μm.

Figure 8.

Proliferating microglial cells synthesize and secrete IGF-1. A–I, Double labeling 72 h after transient MCAO reveals colocalization of Mac-2 (red) and IGF-1 (green; white arrows) in WT transgenic mice treated with GCV and in M-CSF-treated mice 72 h postinjury. J, Densitometry quantification reveals a significant (1.8-fold) decrease in IGF-1 expression in the brain section of transgenic mice treated with GCV compared with the WT-GCV and a significant (1.9-fold) increase in IGF-1 levels in the M-CSF-treated group compared with controls. K–M, Double immunohistochemistry for BrdU and Mac-2 reveals an increase in the number of BrdU and Mac-2 positive cells after M-CSF treatment. Data are expressed as mean ± SEM (BrdU, n = 2, **p = 0.0048; Mac-2, n = 2, *p = 0.0391; IGF-1, n = 2–4, ***p < 0.0001). Scale bar, 25 μm.