Depletion of microglia exacerbates postischemic inflammation and brain injury

Abstract

Brain ischemia elicits microglial activation and microglia survival depend on signaling through colony-stimulating factor 1 receptor (CSF1R). Although depletion of microglia has been linked to worse stroke outcomes, it remains unclear to what extent and by what mechanisms activated microglia influence ischemia-induced inflammation and injury in the brain. Using a mouse model of transient focal cerebral ischemia and reperfusion, we demonstrated that depletion of microglia via administration of the dual CSF1R/c-Kit inhibitor PLX3397 exacerbates neurodeficits and brain infarction. Depletion of microglia augmented the production of inflammatory mediators, leukocyte infiltration, and cell death during brain ischemia. Of note, microglial depletion-induced exacerbation of stroke severity did not solely depend on lymphocytes and monocytes. Importantly, depletion of microglia dramatically augmented the production of inflammatory mediators by astrocytes after brain ischemia . In vitro studies reveal that microglia restricted ischemia-induced astrocyte response and provided neuroprotective effects. Our findings suggest that neuroprotective effects of microglia may result, in part, from its inhibitory action on astrocyte response after ischemia.

Keywords: Microglia; brain ischemia; colony-stimulating factor 1 receptor; neuroprotection.

Figure 1.

PLX3397 treatment eliminated microglia and exacerbated stroke severity in MCAO mice. Wild type C57BL/6 mice were fed PLX3397 or control chow diet for 21 days prior to sham or MCAO surgeries. After surgeries, these mice continued to receive PLX3397 or control diet until they were sacrificed. At day 1 or day 3 after 60 min MCAO and reperfusion, mice were subjected to neurological assessment, MRI, flow cytometry, and pathology staining. (a) Schematic showing the experimental design. (b) Representative flow cytometry plots show the gating strategy of CD11b⁺CD45^int microglial cell population. Bar graphs summarize the results from sham or MCAO mice receiving the indicated treatment at day 1 and day 3 after MCAO reperfusion. (c) Bar graphs illustrate the indicated neurological assessments of sham and MCAO mice receiving PLX3397 or control diet at day 1 or day 3 after reperfusion. (d) Representative MRI images show infarct area (outlined in red) in MCAO mice receiving PLX3397 or control diet after MCAO reperfusion. Bar graphs show the infarct volume of MCAO mice receiving PLX3397 or control diet at indicated time points. n = 15 mice per group. Error bars represent SD; *P < 0.05; **P < 0.01.

Figure 2.

Depletion of microglia augmented brain inflammation and cell death after ischemia. C57BL/6 mice were fed with PLX3397 or control diet for 21 days prior to 60 min MCAO. After MCAO, mice continued to receive PLX3397 or control chow diet until they were sacrificed. (a) At 24 h after ischemia and reperfusion, representative bioluminescence images and quantification analysis show ROS generation in sham and MCAO mice receiving PLX3397 or control diet. n = 15 mice per group. (b) At 24 h after ischemia and reperfusion, brain tissues were obtained from MCAO mice receiving PLX3397 or control diet. Brain tissues from sham operated mice receiving PLX3397 or control diet were used as control. Brain homogenates were analyzed by a Mouse XL Cytokines Array kit. Heat map and cluster analysis show the expression of cytokines, chemokines, and growth factors in brain homogenates from sham and MCAO mice with indicated treatments. A heat map was generated and the relative pixel intensity of spots signal is indicated by the representative color code (red, higher; green, lower). Bar graphs show the significantly altered factors. n = 8 mice per group. (c) Images show immunostaining of neuron (NeuN; green) terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL; red) and 4′,6-diamidino-2-phenylindole (DAPI; blue) in brain sections from sham and MCAO mice treated with PLX3397 or control at 24 h after ischemia and reperfusion. Representative images of four independent experiments with five mice per group each are shown. Scale bars: 40 µm; 20 µm (inset). (d) Quantification of the percentage of NeuN⁺TUNEL⁺ cells in the sham mice and peri-infarct of MCAO mice treated with PLX3397 or control at 24 h after ischemia and reperfusion. Error bars represent SD; *P < 0.05; **P < 0.01.

Figure 3.

Depletion of microglia enhanced leukocytes infiltration after MCAO. Wild type C57BL/6 mice were fed with PLX3397 or control diet for 21 days prior to MCAO. At 24 h after ischemia and reperfusion, single cell suspensions were prepared from brain or spleen tissues of MCAO mice receiving PLX3397 or control diet. (a) Representative flow cytometry plots show the gating strategy of leukocyte subpopulations isolated from brain tissues. Plots show gating of T cells (CD45^highCD3⁺), CD4⁺ T cells (CD45^highCD3⁺CD4⁺), CD8⁺ T cells (CD45^highCD3⁺CD8⁺), B cells (CD45^highCD3^-B220⁺), NK cells (CD45^highCD3^-NK1.1⁺), macrophages (CD45^highCD11b⁺F4/80⁺) and neutrophils (CD45^highCD11b⁺ Ly-6 G⁺). (b) Quantification of CNS-infiltrating lymphocytes, macrophages and neutrophils from sham and MCAO mice receiving the indicated treatments at 24 h after ischemia and reperfusion. (c) Representative flow cytometry plots show the gating strategy of leukocyte subpopulations isolated from the spleen. (d) Quantification of lymphocytes, macrophages and neutrophils in the spleen of sham and MCAO mice receiving indicated treatments at 24 h after ischemia and reperfusion. n = 15 mice per group. Error bars represent SD; *P < 0.05; **P < 0.01.

Figure 4.

Depletion of microglia-induced exacerbation of stroke severity did not entirely depend on lymphocyte and monocyte infiltration. (a)–(b) Rag2^−/−γc^−/− mice were fed with PLX3397 or control diet for 21 days prior to MCAO. After MCAO, these mice continued to receive PLX3397 or control diet until they were sacrificed. Bar graphs illustrate neurological scores of Rag2^−/−γc^−/− mice receiving PLX3397 or control diet at 24 h after MCAO (a). n = 15 mice per group. MRI images show infarct area (outlined in red) in mice subjected to MCAO and indicated treatments at 24 h after reperfusion. Bar graphs illustrate the infarct volume of Rag2^−/−γc^−/− mice receiving PLX3397 or control diet at 24 h after MCAO (b). n = 15 mice per group. (c)–(d) C57BL/6 mice were fed with PLX3397 or control diet for 21 days prior to 60 min MCAO. Starting from immediately after MCAO, bindarit (an inhibitor of monocyte chemotactic protein synthesis) was given orally at a dose of 50 mg/kg twice a day. MCAO mice receiving an equal volume of vehicle carboxymethylcellulose were used as controls. Bar graphs illustrate the infarct size of MCAO mice receiving indicated treatments at 24 h after ischemia and reperfusion (c). n = 15 mice per group. Representative MRI images show the infarct area (outlined in red) in MCAO mice receiving the indicated treatments at 24 h after ischemia and reperfusion. Bar graphs show the infarct volume of MCAO mice receiving indicated treatments at 24 h after ischemia and reperfusion (d). n = 15 mice per group. Error bars represent SD; *P < 0.05.

Figure 5.

Depletion of microglia enhanced astrocyte response after brain ischemia. C57BL/6 mice were fed with PLX3397 or control diet for 21 days prior to MCAO. At 24 h after ischemia and reperfusion, single cell suspensions were prepared from brain or spleen tissues of MCAO mice receiving PLX3397 or control diet. (a) Representative flow cytometry plots and bar graph show astrocytes (GFAP⁺) in MCAO mice receiving PLX3397 or control diet, at 24 h after ischemia and reperfusion. (b)–(c) Representative plots (b) and bar graphs (c) of flow cytometry analysis show the expression of IL-1α, IL-1β, iNOS, CCL2, TNF-α, IL-6 in astrocytes obtained at 24 h after MCAO from mice receiving PLX3397 or control diet. n = 15 mice per group. Error bars represent SD; *P < 0.05; **P < 0.01.

Figure 6.

Microglia restricted ischemia-induced astrocyte response and neural injury. Primary cortical neurons were subjected to 5 min OGD. Immediately after OGD, culture media was replaced with regular neurobasal medium mixed with equal volume of glia-conditioned medium for 24 h. Glia-conditioned medium was obtained from cultured microglia, astrocytes or mixed microglia and astrocytes (microglia/astrocyte) at 24 h after 30 min OGD. (a) Representative images show staining of beta III Tubulin (green), DAPI (blue) and TUNEL (red) in primary cortical neurons exposed to OGD and glia-conditioned medium as indicated. Primary cortical neurons without exposure to OGD or glia-conditioned medium were used as controls. Scale bars: 20 µm. (b) Quantification of TUNEL⁺ cells after exposure to indicated treatment. CM: conditional medium. (c) Bar graphs show the level of released LDH from primary cortical neurons after exposure to indicated treatment. LDH release was expressed by normalizing the released LDH in the medium to the total LDH (released + intracellular). CM: conditional medium. (d) Primary astrocytes were cultured separately or together with microglia (microglia/astrocyte). After 30 min OGD and subsequent 24 h recovery, intracellular staining of GFAP and indicated cytokines was performed and analyzed by flow cytometry. Bar graphs show the percentage of GFAP⁺ cells expressing IL-1α, IL-1β, iNOS, CCL2, TNF-α and IL-6. Error bars represent SD.; *P < 0.05; **P < 0.01.