Temporary microglia-depletion after cosmic radiation modifies phagocytic activity and prevents cognitive deficits

Abstract

Microglia are the main immune component in the brain that can regulate neuronal health and synapse function. Exposure to cosmic radiation can cause long-term cognitive impairments in rodent models thereby presenting potential obstacles for astronauts engaged in deep space travel. The mechanism/s for how cosmic radiation induces cognitive deficits are currently unknown. We find that temporary microglia depletion, one week after cosmic radiation, prevents the development of long-term memory deficits. Gene array profiling reveals that acute microglia depletion alters the late neuroinflammatory response to cosmic radiation. The repopulated microglia present a modified functional phenotype with reduced expression of scavenger receptors, lysosome membrane protein and complement receptor, all shown to be involved in microglia-synapses interaction. The lower phagocytic activity observed in the repopulated microglia is paralleled by improved synaptic protein expression. Our data provide mechanistic evidence for the role of microglia in the development of cognitive deficits after cosmic radiation exposure.

Figure 1

Experimental Design. Animals were exposed to helium radiation (0, 15, 50, 100 cGy) on day 0. Diet changes (+/−PLX) and behavioral analysis are shown relative to radiation exposure. NOR = Novel Object Recognition. EPM = Elevated Plus Maze.

Figure 2

Radiation-induced memory impairments rescued by PLX treatment. Animals were exposed to helium radiation (0, 15, 50, 100 cGy). Beginning 90 days later, animals were tested for memory deficits or anxiety-like behavior. (A) Memory deficits were measured by novel object recognition. Animals were exposed to two identical object, 24 hrs later the animals are exposed to one familiar object and one novel object. Memory deficits are calculated by a deficit in distinguishing the new object. Nv = novel. Fm = Familiar. Two-way repeated measured ANOVA found a significant group (p < 0.0001) and discrimination effect (p < 0.0001). Sidak post hoc analysis revealed differences in discrimination effects. Animals exposed to 15 or 50 cGy of helium radiation were unable to distinguish the objects, denoting memory impairment. (B) Total exploration time with both objects is depicted per group. (C) Arena side preference is determined during the familiarization phase. In which animals are exposed to two identical objects. Time spent exploring each object is measured. L = object on the left side of the arena, R = object on the right side of the arena. (D) Anxiety-like behavior was measured by time spent in the open arms in the elevated plus maze. A one-way ANOVA did not reveal any significant differences between groups. *p < 0.05, **p < 0.01. Individual animal scores represented in dots, bars depict group mean and SEM. N = 12–18 each group.

Figure 3

Radiation-induced gene changes. (A) Gene-expression changes were measured by a 96 chemokine and cytokine target gene array. qPCR analysis found four genes with significant differences between the 50 cGy and 50 cGy + PLX group: (B) CCL2 (C) CD206 (D) DUSP1 and (E) CD163. Unpaired Student t-test revealed significant differences between groups. *p < 0.05, **p < 0.01, ***p,0.001. Individual animal scores represented in dots, bars depict group mean and SEM. N = 11–12 each group.

Figure 4

Flow cytometry analyses of microglia and periphery originated cells in the CNS. (A) Gating for microglia and peripherally derived macrophage population analysis. Microglia (CD11b⁺CD45^low) and peripherally derived macrophage (CD11b⁺CD45^high) populations were identified by their differentially expressed cell surface markers. (B) A graph to show the relative populations of microglia and peripheral cells in the CNS. Data are presented as dot plots (mean +/−SEM) to show percentage of either population in all viable cells. A one-way ANOVA did not reveal any significant differences between groups. N = 4 each group.

Figure 5

Analyses of complement markers in microglia. Mean fluorescence intensity (MFI) was used to compare the protein levels in microglia populations (CD11b⁺CD45^low). (A,B) Comparison of C5aR MFI by surface staining on microglia. PLX5622 treatment after 50 cGy helium irradiation results in significantly reduced C5aR level compared to the 50 cGy-only group, **p < 0.01, One-way ANOVA with Sidak’s multiple comparisons test. (C,D) Comparison of CD11b MFI by surface staining on microglia. Statistics reveals no significant difference in CD11b (CR3A) levels across groups, but there is a trend of decreased MFI in the 50 cGy + PLX5622 group, p = 0.1096, unpaired student t-test. N = 3–4 each group.

Figure 6

Analyses of phagocytic markers in microglia. MFI was used to compare the phagocytic marker levels in microglia. (A,B) Comparison of LAMP-1 (CD107a) MFI by intracellular staining in microglia. PLX5622 treatment caused significantly reduced CD107a compared to the 50 cGy group, *p < 0.05, One-way ANOVA with Sidak’s multiple comparisons test. (C,D) Comparison of CD206 MFI by intracellular staining in microglia. (E,F) Comparison of CD45 MFI by surface staining on microglia. There is no difference in microglial CD45 levels between groups. N = 3–4 each group.

Figure 7

Investigation of neuronal stability markers. Western blot analysis of isolated prefrontal cortex tissues revealed changes in neuronal stability markers: synapsin 1 (A) and PSD95 (B). (A) A significant increase in synapsin 1 levels were observed in the 50 cGy + PLX group when compared to 50 cGy alone. Representative blot depicted below. (B) A significant decrease in PSD-95 levels were observed in the 50 cGy + PLX group when compared to 50 cGy alone. Representative blot depicted below. Full-length blots are presented in Supplemental Fig. 2. Samples were derived from the same experiment and were processed in parallel. Unpaired student t-tests reveal significant differences between groups. *p < 0.05, **p < 0.01. Individual animal scores represented in dots, bars depict group mean and SEM. N = 7–8 each group.