Microglial Depletion with Clodronate Liposomes Increases Proinflammatory Cytokine Levels, Induces Astrocyte Activation, and Damages Blood Vessel Integrity

Abstract

Investigators are increasingly interested in using microglial depletion to study the role of microglia under pathologic conditions. Liposome-encapsulated clodronate is commonly used to eliminate macrophage populations because it causes functionally irreversible inhibition and apoptosis once phagocytized by macrophages. Recent studies have shown that microglia can be depleted in disease models by injecting clodronate liposomes into the brain parenchyma. However, it is unclear whether intracerebral administration of clodronate liposomes is a practical method of eliminating microglia under physiologic conditions or whether microglial depletion induces damage to other brain cells. In this study, injecting 1 μL of clodronate liposomes (7 μg/μL) into the striatum of mice caused ablation of microglia at 1 day that persisted for 3 days. Microglia reappeared in the boundary regions of microglia elimination after 5 days. Importantly, we observed an increase in proinflammatory cytokine levels and an increase in neural/glial antigen 2 and glial fibrillary acidic protein expression in the perilesional region. In contrast, expression levels of myelin basic protein, microtubule-associated protein 2, and postsynaptic protein-95 decreased in the periphery of regions where microglia were depleted. Moreover, clodronate liposome administration decreased the density and integrity of blood vessels in the perilesional regions. In cultured primary neurons, clodronate liposome exposure also attenuated ATP synthesis. Together, these findings suggest that intracerebral administration of clodronate liposomes into brain parenchyma can deplete microglia, but can also damage other brain cells and blood vessel integrity.

Keywords: Clodronate liposomes; Inflammation; Microglial depletion.

Fig. 1

Injection of clodronate liposomes (Clo-Lip) into brain parenchyma depletes microglia starting at 24 h. a, b Series of brain sections at day 1 after injection of 1 μL Dil-Lip (liposomes labeled with red fluorescent dye Dil) into striatum (a) or lateral ventricle (b). Numbers shown indicate anteroposterior ordinate distance to the bregma. Scale bar = 100 μm. c Representative images show depletion of Cx3cr1-GFP⁺ microglia (green) at different time points after striatal injections with Dil-Lip or Clo-Lip. Insets show higher magnification images of microglia. Scale bar = 100 μm, inset, 10 μm. d Quantification of Cx3cr1-GFP⁺ cells in striatum after 1 μL Clo-Lip injection. n = 3–5/group; **p <0.01, ***p <0.001 versus contralateral striatum, two-way ANOVA with Bonferroni multiple comparison test. Data are presented as means ± SEM. e Representative images show that Cx3cr1-GFP⁺ microglia (green) are depleted in corpus callosum (outlined) on day 3 after striatal Clo-Lip injection. Scale bar = 100 μm

Fig. 2

Microglia reappear in the peri-injection region 5 days after striatal clodronate liposome (Clo-Lip) injection. a Representative images of Cx3cr1-GFP⁺ microglia in striatum on days 3 and 5 after 1 μL Clo-Lip injection. The outlined areas indicate the sites of microglial depletion. Scale bar = 100 μm. Asterisk in the right image indicates the fields where microglia repopulated. Inset: high magnification of microglia around the outline, scale bar = 5 μm. Green, Cx3cr1-GFP; red, CD68. b Cx3cr1-GFP⁺ microglia were analyzed by flow cytometry on days 1, 4, and 7 after Clo-Lip injection. Representative dot plots are shown, and the percentages of Cx3cr1-GFP⁺ in the striatal cell population are indicated. Red, Cx3cr1-GFP⁻ cell population; green, Cx3cr1-GFP⁺ cell population. c Graphs show the percentage of remaining Cx3cr1-GFP⁺ population in striatum and cortex after Clo-Lip injection. n = 3–4/group; ***p < 0.001 versus sham group; one-way ANOVA with Bonferroni post hoc test. Data are presented as means ± SD. d Representative images and quantification show that NG2-positive cells (red) increased in striatum at day 7 after Clo-Lip injection. Scale bar = 50 μm. n = 5; ***p <0.001 versus contralateral striatum; Student’s t test

Fig. 3

Microglial depletion upregulates proinflammatory cytokine levels and induces astrocyte activation. a Concentrations of IL-1β, IL-6, and TNF-α in striatum were measured with ELISA on days 1, 3, 5, and 7 after injection of clodronate liposomes (Clo-Lip) or Dil-labeled liposomes (Dil-Lip). n = 3–10/group; e.g., n = 3 in the TNF-α sham group, n = 10 in the Dil-Lip d1 group; **p < 0.01, ***p < 0.001 versus sham group; #p < 0.05 versus Dil-Lip group; one-way ANOVA with Bonferroni post hoc test. Data are presented as means ± SD. Data in the last graph are presented as box-and-whisker plots. The middle horizontal line within the box represents the median, and boxes extend from the 5th to 95th percentile. b Low magnification images show the core injection (outlined in left panel) and region of microglial depletion (outlined in middle panel). Asterisks in the right image indicate the fields where the images in Figs. 3c, d, 4, and 5 were taken. Red, MAP2; green, Cx3cr1-GFP. Scale bar =100 μm. c Representative images of glial fibrillary acidic protein (GFAP) expression (red) on days 3 and 7 after Clo-Lip injection. Scale bar = 50 μm. The graph shows the percent increase in GFAP n = 6; ***p < 0.001 versus contralateral striatum group; #p < 0.05 versus Clo-Lip day 3 group; one-way ANOVA with Bonferroni post hoc test. Data are presented as means ± SD. d Representative images and quantification show downregulation of myelin basic protein (MBP) at 7 days after Clo-Lip injection. n = 6; ***p <0.001 versus contralateral striatum; Student’s t test. Data are presented as means ± SD

Fig. 4

Microglial depletion induces neuronal degeneration. a–c Representative images and quantification of MAP2 (green, a), PSD95 (red, b), and neurofilament (NF; green, c) expression on day 7 after clodronate liposome (Clo-Lip) injection. Scale bars: a 25 μm; b 25 μm, inset 10 μm; c 50 μm. n = 6 per group; **p <0.01, ***p <0.001 versus contralateral striatum; Student’s t test. d Representative images of primary cultured neurons exposed to Dil-labeled liposomes (Dil-Lip) for 18 h. Inset: high magnification of cultured neurons with MAP2 immunofluorescence staining. White, Dil-Lip. Red, MAP2. Scale bar = 25 μm; inset, 10 μm. e Primary cultured neurons were exposed to different concentrations of Dil-Lip or Clo-Lip for 18 h. Then cytotoxicity of neurons was measured by ATP assay. n = 8–12 (n = 8 in the sham group, n = 12 in other groups). **p < 0.01, ***p < 0.001 versus sham group; one-way ANOVA with Bonferroni post hoc test. Data are presented as means ± SD

Fig. 5

Microglial depletion reduces the density and integrity of blood vessels. a Left panel, representative images of CD31 expression (red) on day 7 after Clo-Lip injection. Outlined area indicates injection center. Right panel, higher magnification images of CD31. Scale bars = 50 μm (left panel), 25 μm (right panel). b The density and diameter of blood vessels. n = 6; *p <0.05; unpaired Student’s t test. Data are presented as means ± SD. c Degraded claudin-5 was analyzed by Western blot at each time point after Clo-Lip injection. Claudin-5 (MW, 20 kDa) was increased on days 3 and 7 after Clo-Lip injection. n = 6; ***p < 0.001 versus sham group; one-way ANOVA with Bonferroni post hoc test. Data are presented as means ± SD. d Expression of ZO-1 on days 1 and 3 after Clo-Lip injection. n = 6; ns, not significant. One-way ANOVA with Bonferroni post hoc test. Data are presented as means ± SD