Systemic inflammation regulates microglial responses to tissue damage in vivo

Abstract

Microglia, the resident immune cells of the central nervous system, exist in either a "resting" state associated with physiological tissue surveillance or an "activated" state in neuroinflammation. We recently showed that ATP is the primary chemoattractor to tissue damage in vivo and elicits opposite effects on the motility of activated microglia in vitro through activation of adenosine A2A receptors. However, whether systemic inflammation affects microglial responses to tissue damage in vivo remains largely unknown. Using in vivo two-photon imaging of mice, we show that injection of lipopolysaccharide (LPS) at levels that can produce both clear neuroinflammation and some features of sepsis significantly reduced the rate of microglial response to laser-induced ablation injury in vivo. Under proinflammatory conditions, microglial processes initially retracted from the ablation site, but subsequently moved toward and engulfed the damaged area. Analyzing the process dynamics in 3D cultures of primary microglia indicated that only A2A , but not A1 or A3 receptors, mediate process retraction in LPS-activated microglia. The A2A receptor antagonists caffeine and preladenant reduced adenosine-mediated process retraction in activated microglia in vitro. Finally, administration of preladenant before induction of laser ablation in vivo accelerated the microglial response to injury following systemic inflammation. The regulation of rapid microglial responses to sites of injury by A2A receptors could have implications for their ability to respond to the neuronal death occurring under conditions of neuroinflammation in neurodegenerative disorders.

Keywords: A2A receptors; imaging; microglia; motility; neuroinflammation.

Figure 1

Confirmation of microglial activation in vivo. CX₃CR1^GFP/+ mice treated with 2 mg/kg LPS i.p. were examined for the presence of neuroinflammation 2 days later. A. Expression of the pro-inflammatory cytokines IL-1β and TNF-α, as determined with RT-PCR, increased following LPS treatment. Representative images from one of three PBS- (control, C) or LPS-injected animals for each treatment are shown. B. FluoroJade staining shows that PBS or peripheral LPS treatment (2 mg/kg) do not induce cell death in the cortex (side panels). In parallel, control experiments, pilocarpine (pilo) produced strong cell death (inset). n=3 mice each for LPS and PBS. Scale bar: 50 μm. C. 2D projections of a 30 μm section from the cortex of control (n=9) and LPS-injected (n=11) mice showing altered microglial morphology. Scale bar: 50 μm. Increase in the cell body area (D) and the number of primary processes (E) are morphological changes consistent with microglial activation. Statistics: Student’s t-test, *, p<0.05.

Figure 2

Microglial motility under baseline conditions in vivo. A. The baseline motility of microglia was assessed with time-lapse 2P imaging of control (n=9) or LPS-injected (n=11) CX₃CR1^GFP/+ mice. 2D projections of the cortex spanning ~30 μm vertical distance were analyzed with Imaris software to quantify baseline process dynamics. The figure shows a representative image of microglia in a LPS-injected mouse. Red dots denote objects identified by the software and tracked over time. Scale bar: 50 μm. The average track speed (B) and distance traveled (C) increased in magnitude following microglial activation. Statistics: Student’s t-test, *, p<0.05.

Figure 3

Microglial response to laser-induced tissue damage under resting and pro- inflammatory conditions in vivo. Select images from time-lapse 2P recordings from (A) control (n=9) and (B) LPS-injected (n=11) CX₃CR1^GFP/+ mice show that activated microglia in LPS- treated animals have a delayed response to laser-induced tissue damage. Arrow in first image (t=3 min) points to the location of the laser ablation. Scale bar: 20 μm.

Figure 4

Quantification of microglial response to laser-induced tissue damage in vivo. Time-lapse 2P recordings from control (n=9) and LPS-injected (n=11) CX₃CR1^GFP/+ mice were analyzed with MGPtracker, a custom-written Matlab code to quantify the approach of microglial process to the site of damage. A. An example of detection of microglial processes; the green radial lines divide the image in 36 sectors. The vertices of the red polygon correspond to the microglial processes closest to the ablation in each sector. B. Positions of the vertices of the front-tracking polygon at different time points. C, D. The average distance of the polygon from the ablation site (C), and the average area bound by the polygon (D) over time show different rates of approach to the ablation following microglial activation with LPS. Statistics: two-way ANOVA with Bonferroni’s post hoc test, *, p<0.05 between control and LPS at the indicated time points. E. Proportion of control and LPS-treated animals that displayed initial retraction from the ablation site. Statistics: G-test of independence, *, p<0.05. F. Average time to reach the ablation in control and LPS-injected animals. Statistics: Student’s t-test, n.s., not significant.

Figure 5

Confocal imaging and 3D reconstruction of primary microglia in vitro. Primary microglia from actin-GFP mice grown in Matrigel were treated with 100 ng/mL LPS or HBSS for 24 hr. Confocal imaging over time and 3D reconstructions of the cells at each time point with the Imaris software were used to study cell ramification. The figure shows the effects of ATP treatment on cell morphology of a resting (A) and LPS-activated (B) cell: while ATP induces process extension in the resting cell, it causes retraction of processes in the LPS-activated cell. Scale bar: 10 μm.

Figure 6

Mechanisms underlying microglial process retraction under pro-inflammatory conditions in vitro. 3D cell reconstructions from primary actin-GFP microglia grown in Matrigel were used to determine cell ramification (expressed as surface area-to-volume ratios) in response to different treatments. A. ATP application (20 μM) exerts divergent effects on the ramification of resting and LPS-activated microglia. B. The non-hydrolysable P2Y₁₂ receptor agonist ADPβS (10 μM) increases cell ramification in resting microglia only. C. The A_2A receptor agonist adenosine (10 μM) reduces cell ramification in activated microglia only. D. Summary of the effects of purinergic receptor agonists on cell ramification assessed as the area under the ramification curves. Statistics: two-way ANOVA and Bonferroni’s post hoc test (compared to control cells for each treatment), *, p<0.05. E. The selective A₁ receptor agonist 2′-MeCCPA (1 μM) or the selective A₃ receptor agonist 2-Cl-IB-MECA (0.5 μM) do not affect process dynamics of activated microglia, but 3 μM of the selective A_2A agonist CGS-21680 induces process retraction. F. Summary of the effects of subtype-selective adenosine receptor agonists. Statistics: one-way ANOVA and Tukey’s post hoc test, *, p<0.05. G. The non-selective A_2A receptor antagonist caffeine (100 μM) or the selective A_2A receptor antagonist preladenant (1 μM) both prevent adenosine-induced process retraction in activated microglia. H. Summary of the effects of adenosine A_2A receptor antagonists. Statistics: one-way ANOVA and Tukey’s post hoc test, *, p<0.05. The number of cells for each treatment is shown in parentheses in D, F, and H.

Figure 7

Microglial responses to laser-induced tissue damage following treatment with the A_2A receptor antagonist preladenant in vivo. Representative maximum intensity projections from time-lapse 2P recordings from LPS-injected CX₃CR1^GFP/+ mice before (A) and after preladenant (B) treatment (3 mg/kg, i.p., 1 hr before imaging) at different time points. Arrow in first image (t=3 min) points to the location of the laser ablation. Microglia appear to approach the injury site faster following preladenant injection. Scale bar: 20 μm.

Figure 8

Quantification of microglial responses to tissue damage following preladenant treatment. A, B. Time-lapse 2P sequences from LPS-injected CX₃CR1^GFP/+ mice before and after preladenant injection (n=7 mice) or control PBS injection (n=4 mice) were analyzed with MGPtracker to quantify the radial response to injury. Preladenant treatment induces a significant difference in the average distance of the polygon from the ablation site (A). In contrast, there is no effect of vehicle (B). Statistics: 2way RM-ANOVA, *, p<0.05. C, D. Time to reach the ablation before and after preladenant (C) or PBS (D) treatment. Six out of seven preladenant-treated animals show faster approach after treatment. Only one of four PBS-treated mice shows a decrease in the time to reach the ablation. Statistics: (C, D) Student’s paired t-test. E. Proportion of responses that were accelerated or slowed by treatment. Statistics: G-test of independence, *, p<0.05.