Fig 1.
Elimination of Microglia in the Adult CNS Using CSF1R/c-Kit Inhibition, Followed by Repopulation with New Cells.
A, B) Two month-old wild-type mice were placed on either control (n = 10) or inhibitor diet (PLX3397, provided at 290 mg/kg chow; n = 14) for 21 d, causing the elimination of approximately 99% of microglia brain-wide. A subset of mice were sacrificed to confirm microglial elimination and are shown in (B) (n = 4). C) For the remaining microglia eliminated mice (n = 10), the inhibitor was removed, allowing new microglia to repopulate the entire CNS during a 21d recovery period. Half brain stitches are shown and each microglia is represented with a white dot. D–F) Images of the hippocampal region for each treatment are shown, with IBA1 staining in green. G–I) 10x and 63x z-stack images of the CA1 hippocampal region for each treatment are shown, with NeuN staining in green and GFAP staining in red.
Fig 2.
Impacts of Microglial Repopulation on Behavior, Cognition, and Motor Function.
A) Schematic describing experimental design. Mice were administered either control diet (n = 10) or PLX3397 for 21 d to eliminate microglia, then the inhibitor was removed to allow microglial repopulation (n = 10). From days 14 to 21, behavioral testing was conducted. Mice were given an LPS challenge (0.25 mg/kg LPS or PBS, both administered IP) on the final day of behavioral testing and sacrificed 6 h later (n = 5 per group). B, C) No changes in motor function were observed, as indicated by time on rotarod and average distance moved in the open field, between control and microglia repopulated animals. D) There were no changes in anxiety between treatments, as assessed by time spent in the center or perimeter of the open field arena. E) Both treatments showed similar cortical learning and memory, as indicated by no difference in the time spent investigating the novel object. F) Control and microglia repopulated mice showed similar learning curves during acquisition of the Morris water maze. G) Both treatments spent similar amounts of time in the different quadrants of the Morris water maze, with the most time spent in the correct quadrant (#2). H–J) Following an LPS challenge, there were no changes in distance moved, velocity, or time to locate the platform during reversal testing (21 d repopulation treatment labeled as “Repop” on figure). Data were analyzed using unpaired student’s t-tests and are presented as raw means ± SEM.
Fig 3.
Microglial Modeling for Morphological Analysis.
A–G) Examples of microglial modeling in the hippocampus of control, 14 d repopulation, and 21 d repopulation treatments are shown. 10x zoom images were analyzed with Imaris software. NeuN staining is shown in red and IBA1 in green. A–C) Reference images of the hippocampus for each treatment with microglia are shown in green. D–F) Modeling of the microglia with cell bodies and processes traced in white, as determined by the computer software, show nearly identical overlap with the reference images from (A–C). G, H) Zoom image of the microglial modeling from the 14 d repopulation treatment.
Fig 4.
Morphological Analysis of Control vs. Repopulated Microglia.
A–G) All morphological markers were examined in control (n = 10), 14 d repopulation (n = 5, “14d Repop”), and 21 d repopulation (n = 10, “21d Repop”) treatments to examine the change in microglia morphology over time. A) Changes in the number of microglia were observed in the cortex, but not the hippocampus during repopulation. B) Cell body area was much larger in 14 d repopulation microglia than either controls or 21 d repopulation in both the cortex and hippocampus. C, D) Microglial process diameter (C) and length (D) varied according to brain region and treatment, with some recovery apparent over time. E) Total process length/microglia did not vary due to brain region or treatment. F, G) The number of primary, secondary, and tertiary (or above) branches/microglia varied due to brain region and treatment, where secondary and tertiary (or above) branches were reduced in the cortex at 14 d repopulation, but had recovered by 21 d repopulation (F). While in the hippocampus, a reduction in the number of tertiary (and above) microglial branches were reduced over the course of repopulation (G). Data were analyzed using unpaired student’s t-tests and are presented as raw means ± SEM. Statistical significance is represented as *P<0.05.
Fig 5.
Response of Newly Repopulated Microglia to an Immune Challenge.
Half of the mice from each dietary treatment (control or 21 d repopulation, labeled as “Repop” on figure) were administered either LPS (0.25 mg/kg) or an equivalent volume of PBS via IP injection (n = 5 per group). Mice were euthanized 6 h post-injection (following reversal testing in the Morris water maze), half of each brain was collected and snap-frozen for RNA extraction, cDNA was synthesized, and quantitative RT-PCR performed to determine mRNA gene expression for a variety of inflammatory-related markers. A) Images of the hippocampal region of each treatment are shown. B, C) No changes in microglial number or IBA1+ staining was observed in response to microglial repopulation or LPS challenge. D) The relative mRNA expression of the microglia repopulated brain in response to LPS for a variety of inflammatory gene markers is shown. Data were analyzed as a two-way ANOVA (control vs. repopulated and LPS vs. PBS) and data are presented as raw means ± SEM. Significance is dictated by the following symbols: control + PBS vs. control + LPS*; control + PBS vs. repopulated + PBS†; control + LPS vs. repopulated + LPS#; repopulated + PBS vs. repopulated + LPSϕ (all comparisons, P<0.05). Full P-values can be found in Table 1.
Table 1.
P-values for Statistical Analysis of Inflammatory Profiling.