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Cited by 228Neuroinflammation and Neurodegeneration MechanismsNeurogenesis and neuroplasticity mechanismsTryptophan and brain disorders

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Characterizing Newly Repopulated Microglia in the Adult Mouse: Impacts on Animal Behavior, Cell Morphology, and Neuroinflammation

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Elmore, Monica R. P., Lee, Rafael J., West, Brian L., Green, Kim N.·2015·PLoS ONE
DOI 10.1371/journal.pone.0122912PMC PMC4388515
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Abstract

Microglia are the primary immune cell in the brain and are postulated to play important roles outside of immunity. Administration of the dual colony-stimulating factor 1 receptor (CSF1R)/c-Kit kinase inhibitor, PLX3397, to adult mice results in the elimination of ~99% of microglia, which remain eliminated for as long as treatment continues. Upon removal of the inhibitor, microglia rapidly repopulate the entire adult brain, stemming from a central nervous system (CNS) resident progenitor cell. Using this method of microglial elimination and repopulation, the role of microglia in both healthy and diseased states can be explored. Here, we examine the responsiveness of newly repopulated microglia to an inflammatory stimulus, as well as determine the impact of these cells on behavior, cognition, and neuroinflammation. Two month-old wild-type mice were placed on either control or PLX3397 diet for 21 d to eliminate microglia. PLX3397 diet was then removed in a subset of animals to allow microglia to repopulate and behavioral testing conducted beginning at 14 d repopulation. Finally, inflammatory profiling of the microglia-repopulated brain in response to lipopolysaccharide (LPS; 0.25 mg/kg) or phosphate buffered saline (PBS) was determined 21 d after inhibitor removal using quantitative real time polymerase chain reaction (RT-PCR), as well as detailed analyses of microglial morphologies. We find mice with repopulated microglia to perform similarly to controls by measures of behavior, cognition, and motor function. Compared to control/resident microglia, repopulated microglia had larger cell bodies and less complex branching in their processes, which resolved over time after inhibitor removal. Inflammatory profiling revealed that the mRNA gene expression of repopulated microglia was similar to normal resident microglia and that these new cells appear functional and responsive to LPS. Overall, these data demonstrate that newly repopulated microglia function similarly to the original resident microglia without any apparent adverse effects in healthy adult mice.

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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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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.
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.CC BY · PubMed Central

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Authors
Elmore, M. R. P., Lee, R. J., West, B. L., & Green, K. N.
Affiliations
  • University of California, Irvine (US)
  • Plexxikon (United States) (US)
Corresponding author
Green, Kim N.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2015-04-07
Volume
10
Issue
4
Pages
e0122912
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0122912
PMC
PMC4388515
OpenAlex
W2169700519
Cited by
228
References
73
Citations by year
2026: 10 · 2025: 20 · 2024: 19 · 2023: 19 · 2022: 23 · 2021: 32 · 2020: 26 · 2019: 28 · 2018: 23 · 2017: 18 · 2016: 6 · 2015: 4
FWCI
9.07
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • Plexxikon
  • National Institute on Aging (T32AG000096)
  • National Institute of Neurological Disorders and Stroke (R01NS083801)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
  • Life Sciences › Neuroscience › Biological Psychiatry › Tryptophan and brain disorders
Keywords
microglia, microglial repopulation, PLX3397, CSF1R inhibition, lipopolysaccharide response, neuroinflammation, microglial morphology, cognitive function, motor function, brain immune cells
MeSH terms
Animals, Brain, Cells, Cultured, Central Nervous System Diseases, Cognition, Female, Inflammation, Lipopolysaccharides, Male, Mice, Inbred C57BL, Motor Activity, Nerve Regeneration, Microglia, Maze Learning, Rotarod Performance Test, Cell Shape

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