(A) A field of microglia in an awake mouse imaged over 14 consecutive days. The dark diagonal lines in the center and top right of the image are blood vessels that remain structurally stable and can …
Source data for microglia turnover and migration in the physiological brain.
Source data for CSF1R inhibition in the adult brain.
Source data for the Cytokine panel conducted.
(A–C) Representative maximum intensity projections of confocal images of microglia from the primary visual cortex (V1) in fixed sections of control (A), 7 days PLX (B), and 7 days repopulated (C) …
(A–L) A combination of anti-inflammatory and pro-inflammatory cytokines including IL-1b, IL-4, IL-6, IL-10, and TFNα were tested using a Multiplex Assay. Each experiment was assayed in triplicate. …
(A) A field of microglia during depletion and repopulation imaged in vivo in the same awake mouse. (B) The number of microglia (normalized to control day for each animal) during depletion (PLX) and …
Source data of microglial repopulation after partial depletion.
Source data for the GFAP expression following microglial depletion.
Representative confocal images of astrocytes immunoreacted for GFAP in fixed brain sections from mice in control conditions (A) after PLX treatment (B) and after 30 days of repopulation (C). …
(A) Number of microglia for CX3CR1-GFP (red) and P2Y12-KO (black) animals for each imaging normalized to first control time point. (B–J) There was no significant difference over the course of …
(A) The nearest neighbor quantification for CX3CR1-GFP (red) and P2Y12-KO (black) animals for each imaging time point normalized to the first control time point. (B–J) Differences between genotypes …
(A) Representative in vivo two-photon imaging of microglial division in CX3CR1-GFP awake mice. Inset is a magnified image of the dividing doublet microglia, which appears as an elongated cell body …
Source data for microglial repopulation.
Galectin three expression in microglia after repopulation.
Representative confocal images of microglia in CX3CR1-GFP mice immunoreacted for Galectin 3 (Gal3) in fixed brain sections from mice in control conditions (A) at the peak of repopulation (Day 2) (B).…
We imaged CX3CR1-GFP mice every 4 hr for 24 hr during the peak of repopulation (day 2 day 3). We observed a range of behaviors of existing microglia: (A) no division, (B) rapid division on the time …
Source data for microglia repopulation in mice imaged every 4 hr for 24 hr.
(A–C) Mathematical modeling of microglial repopulation using measured kinetic parameters. Empirical ρvalues from the simulation are plotted for a minimum 4 hr delay (A) and 8 hr delay (B) between …
Code to generate the mathematical model simulation.
Source data for the mathematical modeling of microglial repopulation.
A schematic to demonstrate the steps of the mathematical model and the cells that were used to estimate the total population of cells at the end of 24 hr. In this example, we start N0=4 (there are …
(A) Individual microglia imaged in CX3CR1-GFP and P2Y12-KO mice during control, depletion (7 days of PLX), and repopulation (7 and 30 days). (B) Sholl profiles summarizing the morphology of …
Source data for the morphology and soma area of newly born microglia.
(A–E) Average Sholl profiles of CX3CR1-GFP (red) and P2Y12-KO (black) animals for each imaging time point. (F–J) Quantification of the maximum intersections of the Sholl profiles showing that …
(A) Example of motility analysis showing the original microglia image, (B) Thresholded microglia (C) and a visual representation of microglial motility whereby thresholded imaged from two time …
Source data for newly born microglia motility, surveillance during depletion and repopulation.
(A) The motility index for both CX3Cr1GFP/+ (red) and P2Y12KO (black) animals. (B–F) While there was a slight reduction in motility in P2Y12KO animals in the early stages of repopulation (D1–D3), …
The instability index is the proportion of stable pixels that retracted over the total stable pixels in the first time point. (A) The instability index in both CX3CR1-GFP (red) and P2Y12-KO (black) …
(A). The stability index (the proportion of extended pixels that became stable divided by the total extended pixels in the first overlay) was compared between the two genotypes. (B–F) P2Y12 KO mice …
(A) Microglial surveillance of the parenchyma compared between CX3CR1-GFP (red) and P2Y12-KO (black) animals for each imaging time point. (B–F) P2Y12 KO animals showed smaller surveillance at the …
(A) Graph of surveillance normalized to the extent of microglial coverage in the first time point compared between CX3CR1-GFP (red) and P2Y12-KO (black) animals for each imaging time point. (B–F) …
Example microglial response to focal laser ablation at t=0 min, t=30min and t=55 min after ablation for control (A), 3 days of repopulation (B), 7 days of repopulation (C), and 30 days of …
Source data for newly-born microglia response to acute laser ablation injury.
Two-photon in vivo images were obtained in CX3CR1-GFP awake mice following three consecutive days of training and habituation to the setup with a 1.5x zoom. This video shows a 100-slice z-stack …
Two-photon in vivo images were obtained in CX3CR1-GFP awake mice following three consecutive days of training and habituation to the setup with a 1.5x zoom. This video shows a 100-slice z-stack in …
Imaging was done in CX3CR1-GFP awake with a 1.5x zoom. Here, we compared a laser ablation during control condition, depletion (PLX), and repopulation (D7) and D30. Microglia responded rapidly during …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background | CX3CR1-GFP, C57/Bl6 background (Mouse) | JAX | RRID:IMSR_JAX:005582 Jax stock #005582 | B6.129P2(Cg)-Cx3cr1tm1Litt/J Male and Females used in our study |
Strain, strain background | P2Y12 KO, C57/Bl6 background (Mouse) | Courtesy of Maiken Nedergaard lab via a Materials transfer agreement | Mice were generated by the Conley lab doi:10.1172/JCI17864. Male and Females used in our study | |
Chemical compound, drug | PLX5622 drug | Research Diets, New Jersey, USA | AIN76A-D1001i | PLX5622 drug from Plexxikon Inc, Berkeley, CA, USA |
Chemical compound, drug | Fentanyl (0.05 mg/kg) | University of Rochester Medical Center Pharmacy | ||
Chemical compound, drug | Midazolam (5.0 mg/kg) | University of Rochester Medical Center Pharmacy | ||
Chemical compound, drug | Dexmedetomidine (0.5 mg/kg) | University of Rochester Medical Center Pharmacy | ||
Other | 3 mm Biopsy punch | Integra, Plainsboro, NJ, USA | Cat#: 33–32 | |
Other | 0.5 mm drill bit | FST, Foster City, CA | Cat#: 19007–05 | |
Other | UV Glue | Norland Optical Adhesive, Norland Inc, Cranbury, NJ, USA | Cat#: NOA 61 | |
Other | Loctite 404 Glue | Henkel Corp, Bridgewater, NJ, USA | Cat#: A-A-309 | |
Other | Headplate | Emachine shop | Design courtesy of Mriganka Sur | |
Other | C and B Metabond Dental Cement | Parkell Inc Brentwood, NY, USA | SKU #: S380 | |
Other | Ti: Sapphire | Mai-Tai Spectraphysics Olympics, Center Valley, PA | Custom built two photon microscope | |
Antibody | Anti-Iba-1 (Rabbit polyclonal) | Wako | RRID:AB_839504 Cat # 019–19741 | IF(1:1000) |
Antibody | Anti-GFAP Glial Fibrillary Acidic Protein (Mouse monoclonal) | Sigma | RRID:AB_477010 Cat# G3893 | IF(1:500) |
Antibody | Anti-Galectin3 (Goat polcylonal) | R and D systems | RRID:AB_2234687 Cat# AF1197 | IF(1:1500) |
Antibody | AlexaFlour 488 (Donkey Anti-Rabbit) | Invitrogen | RRID:AB_2762833 Cat# A32790 | IF(1:500) |
Antibody | AlexaFlour 594 (Donkey Anti-Mouse) | Invitrogen | RRID:AB_2762826 Cat# A32744 | IF(1:500) |
Antibody | AlexaFlour 594 (Donkey Anti-Goat) | Invitrogen | RRID:AB_2762828 Cat# A32758 | IF(1:1000) |
Commercial assay or kit | MILLIPLEX MAP Mouse TH17 Magnetic Bead Panel (Mouse) | Millipore, Sigma | MTH17MAG-47K | |
MATLAB | MATLAB and Statistics Toolbox Release 2012b, The MathWorks, Inc, Natick, | Massachusetts, United States | RRID:SCR_001622 | |
GraphPad Prism | GraphPad Software, https://www.graphpad.com’. | San Diego, California USA, | RRID:SCR_002798 | |
R | R Project for Statistical Computing | RRID:SCR_001905 |