Research ArticleNEUROSCIENCE

Repopulating retinal microglia restore endogenous organization and function under CX3CL1-CX3CR1 regulation

See allHide authors and affiliations

Science Advances  21 Mar 2018:
Vol. 4, no. 3, eaap8492
DOI: 10.1126/sciadv.aap8492
  • Fig. 1 Microglial repopulation of the adult mouse retina following genetic depletion occurs in a progressive center-to-peripheral direction originating from the optic nerve head.

    (A to F) The overall distribution of Iba1-immunopositive cells was analyzed in flat-mounted retinal preparations; insets (red boxes) show repopulating cells at high magnification. (A) Young adult (2 months old) CX3CR1CreER-DTA transgenic mice demonstrated a wild-type–like distribution of microglia in the retina under baseline conditions. (B and C) Following the administration of oral tamoxifen (500 mg/kg per dose, two doses administered 1 day apart) to deplete the retina of microglia, few microglia remained at 2 to 9 days DPG. (D to F) Progressive repopulation of the retina occurred thereafter in a central-to-peripheral direction extending to all topographical areas of the retina. Scale bar, 1 mm. (G) The percentage of the total retinal area occupied by Iba1+ cells was measured at different times following tamoxifen-induced depletion. Repopulating cells extended to all retinal areas by 30 DPG. (H) The total number of Iba1+ cells in the retina following depletion recovered progressively to reach the number present before depletion. (I) mRNA expression levels of microglia-enriched genes in the retina, assessed using next-generation sequencing, demonstrated marked decreases at 5 DPG, followed by progressive recovery at 16 and 30 DPG. mRNA levels of each species were normalized to the mean level of expression in the controls. [Graphical data in (G), (H), and (I) are presented as means ± SEM; one-way analysis of variance (ANOVA) and Sidak’s multiple comparison test, n = 3 animals of mixed sex at each time point, except n = 4 in the transgenic (TG) control group. Asterisks (*) indicate comparisons with control for which P < 0.05.]

  • Fig. 2 Microglial repopulation in the retina involves microglial migratory transit in an inner-to-outer direction to reestablish local laminar densities in each plexiform layer.

    (A) The spatial pattern of microglia recolonization in separate retinal lamina following depletion was analyzed in retinal sections; asterisks (*) indicate the optic nerve head, and insets show high-magnification views of the IPL and OPL. Near-complete absence of retinal microglia in all retinal layers was observed at 2 DPG, with few Iba1+ cells found only in the IPL near the optic nerve head. At 16 DPG, significant numbers of Iba1+ cells were present in the IPL but were largely absent in the OPL. Recolonization of the OPL progressed at later time points from central to peripheral retinal areas. Scale bar, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (B) Repopulation dynamics of Iba1-labeled microglia in the IPL and OPL were analyzed additionally in retinal flat-mounts using confocal microscopy at midpoint between the optic nerve head and the retinal periphery. Scale bar, 100 μm. (C) Quantitative analysis of microglial density demonstrated a rapid repopulation of the IPL at 16 DPG (top) that exceeded the numbers of endogenous microglia present at baseline (TG control). At subsequent time points, microglial density in the IPL slowly declined to baseline levels by 150 DPG. Microglial repopulation of the OPL (bottom) demonstrated a slow progressive recovery, reaching baseline levels by 150 DPG. (D) The proportion of interplexiform Iba1+ cells (with somata located between IPL and OPL lamina), expressed as a fraction of all microglial cells in the imaging field, was assessed at (i) baseline (before depletion), (ii) midway through the repopulation process (60 DPG), and (iii) upon the reattainment of baseline distribution (150 DPG). Scale bar, 10 μm. The proportion of interplexiform Iba1+ cells was significantly greater at 60 DPG than at other time points, indicating an inner-to-outer migration of microglia during repopulation. Graphical data in (C) and (D) are presented as means ± SEM (n = 12 imaging retinal fields from three animals of mixed sex at each time point; P values were derived from one-way ANOVA and Sidak’s multiple comparison test).

  • Fig. 3 Iba1+ cells that repopulate the retina following depletion arise from residual endogenous microglia that demonstrate dynamic cellular migration and in situ proliferation.

    (A) Two-month-old CX3CR1CreER/+:Rosa26-flox-STOP-flox-tdTomato transgenic mice (CX3CR1 CreER/+:tdT mice) were administered a bolus dose of tamoxifen (two oral gavage doses of 10 mg, 1 day apart) to induce the expression of tdT in CX3CR1-expressing myeloid cells. They were then maintained under standard conditions for 3 months to allow for the turnover and full replacement of circulating monocytes by newly generated tdT monocytes. Long-lived endogenous microglia in the retina retained their tdT labeling. In vivo fluorescence fundus images centered on the optic nerve were obtained using a 518-nm excitation laser to detect cells expressing red fluorescent protein (RFP). Fundus images revealed extensive tdT expression in retinal microglia before PLX5622-induced depletion at baseline. Following 1 week of PLX5622 administration (day 0), a few tdT-expressing microglia remained (yellow arrows). Fundus imaging at days 7 and 30 demonstrated a gradual repopulation of the retina in a center-to-periphery direction by tdT+ microglia, indicating residual microglia as the source of repopulating cells. Scale bar, 200 μm. (B) Analysis of flat-mounted retina at day 30 of repopulation showed that nearly all Iba1+ (green) repopulating cells were also tdT+ (red). The boxed area in the merged image is shown on the right at higher magnification. Scale bar, 100 μm. (C) Quantification of the proportion of Iba1+ cells that were also tdT+ at baseline (before tamoxifen administration) and at day 30 following depletion indicated that close to 100% of Iba1+ repopulating microglia were tdT+ (n = 5, 3 animals at baseline and at day 30, respectively). n.s., not significant. (D) To detect the contribution of CCR2-expressing monocytes to repopulating Iba1+ cells, CCR2+/RFP mice containing RFP+ monocytes were subjected to PLX5622-induced depletion and analyzed at day 10 following depletion. No expression of CCR2 was noted in repopulating Iba1+ microglia, either at the optic nerve head (shown in the inset at high magnification) or elsewhere in the retina. (E) Microglial repopulation at day 16 of the genetic depletion model involving CX3CR1Cre/+:CCR2+/RFP mice similarly consisted of Iba1+ repopulating cells that were negative for CCR2 expression. Scale bars, 1 mm (D and E). (F) Eleven-week-old CX3CR1+/GFP transgenic mice with GFP-labeled microglia were administered PLX5622 for 7 days to deplete retinal microglia. Time-lapse in vivo fundus imaging was performed at 5 days (top) and 12 days (bottom) following PLX5622 cessation. At day 5 of repopulation, few GFP+ cells were observed in the central retina near the optic nerve head (yellow circle). Time-lapse images taken every 1 to 1.5 hours revealed dynamic and prominent horizontal migration of GFP+ cells across the retina (the red arrow indicates a migrating cell with the red line indicating its migratory track). Binary cellular divisions of repopulating microglia followed by the separation of the two daughter cells (yellow arrows) were also observed. Scale bar, 250 μm. (G) At day 12 of repopulation, when microglial density in the central retina had increased significantly, migration and cellular division were slowed relative to that at day 5 (red arrows show a cell with a relatively stable position). Scale bar, 250 μm. (H) Quantitative comparison of dynamic migration of individual repopulating microglia, demonstrating higher mean migration velocity, higher peak velocity of movement, and greater net displacement of cell position at day 5 when repopulating microglia were lower in density, relative to corresponding values at day 12 (P values from Mann-Whitney test; n = 40 and 32 cells from 4 and 3 animals at days 5 and 12, respectively). (I) The frequency of dynamic cell divisions was higher early in the repopulation process at day 5 relative to that at day 12 (n = 4 and 3 recordings from 4 and 3 animals at days 5 and 12, respectively).

  • Fig. 4 Repopulating microglia recapitulate endogenous microglial functions of dynamic process motility and response to injury signals.

    Ten-week-old CX3CR1+/GFP transgenic mice were administered PLX5622 for 1 week to induce depletion of retinal microglia and then allowed to undergo full repopulation for 60 days. Age-matched CX3CR1+/GFP mice maintained on a standard diet served as controls. (A) Ex vivo live-cell time-lapse imaging was performed in retinal explants to monitor process dynamics of repopulated microglia in PLX5622-fed mice versus endogenous microglia in control mice. Repopulated microglia relative to endogenous microglia demonstrated similar process motility at baseline and increased process motility and process elaboration in response to ATP stimulation (1 mM bath application). Scale bar, 30 μm. Quantitative analysis demonstrated that process motility measures, in terms of rates of process extension and process retraction, at baseline and in response to ATP stimulation were statistically similar between endogenous microglia (white bars) and repopulated microglia (red bars) (P values from Mann-Whitney test; control group = 21 cells from 9 recordings, repopulation group = 23 cells from 11 recordings, 4 animals in each group). (B and C) Response of endogenous versus repopulated cells in an in vivo model of light-induced injury. Wild-type mice were subjected to transient depletion (7 days of PLX5622 administration) and allowed to undergo full repopulation for 30 days and then subjected to light-induced photoreceptor injury. Nondepleted age-matched wild-type mice served as controls. Histological analysis (B) showed that repopulated Iba1+ cells, such as endogenous microglia, responded to photoreceptor injury by migration into the outer retina and the subretinal space and by up-regulation of CD68 expression (insets show the presence of subretinal Iba1+ and CD68+ cells in both groups). Scale bar, 40 μm. (C) Levels of post-injury inflammatory cytokines [interleukin (IL)-1β, IL-10, C-C motif chemokine ligand 3 (CCL3), CCL5, and tumor necrosis factor–α (TNFα)] were similarly elevated in retinas containing endogenous microglia versus repopulated cells (P > 0.05 for all comparisons; Mann-Whitney test, n = 5 retina from individual animals of mixed sex per group).

  • Fig. 5 Microglial repopulation rescues deterioration of retinal function induced by microglia depletion in the CX3CR1CreER-DTA model.

    Twelve-week-old CX3CR1CreER-DTA mice were subjected to sustained microglia depletion for 30 days (light blue line). Following this period, experimental mice were divided into three subgroups: (i) maintained microglial depletion for another 30 days (total of 60 days) (dark blue line), (ii) repopulation for 30 days (pink line), and (iii) repopulation for 60 days (red line). Undepleted CX3CR1CreER-DTA mice served as controls (black dashed line). Sustained microglial depletion resulted in decreases in ERG responses in the a- and b-wave amplitudes that progressed from 30 to 60 days depletion. In the groups undergoing repopulation, these further decrements in ERG responses were halted and were significantly greater than those in animals undergoing 60 days sustained depletion. Upper panels show ERG amplitudes in all subgroups with data points and error bars indicating mean responses ± SEM. Lower panels show statistical comparisons between subgroups with data points and error bars indicating mean differences and ±95% confidence intervals [error bars not crossing x = 0 indicate significant (P < 0.05) comparisons]. Two-way ANOVA with Sidak’s multiple comparison test was used to calculate P values; n = 24 eyes in 12 animals in the control subgroup, and n = 8 eyes in 4 animals for all other subgroups.

  • Fig. 6 Microglia repopulation following depletion in the retina is slowed in CX3CR1 deficiency.

    Eight-week-old signaling-deficient CX3CR1−/− mice and their signaling-sufficient CX3CR1+/− littermates were subjected to PLX5622-mediated microglial depletion for 7 days and allowed to undergo microglial repopulation. (A and B) Microglial repopulation in the central retina was followed for the first 10 days of repopulation using in vivo fundus imaging of GFP-labeled microglia and compared between CX3CR1+/− and CX3CR1−/− littermates. The numbers of residual microglia were comparable in the two groups following depletion (day 0), but CX3CR1-deficient microglia demonstrated significantly slower recovery of microglial numbers at days 7 and 10 (n = 8 CX3CR1+/− and 12 CX3CR1−/− animals). (C and D) Microglial repopulation in the OPL of the mid-peripheral retina was compared in flat-mounted retina. Although microglial densities, revealed by GFP expression, were similar between the two genotypes at baseline, the recovery of microglial density was slower in CX3CR1−/− animals at day 14. At day 32, microglial densities were comparable between the genotypes and approached baseline (n = 9, 12, and 12 imaging fields from 3, 4, and 4 animals for each genotype at baseline, day 14, and day 32, respectively). (E) The proportion of repopulating microglia undergoing active replication at day 14, as measured by Ki67 immunopositivity, was significantly lower in CX3CR1−/− animals (n = 12 imaging fields in 4 animals for each genotype group). (F to J) Morphological analyses of GFP-expressing repopulating microglia in the OPL showed that while the morphology was partially similar between genotypes at baseline, the maturation of ramified morphology at day 14 was significantly slower in CX3CR1−/− animals. By day 32, morphology in both genotypes was comparable and recovered to baseline levels. Besides, the fractional area coverage of synaptic laminar by CX3CR1−/− microglia was significantly lower at day 14. (G to I) n = 27, 36, and 36 cells [(J) n = 9, 12, and 12 imaging fields)] from 3, 4, and 4 animals for each genotype at baseline, day 14, and day 32, respectively. (A to D and J) One-way ANOVA with Sidak’s multiple-comparisons test, (E) unpaired t test with Welch’s correction, and (G to I) Kruskal-Wallis test with Dunn’s multiple-comparisons test. Scale bars, 250 μm (A), 100 μm (C), and 50 μm (F).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/3/eaap8492/DC1

    fig. S1. Microglial repopulation in the adult wild-type mouse retina in a pharmacological (PLX5622) model of microglial depletion demonstrates a center-to-peripheral, vitreal-to-scleral progression.

    fig. S2. Repopulating Iba1+ cells undergo progressive morphological maturation to recapitulate morphologies of endogenous retinal microglia in the CX3CR1CreER-DTA model of microglial depletion.

    fig. S3. Repopulating Iba1+ cells undergo progressive morphological maturation to recapitulate morphologies of endogenous retinal microglia in the PLX5622-mediated model of microglial depletion.

    fig. S4. Repopulating Iba1+ cells demonstrate transient expression of markers of microglial activation and replication in the CX3CR1CreER-DTA model of microglial depletion.

    fig. S5. Repopulating Iba1+ cells demonstrate transient expression of markers of activation and replication in the PLX5622-induced model of microglial depletion.

    fig. S6. Microglial repopulation rescues deterioration of retinal function induced by microglia depletion in the PLX5622 model.

    fig. S7. Intravitreal delivery of exogenous CX3CL1 accelerates microglial repopulation.

    fig. S8. Estimation of cell proliferation dynamics during the repopulation process: Proliferation rates of residual microglia are sufficient to regenerate cells observed during the repopulation process.

    movie S1. Dynamic migration and in situ replication of repopulating microglia in the retina.

    movie S2. Dynamic migration and replication of repopulating microglia slow down as microglial density increases in the retina.

    movie S3. Repopulating microglia demonstrate dynamic motility in their processes at baseline and in response to exogenous ATP that are similar to that in endogenous microglia.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Microglial repopulation in the adult wild-type mouse retina in a pharmacological (PLX5622) model of microglial depletion demonstrates a center-to-peripheral, vitreal-to-scleral progression.
    • fig. S2. Repopulating Iba1+ cells undergo progressive morphological maturation to recapitulate morphologies of endogenous retinal microglia in the CX3CR1CreER-DTA model of microglial depletion.
    • fig. S3. Repopulating Iba1+ cells undergo progressive morphological maturation to recapitulate morphologies of endogenous retinal microglia in the PLX5622-mediated model of microglial depletion.
    • fig. S4. Repopulating Iba1+ cells demonstrate transient expression of markers of microglial activation and replication in the CX3CR1CreER-DTA model of microglial depletion.
    • fig. S5. Repopulating Iba1+ cells demonstrate transient expression of markers of activation and replication in the PLX5622-induced model of microglial depletion.
    • fig. S6. Microglial repopulation rescues deterioration of retinal function induced by microglia depletion in the PLX5622 model.
    • fig. S7. Intravitreal delivery of exogenous CX3CL1 accelerates microglial repopulation.
    • fig. S8. Estimation of cell proliferation dynamics during the repopulation process: Proliferation rates of residual microglia are sufficient to regenerate cells observed during the repopulation process.
    • Legends for movies S1 to S3

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). Dynamic migration and in situ replication of repopulating microglia in the retina.
    • movie S2 (.avi format). Dynamic migration and replication of repopulating microglia slow down as microglial density increases in the retina.
    • movie S3 (.avi format). Repopulating microglia demonstrate dynamic motility in their processes at baseline and in response to exogenous ATP that are similar to that in endogenous microglia.

    Files in this Data Supplement:

Navigate This Article