Neurodegeneration severity can be predicted from early microglia alterations monitored in vivo in a mouse model of chronic glaucoma

Abstract

Microglia serve key homeostatic roles, and respond to neuronal perturbation and decline with a high spatiotemporal resolution. The course of all chronic CNS pathologies is thus paralleled by local microgliosis and microglia activation, which begin at early stages of the disease. However, the possibility of using live monitoring of microglia during early disease progression to predict the severity of neurodegeneration has not been explored. Because the retina allows live tracking of fluorescent microglia in their intact niche, here we investigated their early changes in relation to later optic nerve neurodegeneration. To achieve this, we used the DBA/2J mouse model of inherited glaucoma, which develops progressive retinal ganglion cell degeneration of variable severity during aging, and represents a useful model to study pathogenic mechanisms of retinal ganglion cell decline that are similar to those in human glaucoma. We imaged CX3CR1(+/GFP) microglial cells in vivo at ages ranging from 1 to 5 months by confocal scanning laser ophthalmoscopy (cSLO) and quantified cell density and morphological activation. We detected early microgliosis at the optic nerve head (ONH), where axonopathy first manifests, and could track attenuation of this microgliosis induced by minocycline. We also observed heterogeneous and dynamic patterns of early microglia activation in the retina. When the same animals were aged and analyzed for the severity of optic nerve pathology at 10 months of age, we found a strong correlation with the levels of ONH microgliosis at 3 to 4 months. Our findings indicate that live imaging and monitoring the time course and levels of early retinal microgliosis and microglia activation in glaucoma could serve as indicators of future neurodegeneration severity.

Keywords: Confocal ophthalmoscopy; Cx3cr1GFP/+ DBA/2J; Live image analysis; Microglia activation; Microgliosis; Retinal ganglion cells.

Fig. 1.

In vivo monthly imaging of retinal and ONH microglia and/or peripheral monocytes during early stages of chronic glaucoma. (A) Monthly cSLO image sequence of the same eye, showing GFP⁺ cells within ∼2 mm² of retina around the ONH (circle) in young Cx3cr1^GFP/+ DBA/2J mice. The original greyscale was inverted to improve observation. (B) Multipoint cSLO image spanning approximately one third of the same retina when the mouse was 5 months old. There is a cluster of GFP-labeled cells localized to the ONH (circle), and radial rows of perivascular cells with bipolar shape along vessels (arrows), which interrupt the regular mosaic of parenchymal microglia. Live infrared fundus images of vasculature and optic disc (inset) were acquired for each fluorescence image, to facilitate the alignment of sequential images. (C) High-magnification view of parenchymal and perivascular GFP⁺ cells (arrows). For parenchymal cells, the microglial soma size is readily identifiable, whereas process number and length are better resolved in larger cells. Scale bars: 250 µm (A,B), 50 µm (C).

Fig. 2.

Induced decreases in local microgliosis are detectable by live imaging. (A) Live cSLO images of the same ONH before and after oral minocycline treatment (mouse was 2 and 4 months old, respectively) showing a reduction in GFP⁺ cell clustering, compared to an age-matched untreated control representing moderate microgliosis. (B) Total number of cells per ONH at 2 and 4 months of age (n=9 eyes represented by circles) plotted before and after minocycline treatment showing a significant mean reduction post-treatment (*P<0.05; Student's t-test). Bars indicate mean±s.e.m. at each age (16.9±1.65 cells per 0.05 mm² and 13±1.53, respectively). The graph to the right depicts with lines the drop in microgliosis in 6/9 individual ONHs. (C) Ex vivo immunostaining of the same 4-month-old ONHs shown in A revealing that there is a noticeable downregulation of Iba1 expression, but not of GFP, after minocycline treatment, as visible in the single-channel view of Iba1 and its pseudocolor coding by expression intensity. Each image shows a maximum intensity projection of 50 µm and represents the ONH area. Scale bars: 250 µm (A), 25 µm (C).

Fig. 3.

Eyes show large variability in their levels of ONH microgliosis at pre-neurodegenerative ages. (A) Sequential imaging of the same ONH area at 3, 4 and 5 months revealed dynamic changes in GFP⁺ cell numbers and size; the cross indicates the ONH center. (B) Total number of GFP⁺ cells per ONH at 1, 2, 3, 4 and/or 5 months of age (n=19-59 images per age). Data with eye identification and late nerve pathology are presented in supplementary material Fig. S3. Each bar represents an individual ONH and green bars indicate mean±s.e.m. at each age group (10±1.06, 13.17±1.07, 16.85±0.77, 16.46±0.94 to 19.93±1.53, respectively). The average number of GFP⁺ cells per ONH rises with age and significantly increases between 2 and 3 months (**P<0.01; Student's t-test). Scale bar: 25 µm.

Fig. 4.

Late nerve damage is preceded by early microgliosis at the ONH. (A) Experimental design. (B) Live cSLO images of GFP⁺ cells localized to the ONH at 3 months of age in Cx3cr1^+/GFP DBA/2J mice showing examples of low, medium and high levels of microgliosis. (C) Light-microscopy images of optic nerve cross-sections at 10 months of age for these same eyes, representative of mild, moderate and severe damage, as assessed by visual scoring. (D) Microgliosis level, quantified as total number of GFP⁺ cells per ONH at 1 to 5 months of age (n=19, 31, 60, 36 and 25 eyes per respective age group), plotted for individual eyes and categorized by their corresponding optic nerve damage score at 10 months of age (color-coded as indicated). (E) Box plots of the same dataset illustrate significant changes at 3 and 4 months of age (*P<0.05 and ***P<0.001, respectively; Kruskal–Wallis rank ordered test), most notably in the severe nerves at both ages, and in the moderate nerves at 4 months. Plots indicate the median (thick line), interquartile range (IQR; box height), and data within 1.5 times the IQR (whiskers) or greater (outliers, circles). Scale bars: 250 µm (A) and 50 µm (C).

Fig. 5.

Severity of nerve pathology and early microgliosis show a positive correlation. (A) Threshold analysis of the nerve area occupied by glial cells or scar for the cross-sections in Fig. 4C, and high-magnification views of framed areas. (B) Plot of individual optic nerves from 10-month-old mice sorted by relative glial area and ascending coverage, and classified as low, medium and high gliosis (15.24±0.96, 30.38±1.67 and 59.05±3.22 mean±s.e.m. non-axonal area, respectively). Nerves show a spectrum of glaucomatous damage, with significant increases in mean gliosis across groups (***P<0.001; Student's t-test). (C) Corresponding microgliosis level or total GFP⁺ cell number per ONH at 3 months, plotted by their glial area at 10 months (i.e. same order as in B). Microgliosis is categorized by level as low, medium and high microgliosis (12.63±0.70, 18.43±0.62 and 21.08±0.97 mean cells per ONH, respectively). The mean ONH microgliosis shows significant increases across levels of nerve gliosis (***P<0.001 and *P<0.05; n=41 eyes; Student's t-test). Green lines indicate means and s.e.m. per group. (D) There is significant correlation between the optic nerve glial relative area at 10 months (non-axonal area) and their ONH microgliosis at 3 months (P=0.0001; Spearman's rank test). Scale bars: 50 µm.

Fig. 6.

Early microgliosis is mainly driven by microglia resident in the ONH. (A) Live cSLO images of two 3-month-old retinas (left) and their ONHs (right, cross indicating its center), representative of low and high microgliosis. (B) Ex vivo confocal images of the same ONHs at 4 months of age, shown as maximal intensity projection of the inner 30 µm. Triple-immunostaining detects few cells that are positive for both GFP and sialoadhesin within the ONH, regardless of their different levels of microgliosis and activation, revealed by upregulated Iba1 expression in the ONH with increased microgliosis. (C) Number of cells expressing GFP and/or sialoadhesin per ONH area at 4 months of age. Single- and double-stained cells were quantified in confocal images spanning the central 200 µm of retinal whole mounts, throughout the inner 60 µm (0.8-µm z-slices). (D) Low magnification, single-slice view of the same 4-month-old retina representative of high microgliosis (B, bottom row). (E) Comparable view of a 17-month-old retina, showing sialoadhesin expression in perivascular (arrowheads), parenchymal (arrows) and ONH cells (frame). The ONH area is shown at higher magnification in corresponding insets. Scale bars: 250 µm (A,B,D and E), 25 µm (insets in A, and e).