Local proliferation is the main source of rod microglia after optic nerve transection

Abstract

Microglia are the resident phagocytic cells with various functions in the central nervous system, and the morphologies of microglia imply the different stages and functions. In optical nerve transection (ONT) model in the retina, the retrograde degeneration of retinal ganglion cells (RGCs) induces microglial activations to a unique morphology termed "rod" microglia. A few studies described the "rod" microglia in the cortex and retina; however, the function and origin of "rod" microglia are largely unknown. In the present study, we firstly studied the temporal appearance of "rod" microglia after ONT, and found the "rod" microglia emerge at approximately 7 days after ONT and peak during 14 to 21 days. Interestingly, the number of "rod" microglia remarkably decays after 6 weeks. Secondly, the "rod" microglia eliminate the degenerating RGC debris by phagocytosis. Moreover, we found the major source of "rod" microgliosis is local proliferation rather than the infiltration of peripheral monocytes/hematopoietic stem cells. We for the first time described the appearance of "rod" retinal microglia following optic nerve transection.

Figure 1. Retinal whole-mount immunostaining of microglial cells at different time points after optic nerve transection (ONT).

a, b, d, d, e, f, g, g, i and j showed Iba1 staining (microglia marker) of retina under resting condition, 1 day, 2 days, 3 days, 7 days, 10 days, 14 days, 21 days, 6 weeks and 8 weeks after ONT, respectively. The microglia cells in the retina align to each other at 7 to 21 days after ONT, showing “rod” morphology; while the activation tuned down at 6 weeks after ONT. Scale bar: 200 μm.

Figure 2. Rod microglia are specific to ganglion cell layer and are phagocytic.

a: Whole retina staining of Iba1 3 weeks after ONT. Scale bar: 200 μm. b: Rod-morphology microglia at ganglion cell layer (GCL) layer of retina. Scale bar: 50 μm. c: High magnification image of Rod-morphology microglia at GCL. Scale bar: 10 μm. d: DAPI staining of retina (view from laterally) showing different cellular layers. Scale bar: 50 μm. e: Resting morphology microglia at Inner nuclear layer (INL). Scale bar: 50 μm. f: High magnification image of resting-morphology microglia at INL. Scale bar: 10 μm. g: Whole retina staining of Iba1 (green) and tubulin (red) 2 weeks after ONT. Scale bar: 200 μm. h: Close relationship between microglial processes and the nerve fibers. Scale bar: 50 μm. i: Z-stack view of close relationship between microglial processes and the nerve fibers (white triangle). Scale bar: 50 μm. j, k and l: Whole retina staining of Iba1 (green, 2J) and Fluoro-Gold (FG) (purple, 2K) 2 weeks after ONT. 2L is the merged picture. The arrow shows a survived ganglion cell from ONT. Scale bar: 50 μm. m: Z-stack view showing that FG particles were phagocytized by microglia after ONT (white triangle). Scale bar: 10 μm.

Figure 3. Rod microglia rarely originated from peripheral infiltrating macrophages.

a: Pairs of C57BL/6 wild-type (WT) mouse and Cx3cr1^+/GFP mouse were joined for parabiosis model for at least 14 days to allow the mixed circulation. Then the ONT was performed on the WT mouse, and the tissues were harvested at 7 to 10 days after the ONT. b: 7 days after ONT, the mouse retina exhibited rod microglia as well, however only one cell in the view showing GFP signal. Green: GFP. Red: Iba1 staining. Blue: DAPI. Scale bar: 50 μm. c: 10 days after ONT, the mouse retina exhibited rod microglia as well, however only one cell in the view showing GFP signal. Green: GFP. Red: Iba1 staining. Blue: DAPI. Scale bar: 50 μm.

Figure 4

Rod microglia are proliferating in situ. a, b, c and d: Whole retina staining of Iba1 (Green) and BrdU (Red) at 3 days, 7 days, 10 days and 14 days after ONT. Scale bar: 50 μm. e, f, g, and h: Co-localization of BrdU (red) and Iba1 (Green) staining in a microglial cell at 7 days after ONT (white triangle). Scale bar: 50 μm. 3I: Z-stack view showing the proliferating microglia cell (white triangle). Scale bar: 50 μm.