Activation of retinal microglia rather than microglial cell density correlates with retinal neovascularization in the mouse model of oxygen-induced retinopathy

Abstract

Background: Retinal neovascularization has been intensively investigated in the mouse model of oxygen-induced retinopathy (OIR). Here, we studied the contribution of microglial cells to vascular regression during the hyperoxic phase and to retinal neovascularization during the hypoxic phase.

Methods: Mice expressing green fluorescent protein (GFP) under the Cx3cr1 promoter labeling microglial cells were kept in 75% oxygen from postnatal day 7 (P7) to P12. Microglial cell density was quantified at different time points and at different retinal positions in retinal flat mounts. Microglial activation was determined by the switch from ramified to amoeboid cell morphology which correlated with the switch from lectin negative to lectin positive staining of GFP positive cells.

Results: Microglial cell density was constant in the peripheral region of the retina. In the deep vascular layer of the central region, however, it declined 14 fold from P12 to P14 and recovered afterwards. Activated microglial cells were found in the superficial layer of the central avascular zone from P8 to P12 and from P16 to P18. In addition, hyalocytes were found in the vitreal layer in the central region and their cell density decreased over time.

Conclusion: Density of microglial cells does not correlate with vascular obliteration or revascularization. But the time course of the activation of microglia indicates that they may be involved in retinal neovascularization during the hypoxic phase.

Figure 1

Retinal zones and layers used for quantifying microglia. (A) Retinal flat mount stained with lectin showing the regions used for evaluation. Vascular tufts are located at the border of the central avascular zone and the peripheral zone. (B) Cryosection of the eye showing the superficial layer (s) and the deep layer (d) of microglia (GFP, green). The nuclei (DAPI, blue) of the retinal ganglion cells (RGC), inner nuclear layer (INL) and outer nuclear layer (ONL) are shown for orientation. Macrophages in the choroid (c) and sclera express GFP, too. Vitreous (v).

Figure 2

Lack of microglia in the deep layer of the central avascular zone in OIR at P14. Flat mounts (A - D) and cryosections (E, F) show resting microglial cells with ramified processes in the central zone of the deep retinal layer (d) expressing GFP (green) under the control of the Cx3cr1 promoter. Large, blurred, green dots are microglial cells of the superficial layer (s). Note that the microglial cell density in the deep layer is much smaller at P14 compared to P12. Vessels of the deep vascular layer are stained with lectin (red). No vessels are visible in P12 and P14 OIR images as these images are taken from within the avascular central zone of the retina. Vitreous (v), choroid (c).

Figure 3

Cell densities of retinal microglial cells and hyalocytes. Solid lines are from OIR mice, while dashed lines are from controls without oxygen treatment. Red lines label microglia data from the superficial layer and violet lines label hyalocytes from the peripheral zone, respectively. While microglial cell densities of the peripheral zone were almost constant over time, a marked drop was observed in the deep layer of OIR mice after return to normal air. Activated microglia (yellow line) were found in the superficial layer only and peaked at P10 and at P17. The cell density of hyalocytes decreased over time. Error bars indicate standard errors. Significant differences were found (1) in the deep layer of the central avascular zone in OIR between P12 and P14, (2) in activated microglia in the superficial layer of the central avascular zone between P7 and P10, and (3) between P14 and P17.

Figure 4

Activated microglial cells in the central avascular zone at P17. These cells are found in the superficial layer (s) of the central avascular zone (see cryosections). Activated microglial cells express GFP (green) under the control of the Cx3cr1 promoter and are additionally positive for lectin (red). Their morphology had changed from ramified cells to cells with short and broad processes (see flat mounts). E and F are the same as C and D, respectively, with the red channel omitted.

Figure 5

Distribution of retinal microglia at vascular tufts at P17. Vascular tufts and other vessels (lectin staining, red) are located at the border of the vascularized and the avascular central zone in the superficial layer (s). Microglial cells (GFP, green) near vascular tufts are not activated as they are ramified and not positive for lectin. Flat mounts show a rather even distribution of microglia. Activated microglia were found in the central avascular zone (A). Yellow staining in the sections (C and D) comes from super-position of microglia and endothelial cells but not from microglia activation. Arrow heads point to hyalocytes in the vitreous (v). E and F are the same as C and D, respectively, with the red channel omitted.

Figure 6

Hyalocytes in the central avascular zone of the retina at P8. Hyalocytes are positive for lectin (red) and GFP (green) expressed under the control of the Cx3cr1 promoter. They are located in the vitreous (v) near the retina (see arrow heads in the cryosection). In contrast to the microglial cells, they have a larger round cellular body and no processes (see retinal flat mount). Cells with GFP expression but without lectin staining are microglial cells. Vessels remnants are stained with lectin, too (arrows).