Synaptic changes and the response of microglia in a light-induced photoreceptor degeneration model

Abstract

Purpose: To explore synaptic changes and the response of microglia in a light-induced photoreceptor degeneration model.

Methods: Sprague-Dawley rats were euthanized 1 h, 1 day, 3 days, 7 days, and 14 days after being exposed to intense blue light for 24 h. Hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were used to evaluate changes in the outer nuclear layer (ONL). Transmission electron microscopy (TEM) was applied to observe the ultrastructural changes in the synapses between the photoreceptors and second-order neurons. Western blotting was conducted to evaluate specific proteins, including postsynaptic density-95 (PSD-95), metabotropic glutamate receptor 6 (mGluR6), synapsin I, and synaptophysin. Immunofluorescence of CD11b and PKC-α or mGluR6 was used to explore the spatial relationships between microglial processes and synaptic elements. Immunoelectron microscopy of PSD-95 was performed to further confirm its engulfment of synaptic materials.

Results: H&E and TUNEL staining showed that the thickness of the ONL decreased markedly, and the number of apoptotic photoreceptors peaked at day 1. TEM revealed darkened photoreceptor terminals and that ribbons of them were floating in the cytoplasm, coinciding with the downregulation of PSD-95 and mGluR6. Downstream synaptic protein synapsin I and synaptophysin exhibited upregulation in the inner plexiform layer. Activated microglia migrated to the outer retina, and their processes were found in close proximity to synapses in the outer plexiform layer under light and electron microscopy levels. Double immunostaining of CD11b and mGluR6 showed colocalization. PSD-95-immunoreactive electron-dense materials were observed inside the microglia suggesting engulfment of synaptic components.

Conclusions: The study showed that there are early synaptic impairment and late compensatory changes in downstream synapses in this photic injury model. Activated microglia touched and directly engulfed synaptic materials. Microglia may play a role or a partial role in synaptic changes.

Figure 1

H&E and TUNEL staining of the retina. A–F: After light exposure, the number of photoreceptors decreases markedly. The thickness of the outer plexiform layer (OPL) also decreases gradually (arrow). G: In the control retina, positive staining is seldom observed. H: At 1 h after light exposure, positive cells start to appear in the outer nuclear layer (ONL). I: At day 1, the number of positive cells peaks. J–L: The number of positive cells gradually decreases. M: Quantification of the thickness of the ONL. n = 4. **p<0.01. N: Quantification of the thickness of OPL. n = 4. *p<0.05, **p<0.01. Scale bar: 50 μm.

Figure 2

Transmission electron microscopy pictures of the retina show thinning of the OPL and degeneration of synapses in the OPL. A, B: The outer plexiform layer (OPL) is thinner in the light exposed animals at day 14 after light exposure than in the control animals. C: At day 1, many degenerated rod spherules (RSs) in the OPL have become very dark containing several vacuoles (arrowhead). D, E: At day 1, presynaptic ribbons float in the cytoplasm of the RSs and cone pedicle (arrow). F: At day 14, the OPL is occupied by a sponge-like structure (star). G: At day 14, the RSs are observed to retract into the outer nuclear layer (ONL; box). H: Magnification of the box area in G. CP, cone pedicle; h, horizontal cell; P, photoreceptor; RS, rod spherule. Scale bar: 5.0 μm for A and B, 0.5 μm for C–H.

Figure 3

Analysis of synaptic proteins of first synapses in the retina. A: Western blotting of postsynaptic density-95 (PSD-95). B: Western blotting of metabotropic glutamate receptor 6 (mGluR6). n = 4. *p<0.05, **p<0.01. C: In the control animals, mGluR6 immunoreactivity is observed in the outer plexiform layer. D–H: After light exposure, the staining is diminished during the early course of the degeneration. Scale bar: 50 μm.

Figure 4

Analysis of synaptic proteins of the second synapses in the retina. A: Western blot of synapsin I and synaptophysin. n = 4. *p<0.05. B: In the control retina, intense labeling of synaptophysin is observed in the outer plexiform layer (OPL) and the inner plexiform layer (IPL). C–F: After light exposure, the intensity of the fluorescence of synaptophysin in the OPL starts to weaken. G: At day 14, the staining of synaptophysin is almost undetectable in the OPL. H: Quantification of the staining intensity of synaptophysin in the IPL. n = 6. **p<0.01. Scale bar: 50 μm.

Figure 5

Immunofluorescence against CD11b after light exposure. A: In the control retinas, ramified microglia are observed (arrow). B: One hour after light exposure, activated microglia migrate toward the outer retina. In addition, some microglia terminate their processes in the outer plexiform layer (OPL; arrow). C: At day 3, some microglia are still terminating and stratifying their processes in the OPL (arrow). Some activated microglia are observed in the subretinal space (arrowhead). D: Quantification of the number of microglia in the outer retina (including the OPL, outer nuclear layer, and subretinal space) during retinal degeneration. n = 6. **p<0.01. Scale bar: 50 μm.

Figure 6

Double immunostaining against CD11b (green) and PKC-α (red). A: One hour after light exposure, tight interaction between microglial processes and dendrites of bipolar cells is observed (arrow). B: At day 3, some activated microglia are observed in the subretinal space (arrowhead), and tight interaction between microglia and bipolar cells can still be observed. Scale bar: 50 μm.

Figure 7

Transmission electron microscopy pictures shows a direct connection between the microglia and the synapses. A: At day 1, microglia are found in the outer plexiform layer (OPL). B: The direct juxtaposition between the microglia and synaptic elements (arrow) is observed at day 3 (box). C: At day 14, microglia are found to extend their processes between sponge-like structures (star) in the OPL. M, microglia. Scale bar: 5.0 μm for A, 0.5 μm for B and C.

Figure 8

Double immunostaining against CD11b (green) and CD68 (red). A: In the control retinas, CD68 signaling is seldom observed. B: At day 3, there is upregulation of CD68 in microglia in the outer plexiform layer (OPL) after light exposure (arrow). Scale bar: 50 μm.

Figure 9

Double immunostaining against CD11b (green) and mGluR6 (red). A: In the control animals, metabotropic glutamate receptor 6 (mGluR6) immunoreactivity is observed in the outer plexiform layer. B: One hour after light exposure, punctate staining of mGluR6 appears elsewhere, colocalizing with CD11b (arrow). C: Magnification of the box area in B. Scale bar: 50 μm.

Figure 10

Immunoelectron microscopy shows engulfment of synaptic material by the microglia. A: In the control animals, microglia are observed in the inner part of the retina. Postsynaptic density-95 (PSD-95)-immunoreactive electron-dense material is seldom seen inside the microglia. B, C: Magnification of the box area in A. No PSD-95-immunoreactive electron-dense material is observed in the cytoplasm of the microglia. D: A representative picture of a negative control at day 1 (without primary antibody). E, F: Magnification of the box area in D. No PSD-95-immunoreactive electron-dense material is observed in the cytoplasm of the microglia. G, H: At day 1, microglia are occasionally presented in the outer plexiform layer (OPL), and several 10 nm gold particles are observed inside the microglia (arrow). H: Magnification of the box area in G. M, microglia; P, photoreceptor.