Figure 1.
Illustration of the transwell co-culture system and the chemotaxis assay.
The transwell consists of two chambers separated by a porous membrane. The 661W cells were placed on the bottom of the lower chamber while the microglial cells were placed on the membrane of the upper chamber.
Figure 2.
Hematoxylin–eosin and TUNEL staining of SD rat retinas showed progressive changes in the ONL thickness and photoreceptor apoptosis in the in vivo light-induced photoreceptor degeneration model.
(A–E) Compared with the normal group, the photoreceptor number and ONL thickness decreased markedly at different time points after exposure to light (black arrow). (F–I) A few hours after exposure to light, TUNEL-positive cells began to appear in the ONL. (J–K) At 1 day, the number of TUNEL-positive cells reached a peak in the ONL. (L–O) TUNEL-positive cells gradually decreased at 3 and 5 days. (P–Q) At 7 days, TUNEL fluorescence was almost completely absent.
Figure 3.
The effect of intense blue light on 661W photoreceptor cell morphology and apoptosis.
(A) In the normal control, 661W had a flattened appearance with few intercellular spaces. (B) After light exposure, the cells became spindled with large intercellular spaces. (C–E) 661W photoreceptors underwent apoptosis, as determined by TUNEL assay. (F) Statistical analysis showed that the TUNEL positivity was significantly increased at 0 hour, 24 hours and 48 hours after light exposure.
Figure 4.
Immunolabeling with the OX42 antibody showed the activation and migration of retinal microglia.
(A) In normal retinas, labeled microglia existed only in the GCL and IPL. (B) At 3 days after exposure to light, plenty of microglia migrated to the outer retina. (C–D) The microglial morphology changed from the resting ramified state with long processes to the activated amoeboid appearance at 3 days after photic injury.
Figure 5.
Immunofluorescence analysis of fractalkine in the retina at different time points after exposure to light.
(A) In the normal retina, fractalkine immunoreactivity showed a weak but widely distributed fluorescence. (B–F) At 6 hours, 1, 3, 5, 7 days after light exposure, fractalkine staining increased in the photoreceptors.
Figure 6.
Double immunofluorescence analysis of CX3CR1 and fractalkine at the 3-day time point after light exposure.
(A–C) Double staining for OX42 and CX3CR1 revealed that CX3CR1-positive cells were reactive for the microglial marker. (D–I) Photoreceptors were labeled by recoverin antibody and microglial cells were labeled by OX42 antibody. The two merged images showed that photoreceptors were the source of increased fractalkine as well as the direction of microglial migration.
Figure 7.
Western blot and real-time PCR analyses for fractalkine/CX3CR1 expression in the retinas after exposure to light.
(A–C) The protein expressions of fractalkine and CX3CR1 began to increase at two hours after exposure to light and peaked at 1 day and 3 days respectively, then decreased. (D–E) The mRNA results were normalized relative to levels in the normal control retinas.
Figure 8.
The changes in microglial morphology in the co-cultures after exposure to light.
(A, C, E, G) Primary retinal microglia grown in basal media showed ramified shapes with processes. (B, D, F, H) After exposure to light, the microglia in the co-cultures became rounder and took on a characteristic amoeboid shape as they were activated.
Figure 9.
The soluble fractalkine release after exposure to light.
ELISA analysis revealed that the levels of soluble fractalkine were increased in the 661W control group and the 661W+MG co-culture group. The difference between them is not statistically significant at 6-hour and 12-hour time points. At other time points, the release of soluble fractalkine in the 661W control group was less than that of the co-culture group due to the further degeneration of remaining photoreceptors caused by activated microglia.
Figure 10.
The expression of IL-1β and TNF-α after exposure to light.
(A–C) The protein expressions of IL-1β and TNF-α in the retina began to increase at 6 hours after exposure to light and peaked at 3 days. (D–E) The mRNA expression of IL-1β and TNF-α in the retina was increased, and peaked at 1 day after photic injury as detected by real-time PCR. (F–G) ELISA assay detected the upregulated secretion of IL-1β and TNF-α in the 661W/microglia co-culture supernatant. (H–I) Compared with untreated co-cultures, the group pretreated with neutralizing CX3CR1 antibody showed significantly decreased expression of IL-1β at 12 hours and 24 hours after exposure to light. For TNF-α, statistical analysis showed significantly reduced production in the treated cultures at 12 hours. No difference was found between treated and untreated co-cultures at 24 hours.
Figure 11.
Statistical analysis of TUNEL assay in different co-culture groups at 24 hours after exposure to light.
Figure 12.
Soluble fractalkine-induced microglial migration in the co-culture of retinal microglial cells with 661W cells.
Results are presented as numbers of migratory cells. Microglia exhibited stronger migratory activity in the co-cultures with additional soluble fractalkine than in other groups and there was a significant reduction of microglial migration in the co-cultures with neutralizing CX3CR1 antibody at most time points. The most obvious result was obtained at the 6-hour time point after the beginning of light exposure.
Figure 13.
The effect of membrane-bound fractalkine on 661W photoreceptor survival.
(A–C) At 24 hours after exposure to light, a large number of TUNEL-positive photoreceptors was detected in the untreated co-culture. (D–F) TUNEL-positive cells were less abundant in the co-culture treated with exogenous membrane-bound fractalkine. (G) The expression of anti-inflammatory cytokine, IL-10, was increased in the recombinant full-length fractalkine treated groups.