Authors: Tang Huanyu, Lu Ziyang, He Juncai, Gao Yuan, Yin Zhengqin
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To explore the immune-microenvironment of the retinas at different stages of retinal degeneration in Royal College of Surgeon (RCS) rats.
RCS-rdy–-P+ (RCS) rats at early stage (P20), middle stage (P40) and late stage (P60) were involved, 12 rats at each post-natal day, RCS-rdy+ -P+ rats severed as control.Relative concentrations of rat cytokines in rat retina homogenate were detected by using Bio-Plex Suspension Array System.Relative expressions of interleukin-2 (IL-2), C-C motif ligand 2 (CCL2), chemokine (C-X-C motif) ligand 9 (CXCL9), CXCL10, CXCL11 and interferon-γ(IFN-γ) mRNA in rat retina were analyzed by real-time PCR.Expressions of IFN-γ and immune cells surface marker CD4, CD8 and CD161 in the retinas were detected by immunohistochemical staining.Percentage of IFN-γ positive T lymphocytes and natural killer(NK) cells in rat retina were analyzed by flow cytometry.The concentrations of IFN-γ in rat retina homogenate were evaluated by enzyme-linked immunosorbent assay (ELISA). The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.
Lymphocytes related cytokines and chemokines mRNA expression levels in the RCS rat retinas showed increase trends with the extension of time.The expression levels of IL-2, CCL2, CXCL9, CXCL10, CXCL11 and IFN-γ mRNA in P60 RCS rat retinas were significantly increased than those in the P20 RCS rat retinas and the control rat retinas (all at P<0.05). The positive rates of CD4, CD8 and CD161 cells in the retinas of P60 RCS rats was (9.09±0.89)%, (18.77±0.38)% and (9.41±0.38)%, respectively.The proportion of IFN-γ positive cells in the retinas of P60 RCS rats was (8.29±0.27)%, which was significantly higher than that of the control rats ([0.28±0.02]%), with a significant difference between them (t=29.03, P=0.00). CD4+ , CD8+ and CD161+ lymphocytes were mainly distributed in the retinas of P60 RCS rats, and the expressions of IFN-γ were co-located with lymphocyte surface markers.There were significant differences in the concentrations of IFN-γ in the retinas of RCS rats and control rats at different day ages (Fgroup=16.49, P<0.01; Ftime =21.05, P<0.01), the concentration of IFN-γ in retinas of P60 RCS rats was significantly higher than that of P20 RCS rats, P40 RCS rats and control rats, and the differences were statistically significant (all at P<0.05).
Along with the process of retinal degeneration, immune privilege balance in the retinas is disrupted, the expressions of lymphocytes related chemokines and cytokines are elevated.Lymphocytes infiltration and activation are appeared in the retina highly activated at the late stage of RP, leading to the significant up-regulation of inflammatory cytokine IFN-γ in microenvironment, which indicates that lymphocytes mediated immune response may take part in retinal degeneration.