Effects Of γ-flicker Light On Retinal Aging Of Mice

[Objective]1.To explore the effect ofγ-flicker light（γFL）on retinal aging features in mice and to probe the underlying mechanisms.2.To explore the effect ofγFL treatment on mouse retinal immune state during aging.[Methods]1.Eight-week-and 20-month-old mice were treated withγFL 1 hour per day for 6 consecutive days.Retinalβ-galactosidase（β-gal）staining was performed to count retinal senescent cells.Then retinal flat-mount with vascular staining was carried out to observe the intensity of autofluorescence（AF）around vessels for assessing retinal lipofuscin level.And retinal section immunofluorescence tests for complement factor 3（C3）,microtubule-associated protein 1 light chain 3 beta（LC3B）,catalase（CAT）,carboxymethyl lysine（CML）,glial fibrillary acidic protein（GFAP）,S100-A10,and activating transcription factor 4（ATF4）were applied to analyze changes of complement,autophagy,oxidative stress,astrocytes,and unfolded protein response（UPR）in the neural retina and retinal pigment epithelium（RPE）tissue.Western blot tests were also carried out to quantify the levels of LC3B,Beclin-1,CAT,CML,GFAP,aquaporin（AQP-4）,ATF4,and C3.The potential biological processes underlyingγFL effects were evaluated by RNA sequencing,with a part of core genes verified by RT-q PCR and western blot.2.Twenty-month-old Apoe^-/-mice were treated withγFL 1 hour per day for 30 consecutive days.The RPE-Bruch membrane（BM）complex was observed through transmission electron microscopy.3.The data of RNA sequencing were analyzed again,with immunity-associated processes focused.After a ranking with P value,the first process,namely,antigen presentation via MHC-Ⅱ,was gained.The genes with statistical significance in this pathway were then quantified by RT-q PCR.4.γFL treatment（2 hours per day,1 hour in the morning,another hour in the afternoon,for 6 consecutive days）was applied to mice of 8 weeks old,9 months old,and 18 months old.Retinal flat-mounts were performed,with immunostaining for GFAP to count astrocytes and for ionized calcium binding adapter molecule 1（IBA-1）to observe microglia morphology,as well as for MHC-Ⅱ,IBA-1,and vessel markers to assess inner-and sub-retinal antigen presentation.The retinal CD74 expression level of 18-month-old mice was then examined in western blot.5.Eight-week-and 9-month-old mice were intravitreally injected with0.2-μg amyloidβ_1-40(Aβ_1-40)oligomers,followed by aγFL treatment（2hours per day,1 hour in the morning,another hour in the afternoon,for 3consecutive days）.CD74 and CD68 expression levels were tested through western blot,representing the antigen presentation and microglia activation,respectively.Retinal flat-mounts of 8-week-old mice were co-stained with anti-CD68,MHC-Ⅱ,and Aβ_1-40antibodies to evaluate microglia activation,MHC-Ⅱ expression,and Aβphagocytosis.An IB4 re-staining was then carried out to confirm the distributions of microglia and the retinal blood vessels.[Results]1.Large numbers ofβ-gal-positive cells and AF dots were found in the inner retina and around the blood vessels of aging mice retina,respectively,whereas becoming less afterγFL treatment.2.γFL treatment also ameliorated other retinal aging characteristics including the expression of LC3B,C3,ATF4,and CML/CAT,which checked in immunofluorescence and western blot.3.RNA sequencing revealed mitochondrial function,chaperone-mediated protein folding,and other significant processes involving inγFL effects.4.Transmission electron microscopy results showed reduced thicknesses of basal infolding layer（BI）and BM for the RPE-BM complex in aged Apoe^-/-mice.5.Re-analyzed for RNA sequencing and ranked with P value,the fist biological process,antigen representation via MHC-Ⅱ,was gained.6.The retinal flat-mounts of 8-week-,9-month-and 18-month-old mice not only showed no significant differences in retinal astrocytes count,microglia count,microglia average process length,and microglia cell body diameter afterγFL treatment;but also exhibited reduced astrocyte numbers,increased microglia numbers,and decreased microglia average process lengths from 8-week-to 9-month-to 18-month-old mice.7.Stained with anti-MHC-Ⅱ antibody,the retinal flat-mounts of 8-week-,9-month-and 18-month-old mice displayed below results:（1）increased MHC-Ⅱ-positive dots inγFL-treated 18-month-old mouse retina,the dots which were distributed in a radial axis with the optic disc as the center and overlapped with para-venous retinal microglia;（2）an elevated number of MHC-Ⅱ⁺and IB4⁺microglia in the subretinal space ofγFL-treated 18-month-old mice,the cell body diameter was also increased.Western blot also revealed a higher level of CD74 expression in the retina of 18-month-old mice afterγFL treatment.8.Higher expression levels of CD74 and CD68 were found in the retina of 8-week-and 9-month-old mice after Aβinjection andγFL treatment.In the retinal flat-mounts for the 8-week-old mice,the CD68⁺cells with positive Aβ_1-40 staining were significant increased afterγFL treatment,and the CD68⁺with MHC-Ⅱ⁺cells were also increased around the veins.[Conclusions]1.γFL can effectively regulate most of the aging pathological features,which involve senescent cells,lipofuscin deposition,autophagy,complement,oxidative stress,and UPR;thought it has no obvious effect on the morphology of retinal astrocytes and microglia from superficial to deep layer of the retina.2.γFL can effectively improve the structure of RPE-BM complex in aged Apoe^-/-mice,namely,decreased thicknesses of BI and BM.3.γFL is able to increase MHC-Ⅱ expression in microglia around retinal veins of mice under natural aging condition.In addition,γFL can increase the number of MHC-Ⅱ positive subretinal microglia and enlarge the cell body diameter.There are no obvious effects on the above indexes in young mice.γFL can also increase the expression of CD74 in the neuroretina of naturally aged mice.4.Aβcan partially activate retinal microglia in young mice,but has no obvious effect on MHC-Ⅱ expression.γFL treatment can further activate microglia,enhance microglia phagocytosis of Aβ,and promote the MHC-Ⅱ-mediated antigen presentation of para-vascular microglia.In conclusion,γFL can effectively repair a variety of aging-related pathological features of the retina in aged mice,and increase the microglial antigen presentation under the condition of retinal aging to facilitate the clearance of Aβand other aging proteins.γFL therapy is expected to be one of noninvasive,convenient and tissue-specific retinal aging interventions.