| Objective:To investigate the morphological and functional changes of retinal photoreceptors,and the changes of apoptosis,necroptosis,autophagy and RHO protein folding afterαA-orαB-crystallin knockout in P23H mice,as well as the changes of retinal inflammation before and afterαA-orαB-crystallin knockout in P23H mice.Methods:AKO/P23H and BKO/P23H mice were obtained by crossingαA-crystallin knockout(AKO)andαB-crystallin knockout(BKO)mice with P23H mice,respectively.Western blot was used to detect whetherα-crystallin was totally knocked out in P23H retina.Firstly,the effects ofα-crystallin protein on the structure and function of P23H retina were detected:(1)Using optical coherence tomography(OCT)to measure the thickness of the outer nuclear layer(ONL)in AKO/P23H,BKO/P23H and P23H mice at 2,3 and 4 months of age.(2)Using HE staining to histologically and qualitatively observe the retinal morphological changes of 4-month-old AKO/P23H,BKO/P23H and P23H mice.(3)Using electroretinography(ERG)to quantitatively detect the retinal function of 4-month-old AKO/P23H,BKO/P23H and P23H mice at the intensity of 0.01 cd.s/m~2,10 cd.s/m~2 and 32 cd.s/m~2.(4)Using immunofluorescence to qualitatively observe the distribution and expression of rhodopsin(RHO)and M-opsin in the 4-month-old AKO/P23H,BKO/P23H and P23H mice.Secondly,the effects ofα-crystallin protein on the cell death pathways of P23H retina were detected:(1)TUNEL-positive cells in ONL layer of AKO/P23H,BKO/P23H and P23H mice were counted on postnatal day 15(P15).(2)The transcriptional levels of caspase-8 in the retina of 2-month-old AKO/P23H,BKO/P23H,P23H and C57 mice were measured by RT-PCR,while the activity of caspase-8 was detected by the caspase-8 enzymatic activity kit.(3)The transcription levels of RIPK1,RIPK3 and MLKL in the retina of 2-month-old AKO/P23H,BAKO/P23H,P23H and C57 mice were detected by RT-PCR;Protein expression levels of p-RIPK3 and RIPK3 in retina of 2-month-old BKO/P23H,AKO/P23H,P23H and C57mice were detected by western blot.(4)The protein expression of retinal autophagy markers,LC3 and P62,in 2-month-old AKO/P23H,BKO/P23H,P23H and C57 mice was detected by western blot.(5)Soluble/insoluble RHO protein expression levels in retina of 2-month-old AKO/P23H,BKO/P23H and P23H mice were detected by western blot to observe whetherα-crystallin exerted the function of molecular chaperone.At last,the inflammatory state before and afterα-crystallin knockout in P23H retina was observed:(1)RT-PCR was used to detect the transcriptional levels of inflammatory factors,including CCL2,CCL3,TNF-α,IL-1βand IL-6 in 2-month-old P23H and C57 retina.(2)The infiltration of Iba-1 positive cells in GCL,INL,ONL and subretinal space in the retina of P23H and C57 mice was observed by retinal whole flat-mount and Iba-1 immunofluorescence.(3)RT-PCR was used to detect the transcriptional levels of inflammatory factors,including CCL2,IL-1βand IL6,in 2-month-oldα-crystallin-knockout P23H mice.(4)The infiltration of Iba-1 positive macrophage/microglia in all retinal layers of2-month-old AKO/P23H,BKO/P23H and P23H mice was observed by30μm retinal section and immunofluorescence staining.The number of Iba-1 positive macrophage/microglia infiltrated in ONL layer and subretinal space,was quantified by retinal whole flat-mount and immunofluorescent staining.Results:Compared with C57 and P23H mice,western blot showed no bands ofαA-crystallin in AKO/P23H mice,and no bands ofαB-crystallin in BKO/P23H mice,indicating complete deletion of retinalα-crystallin.The results of the morphological and functional changes were as follows:(1)OCT showed that AKO/P23H and BKO/P23H retina had thinner ONL than age-matched P23H retina,especially in the inferior retina,but there was no significant difference in ONL layer thickness between AKO/P23H mice and BKO/P23H mice.(2)Retinal HE staining of 4-month-old mice showed the thinning of ONL layer and shortening of photoreceptor inner segment and outer segment in AKO/P23H and BKO/P23H retina,especially in the inferior retina.(3)Retinal ERG showed that the amplitude of dark-adapted a and b waves in 4-month-old AKO/P23H or BKO/P23H mice was significantly lower than that in P23H mice,but AKO/P23H and BKO/P23H mice had no significant difference in dark-adapted ERG function.(4)Immunofluorescence showed that the expression levels of RHO and M-opsin in the outer segment of photoreceptor,which was more shortened and distorted,decreased in 4-month-old AKO/P23H and BKO/P23H mice as compared to P23H mice.Secondly,the effects ofα-crystallin on P23H retinal cell death pathways were detected:(1)Loss ofαA-orαB-crystallin in P23H retina resulted in a significant increase of TUNEL-positive cells in the ONL layer at P15.(2)In the caspase-dependent apoptosis pathway,the transcriptional levels of caspase-8 and caspase-8 enzymatic activity were significantly increased in 2-month-old AKO/P23H and BKO/P23H mice compared with the age-matched P23H control group.(3)Among the key markers of necroptosis,the transcriptional levels of RIPK1 and RIPK3 in the retina of AKO/P23H mice were significantly increased compared with the control group,while the transcriptional levels of RIPK1,RIPK3 and MLKL in the retina of BKO/P23H mice were significantly increased compared with the control group.The phosphorylation of RIPK3(p-RIPK3)protein in AKO/P23H and BKO/P23H mice was also significantly increased compared to the control group,which was consistent with the activation of necroptotic pathway.(4)The autophagic proteins,P62 and the ratio of LC3I to LC3-II,did not significantly change in retina of AKO/P23H and BKO/P23H mice compared to P23H control group.(5)The ratio of insoluble/soluble RHO in the retinas of AKO/P23H and BKO/P23H mice was not significantly different from that of P23H retina.Finally,the status of P23H retinal inflammation and the effects ofα-crystallin on P23H retinal inflammation were detected:(1)RT-PCR results showed that the transcriptional levels of CCL2 and IL-1βin retina of 2-month-old P23H mice were higher than those of C57 mice,the transcriptional levels of IL-6 were decreased,and levels of CCL3 and TNF-αhad no significant changes.(2)Compared with C57 mice,Iba-1positive macrophage/microglia in 2-month-old P23H mice infiltrated and migrated from the inner layer of the retina to the outer layer of the retina.Of particular note,these Iba1-positive cells had amoeba-like cell bodies and shortened branches.The number of Iba-1 positive macrophage/microglia in the ONL layer and subretinal space of P23H mice was significantly increased.(3)The transcriptional levels of CCL2and IL-1βin the retina of 2-month-old AKO/P23H and BKO/P23H mice were significantly increased compared to the control group,while the transcription levels of IL-6 were significantly decreased.(4)The number of Iba-1 positive macrophage/microglia infiltration in ONL layer and subretinal space of AKO/P23H and BKO/P23H mice were significantly increased compared with the control group.Conclusion:αA-orαB-crystallin knockout in P23H mice may further impair the morphology and function of retinal photoreceptor cells,and there is no significant difference in the effect ofαA-orαB-crystallin knockout.Knockout ofα-crystallin may lead to further apoptosis and necroptosis of P23H retinal cells,but it does not regulate the autophagic activity of retinal cells or change the RHO protein folding.In P23H mice,the retina is in an inflammatory state,including a large number of infiltrated macrophages/microglia and activated inflammatory factors.Loss ofαA-orαB-crystallin may lead to further recruitment and activation of macrophages/microglia and increase expression of inflammatory factors in P23H retina. |
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