Experimental Study On The Mechanism Of Acute Retinal Injury Induced By MMS

Background and purposeVision is one of the dominant sources of external information for human beings to obtain external information,and the retina is very important for the visual perception.As a part of the central nervous system,the retina has a complex structure,which is responsible for transducing environmental light signals into electrical signals,then transmit visual information to the brain for image processing to produce vision.The retina is mainly composed of photoreceptor cells,bipolar cells,horizontal cells,ganglion cells,amacrine cells,pigment epithelial cells,microglia and Müller cells.Once the retina is damaged,abnormalities of visual function can result in permanent decrease or loss of vision,which will have an inestimable impact on the society and the family.Cancer which has a high incidence rate is the 2nd lethal diseases only followed by cardiovascular disease in China.Cancer has severe effects on psychological and physical health of patients.Cancer places an enormous medical and economic burden on society.Cancer treatment,today,consists of surgery,immunotherapy,chemotherapy,radiation and so on.Most anticancer chemotherapeutics have high toxicity and side effects.Conventional chemotherapy agents that aim to kill tumor cells may also damage normal cells as it cannot tell normal cells apart from cancer cells and kills both.Research query found that some anticancer chemotherapeutics,in fact,can induced retinal toxicity.Therefore,it is particularly important to find ways to reduce the acute retinal toxicity caused by chemotherapy drugs.Alkylating agents,is often a first-line therapeutic approach for the treatment of a wide variety of cancers.They kill cancer cells mainly through induction of DNA damage.Available literature shows,SN2 alkylating agents,Methyl methanesulfonate(MMS)can damage the retina of chicken embryos,and induce photoreceptor cells undergo degeneration and apoptosis.MMS was peritoneally injected to mice for retina injury induction.MMS can site-specific damage the retinal photoreceptor cells.The aim of this study was to construct a mouse model of drug-induced retina acute injury.The C57BL/6J mice received single intravenous injection of MMS(60 mg/kg).Behavioral,function,immunofluorescent and histological assays were performed during acute retinal injury.And the altered expression levels of key pathway proteins were validated by western blotting.In this study,we aimed to found a potential target for the intervention to prevent and treat the acute drug-induced retinal injury.Methods1.Electroretinogram(ERG)examination: On day 1,3,5 and 7 after MMS single intravenous injection at a dose of 60 mg/kg,the retinal electrophysiological function was tested in each group.The a-wave and b-wave amplitude variation of electroretinogram of mice in each group were calculated to evaluate the changes of electrophysiological function.2.Optokinetic behavioral test: In order to evaluate the changes in visuospatial functions of mice after MMS administration,a double-blind crossover test was conducted on day 1,2,3,4 and 5 after MMS administration in each group.3.Open field test: On day 1,3,5 and 7 after MMS administration,behavioral changes in open field was tested.Mice in each experimental group and normal control group were tested in a crossover order.Each mouse was tested for 10 minutes.The behavioral changes of visual function were evaluated by observing the total distance of movement,the Times of entering the central area and the duration of staying in the central area.4.Light/Dark Transition Test: On day 1,3,5 and 7 after MMS administration,behavioral changes in light-dark box were tested.Mice in each experimental group and normal control group were tested in a crossover order.Each mouse was tested for 10 minutes.The behavioral changes of visual function were evaluated by observing the percentage of time spent in the white box and the number of times from the black box to the white box.5.Hematoxylin and eosin(HE)staining: On day 1,2,3,4,5,7 and 10 after MMS administration,fixed eyeballs were embedded in paraffin,sectioned for the subsequent hematoxylin-eosin(HE)staining.The thickness of the outer retinal nucleus layer was observed and the quantitative statistical analysis was conducted to evaluate the morphological changes of the photoreceptor cells in the retina of mice in the experimental group.6.Immunofluorescence Staining: On day 3,4,5 and 10 after MMS administration,rhodopsin immunofluorescence staining was performed on paraffin sections of retina of mice in each group.And observed the changes of rhodopsin in retinal photoreceptor cells.7.Terminal Deoxynucleotidyl TUNEL Assays: On day 1,2,3,4,5 and 7 after MMS administration,a TUNEL assay was used to evaluate the apoptosis of retinal photoreceptor cells.Whether apoptosis occurred in retinal photoreceptor cells was observed.8.Caspase 3/7 activity apoptosis assay: On day 3 and 7 after MMS administration,quantification of the relative caspase-3/7 activity in total retinal protein extraction was performed to evaluate the apoptosis of retinal photoreceptor cell.9.Western Blot Analysis: On day 1,3 and 7 after MMS administration,western blot Analysis as performed to evaluate the altered expression levels of key pathway proteins.The expression levels of key proteins in JAK/STAT,PI3K/ Akt/m TOR and GSK3β/NFκB signaling pathways were observed during acute retinal injury.Results1.Electroretinogram(ERG)examination: On day 1 after MMS administration,the awave and b-wave amplitude variation of electroretinogram of mice in experimental group all decreased to almost disappeared.And on day 3,5 and 7,they decreased to the baseline.2.Optokinetic behavioral test: On day 1 after MMS administration,visuospatial functions of mice all decreased.And the decrease increased gradually with time compared with normal control mice.3.Open field test: On day 1,3,5 and 7 after MMS administration,the total movement distance and the number of entering the central area in the experimental group were increased compared with the normal control group.4.Light/Dark Transition Test: On day 1,3,5 and 7 after MMS administration,the percentage of time spent in the white box and the number of times from the black box to the white box in the experimental group were increased as compared with those in the normal control group.5.Hematoxylin and eosin(HE)staining: On day 1 after MMS administration,the thickness of the outer nuclear layer of mice in the experimental group gradually became thinner with the prolongation of administration time.The photoreceptor nucleus of the retina had only 2-3 layers on the 5th day and 1-2 layers on the 7th day.The photosensitive nuclei of the retina were arranged disorderly and the space was enlarged.The outer segment of the photoreceptor cells in the retina is damaged.6.Immunofluorescence Staining: The expression of rhodopsin was high in the photoreceptor cells of normal mouse retina.The expression level was significantly decreased on the 3rd day,and the fluorescent marker of rhodopsin disappeared on the 7th day after MMS administration.7.Terminal Deoxynucleotidyl TUNEL Assays: On day 1 after MMS administration,scattered green fluorescence appeared in the outer nuclear layer of the retina.There was a large amount of green fluorescence in the outer nuclear layer of retina from the 2nd day to the 5th day.On the 7th day,there was only one layer of photoreceptor cells in the outer retinal nucleus and all of them showed green fluorescence.8.Caspase 3/7 activity apoptosis assay: On day 3 and 7 after MMS administration,quantification of the relative caspase-3/7 activity in the retina was increased compared with the control group,and the enzyme activity was higher on the 3rd day.9.Western Blot Analysis: On day 1 after MMS administration,the expression of STAT3 and NFκB phosphorylated protein in retina was significantly increased.The total protein expressions of Akt,m TOR and GSK3β in the retina were significantly increased on day 3 and 7.Conclusions1.In this study,the animal model of acute drug-induced retinal injury is successfully established.C57BL/6J mice receive a single intravenous injection of MMS(60mg/kg).This model is repeatable,reliable and stable.2.MMS causes visuospatial function loss,morphologic changes in the retina and reduce retinal function during acute retinal injury induced by MMS.Expression levels of key pathway proteins is also significantly altered.3.MMS induces rapid and massive apoptosis in retinal photoreceptor cells during acute retinal injury.4.MMS activates JAK/STAT and PI3K/AKT/m TOR signaling pathways during acute retinal injury.