Research On The Role Of Drp1 Dependent Mitochondrial Fission In Photoreceptor Death After Retinal Detachment

【Purpose】Photoreceptor cell death is one of the major causes of decreased visual function.The photoreceptors’inner segments are rich in mitochondria,while the outer segments are rich in a large number of membrane disks.The energy required for this process is mainly from the mitochondria.When the energy metabolism of the mitochondria is impaired,the structure of the photoreceptor cells was damaged accordingly.Some scholars speculate that the photoreceptor impaired metabolism and cell edema may be an important reason for the destruction of the ellipsoidal structure.Optical coherence tomography（OCT）showed OS（Outer Segment）and IS（Inner Segment）continuous disrupted and the interruptions often indicate a poor prognosis of vision.Therefore,a deep understanding of mitochondrial metabolism and function of photoreceptor degeneration is of great significance.Based on the results of metabolism results of vitreous and subretinal fluid in patients with retinal detachment,we hypothesized that there are energy metabolism disorders in mitochondria.Recently,the edema mitochondrial was observed after RD.We concluded that mitochondrial dysfunction of photoreceptor is an important reason for cell death.While,the function of mitochondria is closely related to the morphology of mitochondrial.The change of mitochondrial morphology may be an early change of mitochondrial dysfunction,mainly as the increased fission of mitochondrial.If the mitochondrial dysfunction caused mitochondrial fission after RD is unknown,this topic is trying to investigate the role of Drp1-mediated mitochondrial division in photoreceptor death after RD.【Method】1.We analyzed the significant metabolism products from the previous metabolism results of vitreous and subretinal samples from retinal detachment patients and cornea donors.a P value less than 0.05 was regarded as significant difference.2.Varification of disease model:We observed the mitochondrial structure by TEM（Transmission Electron Microscope）and measured the mitochondrial length 3 days after successful retinal detachment model.To examine the expression of Drp1 at 1d,3d,5d,7d after RD by Western blotting.To observe mitochondrial morphology by V-βimmunofluorescence and classified them into three shapes including tubular,fragmented and intermediated shape.Finally,to analyze the difference in groups by one-way ANOVA and Chi-square test.a P value less than 0.05 was regarded as significant difference.3.We used SD rat to established the disease models,to verify the effect of inhibition of Drp1 on the photoreceptor,Mdivi-1,a selective inhibition of Drp1 GTPase was used to investigate the effect after RD.We injected 5μL of Mdivi-1（2.4mg/ml）（treated group）or 5ul DMSO（Vehicle control group）into subretinal space after retinal detachment.TUNEL staining,HE staining and electroretinagram were to evaluate the effect of Mdivi-1 treatment.One-Way ANOVA was used to compare the different groups.a P value less than 0.05 was regarded as significant difference.4.To investigate the exact mechanism of Drp1 in photoreceptor death after RD.We divide into vivo and vitro experiments.In vivo.We measured the main apoptosis related proteins and Drp1 expression by Western blotting.（1）The major way of photoreceptor death after retinal detachment is apoptosis and it peaks at 3 days after RD.So we detected the proteins associated with cell death by Western blotting;（2）To get deep into the upstream of Drp1,we used N-acetylcysteine（NAC）to reduce ROS,NAC（100μM）,（200μM）5μl were injected nto subretinal space at the time of retinal detachment,the same volulme of saline was injected into the subretinal space as control group.Finally,we detect the Drp1 expression by Western blotting and measured the photoreceptor death using TUNEL staining.One-Way ANOVA was used to analyze the difference in each group,a P value less than 0.05 is regarded as significant difference.5.To mimic the oxidative micro-environment of photoreceptor after RD in vivo,we used H₂O₂（500μm）to stimulates the 661 W cells for 24 with the treatment of Mdivi-1or siDNML1,we compared the distributions of different mitochondrial morphology in different groups by V-βstaining for mitochondrial.The cell viability was measured by CCK8.Photoreceptor death was detected by TUNEL staining and the mitochondrial membrane potential was measured by JC-1staining.One-Way ANOVA was used to compare the results in different groups,a P value less than 0.05 was regarded as significant difference.【Result】1.There were 8 significant metabolism products after RD.The lactate increased while pyruvate and glutamine reduced after retinal detachment,they are important sources for TCA,It is suggested that mitochondrial dysfunction occurs after retinal detachment.2.Mitochondrial of photoreceptor became shorter after RD by transmission electron microscope.The mitochondrial length in RD was shorter than normal group,^***P<0.0001;mitochondrial fission protein Drp1^S616 increased 1 day,3 day,5 day,and peaked 1 day after RD,**P<0.01,**P<0.01,*P<0.05 versus normal.Mitochondrial morphology after RD is mainly on fragmented shape,fragmentation ratio and the distribution of fragmented mitochondrial morphology increased after RD,***P<0.001,***P<0.001 versus normal,Chi-square test.Based on the previous results,we inferred that mitochondrial fission may play an important role in the retinal detachment.3.To verify the previous hypothesis,we used a selective inhibitor of Drp1 named Mdivi-1 to carry on the intervention experiment.The TUNEL positive staining number in Mdivi-1 treated group is lower than control group 3 day after RD,^*P<0.01,ONL/INL ratio in Mdivi-1treatment group is higher han control group,^***P<0.0001,the amplitude of scotopic a or b wave decresed either in Mdivi-1treatment group or DMSO group,^***P<0.001,^**P<0.01,the amplitude of scotopic a wave in Mdivi-1 treated group is higher than control group,^*P<0.05,the amplitude of scotopic b wave in Mdivi-1treated group is higher than control group,^*P<0.05.4.To get deep into the exact mechanism of neuroptotection with Mdivi-1 treatment.We measured the proteins expression in retinal and mitochondrial.The results indicated that Drp1^S616/tDrp1,caspas3 and Bax increased after RD,^*P<0.05,^***P<0.001,^***P<0.001 versus normal group;separately,while Mdivi-1 treatment reduced the effect,^*P<0.05,^*P<0.01,^**P<0.01 versus control group（RD+DMSO）,Drp1^S616/tDrp1,BAX increased after RD,^***P<0.001,^**P<0.01 versus normal group,while Mdivi-1treatment reduced the effect,^***P<0.001,^**P<0.01,versus control group（RD+DMSO）,separately;5.The vitro experiment indicated that Drp1^S616616 expression in 500μM and 1mM H₂O₂treatment group is higher than normal group,^**P<0.01,^**P<0.01,Fragmented mitochondria increased after H₂O₂ treatment,^***P<0.001 versus normal group,while Mdivi-1 or siDNML1 reduced the effect,^**P<0.01,^***P<0.001 versus H₂O₂ group,separately;H₂O₂ treatment induced cell death,^***P<0.001,versus normal group,Mdivi-1 or siDNML1 attenuated photoreceptor death,^***P<0.001,^***P<0.001 versus H₂O₂group,while^***P<0.001 for Mdivi-1 versus siDNML1;Mitochondrial membrane potential results indicated that the ratio of red/green intensity decreased after H₂O₂ and CCCP treatment,^**P<0.01,^***P<0.001 versus normal group;while Mdivi-1 rescued H₂O₂-induced loss of mitochondrial membrane potential and increased the red/green ratio,^**P<0.01,versus H₂O₂ treatment group,【Conclusion】Retinal detachment can cause mitochondrial dysfunction and increased mitochondrial fission.Inhibition of mitochondrial fission can attenuate photoreceptor death,Drp1 is a new neuroprotection target for retinal disease.