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چکیده
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Introduction: Leber's hereditary optic neuropathy (LHON) is a mitochondrial and neurodegenerative disease that mainly affects retinal ganglion cells (RGCs). Complex 1 deficiency due to mitochondrial DNA mutation leads to visual failure. LHON selectively targets RGCs, which, in contrast to most other cells, depend highly on mitochondrial performance. The pathology of LHON is characterized by a decrease in retina layer thickness and loss of RGCs. Nowadays, there is no available treatment for improving the final visual outcome in LHON. Rotenone, a mitochondrial complex I inhibitor, causes retinal degeneration via unknown mechanisms. This poison inhibits the reduction of ubiquinone and when administered intravitreally to mice, causes biochemical, structural, and functional retinal deficits resembling those observed in LHON patients. Rotenone induced an increase in mitochondrial-derived free radicals and lipid peroxidation in primary rat retinal cultures. The goal of current study was to develop an improved and highly reproducible rotenone model of LHON. Method: In this study, male C57BL/6J (7-8 weeks old) were administered rotenone (2ul) in a specialized vehicle by intravitreal injection. Modeling is done only in one eye and the other eye is considered as a control. Rotenone solution in dimethyl sulfoxide (DMSO) was freshly prepared before each treatment. After rotenone injection, vision test and morphometric test were performed at four time points (1h, 24h, 48h and 7days). Visual acuity was evaluated by optokinetic response (OKR). As mentioned above, LHON is characterized by decreasing retinal layers thickness and loss of RGCs, therefore the thickness of the retinal layers is measured by DAPI staining, and also RGCs viability is evaluated by immunohistochemistry technique. Finally, the results of different time points were compared. Results: Images from morphometric tests were analyzed by ImageJ software. Surprisingly, the results obtained from morphometric tests was compromised by the visual tests. The thickness of retinal layers and the survival rate of retinal cells were significantly decreased 24 hours following intravitreal injection of rotenone as compared to control groups. Visual- and retinal- deficit induced by 48 hours and seven days after rotenone treatment was too severe. These results suggest that the mice received intravitreal administration of rotenone after 24 hours was considered the most suitable model of LHON. Conclusion: Here, we established a mouse model of LHON by injecting rotenone in mice's eyes. Eyes exposed to rotenone displayed significantly loss of vision and alterations in retinal layers specially in inner plexiform layer. We demonstrated that rotenone induces retinal cell death by apoptosis resembling those observed in LHON patients. In summary, this version of the rotenone model is highly reproducible and may provide an excellent tool to test new neuroprotective strategies.
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