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Pawan Kumar Singh, Ajay Kumar, Ashok Kumar; Molecular mechanisms of retinal cell death in Staphylococcus aureus endophthalmitis. . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):872. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Severe vision loss in bacterial endophthalmitis is associated with the death of retinal cells including photoreceptors. In the present study, we investigated the molecular mechanisms of cell death triggered by Staphylococcus aureus using both in vivo and in vitro experimental models.
For in vivo studies, endophthalmitis was induced in C57BL/6 mice by intravitreal injection of 5000 cfu of S. aureus (strain RN6390). TUNEL and Annexin V & PI staining were used to determine cell death using microscopy and flowcytometry, respectively. qRT-PCR was used to determine the induced expression of pathway-specific genes and death domain receptors. Western blot analysis was performed to assess the activation of caspases and other executioner proteins (cytochrome C, AIF, PARP). Human Müller glia (MIO-M1 cells) and mouse cone photoreceptor cells (661W) were used for in vitro studies. The mitochondrial membrane potential was studied by JC1 staining using fluorescence microscopy, as well as by flowcytometry. The caspase and PARP1 inhibitors were used for the inhibition study.
S. aureus caused significant cell death in the mouse retina and cultured MIO-M1 and 661W cells as evidenced by increased number of TUNEL positive cells. S. aureus-challenged retina and retinal cells exhibited increased generation of ROS, reduced mitochondrial membrane potential (JC1 staining), the release of cytochrome C into the cytoplasm and the activation of caspase 3. We also observed the cleavage of PARP-1 and the release of Apoptosis Inducing Factor (AIF) from mitochondria in S. aureus-infected cells. Our inhibition studies showed that pancaspase inhibitor Q-VD-OPH and PARP-1 inhibitor DPQ reduced retinal cell death.
Our data demonstrate that in bacterial endophthalmitis, retinal cells undergo apoptosis in both caspase-dependent and caspase-independent manner and mitochondria seems to play a central role in this process. These insights prompt further study to assess potential therapeutic candidates for retinal protection against bystander damage induced by microbial pathogens.
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