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Huiyang Zeng, Xingxing Chen, ; The Role Of RANTES And Chemokine Receptors In The Retinal Degeneration In Rd Mice. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3246.
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Our previous study reported activation of microglia might play a major role in the retinal degeneration in rd mice and CC chemokines were involved in the activation of microglial cells. In this study we further investigate the expression of RANTES and its receptors in the rd retina and their signal transduction pathway in the photoreceptor degeneration.
The expression of RANTES,CCR1,CCR3 and CCR5 in the rd retinas at different age group was determined by RT-PCR and immunostaining . Expression of gp91phox was studied by western blot and immunostaining. For in-vitro experiments,intracellular calcium levels in the BV-2 murine microglial cells were determined by using calcium probe Fura-2 when the cells were stimulated with 50ng/ml RANTES. A murine photoreceptor cell line (661W cells) was exposed to RANTES-treated BV-2 microglial cells conditioned medium(MGCM), and the cell viability was assayed by TUNEL and MTT assay. Production of superoxide in the activated BV-2 cells supernatant was also determined by measuring WST-1 .met-RANTES was used to inhibit microglial activation and photoreceptor apoptosis in-vivo and in-vitro .
RANTES,CCR1 and CCR5 mRNA was moderately elevated in the rd retina peaking at P 12 and P14. Immunoreactivity of CCR1 on the photoreceptors and CCR5 on the activated retinal microglial cells were found to be increased in the rd mice. Elevated expression of gp91phox were found in the rd retinas at P8 and reached peak at P12 and P14. gp91phox was seen on the microglial cells. Most of gp91phox were co-localized with CCR5 in the microglial cells. Compared with controls, the thickness of the outer nuclear layer was significantly increased when intravitreous injection of met-RANTES was applied in the rd mice at P7. Marked elevation of intracellular calcium flow was observed in the BV-2 cells expressing CCR5 when the cells were stimulated by RANTES. 33% of 661W cells underwent apoptosis following exposure to MGCM for 24hrs. MGCM-induced cell death was associated with increased ROS release by activated microglial cells and could be inhibited by met-RANTES.
RANTES may play a important role in the retinal degeneration in rd mice through activation of microglial cells killing mechanism ( activation of CCR5 and release ROS). Inhibition of CCR5 activation by met-RANTES may rescue or delay photoreceptor cell death in the degenerative process.
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