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Guzel Bikbova, Toshiyuki Oshitari, Shuichi Yamamoto; Both caspase-dependent and capase-independent cell death pathways are involved in neuronal death in rat retinas exposed to AGEs. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1272.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether the neuroprotective and regenerative effects of neurotrophin-4 (NT-4) is correlated with a reduction of caspase-9 and AIF expression in rat retinas exposed to AGEs.
All of the procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Retinal explants of 4 adult SD rats were three-dimensionally cultured on collagen gels and incubated in; serum free control culture media, 10 μg/ml glucose-AGE-BSA media, 10 μg/ml glycolaldehyde-AGE-BSA media, 10 μg/ml glyceraldehyde-AGE-BSA media, glucose-AGE+100 ng/ml NT-4 media, glycol-AGE+100 ng/ml NT-4 media, or glycer-AGE+100 ng/ml NT-4 culture media. After 7 days, the number of regenerating neurites from the explants was counted under a phase-contrast microscope. After counting, the retinal explants were fixed, cryosectioned, and stained by TUNEL and DAPI. The ratio of TUNEL-positive cells to the number of DAPI-staining nuclei in the ganglion cell layer was calculated. Immunohistochemistry for the active form of caspase-9 and apoptosis-inducing factor (AIF) was performed. Statistical analyses were performed by one-way ANOVA.
In retinas incubated with AGEs (glucose-AGE, glycol-AGE, and glycer-AGE), the number of regenerating neurites was fewer than in retinas without AGE (P=0.0033, P=0.0044, P=0.0238). The number of TUNEL-positive cells (P<0.0001, P<0.0001, P<0.0001) and caspase-9-positive cells (P<0.0001; P<0.0001; P<0.0001) and AIF-positive cells (P=0.0004, P=0.0002, P=0.056) in the ganglion cell layer was higher than in controls. NT-4 supplementation increased, the number of regenerating neurites (P<0.0001, P<0.0001, P<0.0001). The number of TUNEL-positives cells (P<0.001, P=0.005, P=0.0003), caspase-9-positive cells (P<0.0001, P<0.0001, P<0.0001), and AIF-positive cells (P=0.026, P=0.026, P=0.016) was significantly fewer than in glucose-AGE without NT-4 and in glycol-AGE without NT-4 and in glycer-AGE without NT-4.
Both caspase-dependent and caspase-independent cell death pathways are associated with retinal neuronal cell death in low dose AGE-BSA exposed rat retinas. Neuroprotective and regenerative effect of NT-4 is correlated with a reduction of caspase-9 and AIF activation.
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