June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Both caspase-dependent and capase-independent cell death pathways are involved in neuronal death in rat retinas exposed to AGEs
Author Affiliations & Notes
  • Guzel Bikbova
    Ophthalmology & Visual Science, Chiba Univ Grad School of Medicine, Chuo-ku, Chiba, Japan
  • Toshiyuki Oshitari
    Ophthalmology & Visual Science, Chiba Univ Grad School of Medicine, Chuo-ku, Chiba, Japan
  • Shuichi Yamamoto
    Ophthalmology & Visual Science, Chiba Univ Grad School of Medicine, Chuo-ku, Chiba, Japan
  • Footnotes
    Commercial Relationships Guzel Bikbova, None; Toshiyuki Oshitari, None; Shuichi Yamamoto, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1272. doi:
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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)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 615 neuroprotection • 499 diabetic retinopathy • 694 retinal culture  
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