June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Apoptosis and necroptosis is induced in cone photoreceptor cells subjected to cold storage and rewarming
Author Affiliations & Notes
  • Swati Jain
    University of Colorado, Denver, Colorado, United States
  • Dor Yoeli
    University of Colorado, Denver, Colorado, United States
  • Neil Khatter
    University of Colorado, Denver, Colorado, United States
  • An-Jey Su
    University of Colorado, Denver, Colorado, United States
  • Evan Farkash
    University of Michigan, Ann Arbor, Michigan, United States
  • Christene Huang
    University of Colorado, Denver, Colorado, United States
  • Kia Washington
    University of Colorado, Denver, Colorado, United States
  • Alkesh Jani
    University of Colorado, Denver, Colorado, United States
  • Footnotes
    Commercial Relationships   Swati Jain None; Dor Yoeli None; Neil Khatter None; An-Jey Su None; Evan Farkash None; Christene Huang None; Kia Washington None; Alkesh Jani None
  • Footnotes
    Support  This work is funded by a Department of Defense award (W81XWH2010858)
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3114. doi:
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      Swati Jain, Dor Yoeli, Neil Khatter, An-Jey Su, Evan Farkash, Christene Huang, Kia Washington, Alkesh Jani; Apoptosis and necroptosis is induced in cone photoreceptor cells subjected to cold storage and rewarming. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3114.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Blindness afflicts 39 million people worldwide. Retinal ganglion cells are unable to regenerate, making this condition irreversible in many cases. Whole-eye transplantation (WET) may be a viable treatment option. To further enhance our knowledge of WET, individual cell types response to the transplant must be studied. 661W cone photoreceptor cells, derived from mouse, have been widely used as a model for studying macular degeneration. Here, we characterize the 661W cell line for studying the effect of cold ischemia/warm reperfusion injury that is relevant to WET.

Methods : To understand the phenotype of injury during in-vivo cold ischemia/warm reperfusion injury, 661w cells were subjected to cold storage and rewarming (CS/REW). First, cells were subjected to cold storage (CS) in cold saline solution for 24 h at 4°C. Next, cells were rewarmed (REW) in normal growth media at 37°C for 24 h. Control cells were kept at 37°C. A dose response analysis was performed to determine the optimal concentration of pan-caspase inhibitor (Q-VD-OPh) and necroptosis inhibitor (Necrostatin-1) to completely inhibit aoptosis and necroptosis.

Results : Cell death percentage, measured as Annexin V positive and/or PI positive cells by flow cytometry was significantly increased in the 661w cells subjected to CS/REW compared to control cells (Figure 1). We looked for the expression of key integrators of signaling pathways that mediate apoptosis and necroptosis. 661w cells subjected to CS/REW have activation of apoptosis as determined by increased expression of cleaved caspase-3 and increased TUNEL staining. Moreover 661w cells exposed to CS/REW also showed activation of necroptosis as determined by increased expression of RIP3 and pMLKL. The effect of Q-VD-OPh and Necrostatin-1 on caspase-3 protein expression and pMLKL was dose-dependent. Q-VD-OPh treatment at 50 µM inhibit the apoptosis, whereas Necrostatin-1 treatmnet at 100 µM inhibit the necroptosis.

Conclusions : Our results suggest that the inclusion of cold storage followed by warm reperfusion during WET will likely induce cell death by both apoptosis and pnecroptosis. Apoptotic cell death and necroptosis can be successfully prevented using a pan-caspase I inhibitor and Necrostatin-1. Future studies: We will also study the collective effect of inhibiting apoptosis and necroptosis in 661w cells after CS/REW by using a cocktail of Q-VD-OPh and Necrostatin-1.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

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