June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
DEVELOPMENT OF A NOVEL APPROACH FOR ASSESSMENT OF MITOCHONDRIAL RESPIRATION IN DIABETIC RETINA
Author Affiliations & Notes
  • Anand Rao-Venkata Saripalli
    Physiology/Chemistry/Medicine, Michigan State University, East Lansing, Michigan, United States
    Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, United States
  • Yan Levitsky
    Physiology/Chemistry/Medicine, Michigan State University, East Lansing, Michigan, United States
  • Elahe Crockett
    Physiology/Chemistry/Medicine, Michigan State University, East Lansing, Michigan, United States
  • Evangelyn Alocilja
    Engineering, Michigan State University, East Lansing, Michigan, United States
  • Julia V Busik
    Physiology/Chemistry/Medicine, Michigan State University, East Lansing, Michigan, United States
  • Denis Proshlyakov
    Physiology/Chemistry/Medicine, Michigan State University, East Lansing, Michigan, United States
  • Footnotes
    Commercial Relationships   Anand Saripalli, None; Yan Levitsky, None; Elahe Crockett, None; Evangelyn Alocilja, None; Julia Busik, None; Denis Proshlyakov, None
  • Footnotes
    Support  This work was supported by NIH-R25-HL108864 award to Crockett, and NIH/NEI grant 2 R01 EY016077-08 to Julia Busik/Proshlyakov. Anand was a REPID scholar and his research training was supported by NIH-R25-HL108864 award.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2525. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Anand Rao-Venkata Saripalli, Yan Levitsky, Elahe Crockett, Evangelyn Alocilja, Julia V Busik, Denis Proshlyakov; DEVELOPMENT OF A NOVEL APPROACH FOR ASSESSMENT OF MITOCHONDRIAL RESPIRATION IN DIABETIC RETINA. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2525.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Despite recent success in treatment approaches, diabetic retinopathy (DR) remains a leading cause of progressive vision loss and blindness. It is well accepted that mitochondrial dysfunction contributes to the development of DR, however the mechanism(s) of mitochondrial damage are not well understood. Recent studies demonstrate that there is an intricate connection between ceramide and mitochondrial function. We have previously demonstrated that activation of acid sphingomyelinase (ASM) and ceramide production is an important early event in the pathogenesis of DR, however the role of ceramide-induced mitochondrial dysfunction in DR is unknown due to the lack of sufficiently sensitive tools. This study aims to design the tools for evaluation of the role of mitochondria in ceramide-induced damage in diabetic retinas.

Methods : Using differential centrifugation and magnetic nanoparticles (MN) conjugated to anti-translocase of the outer membrane of mitochondria-22 (anti-TOM-22) antibodies, mitochondria were isolated from mouse livers, retinas, and retinal pigment epithelium (RPE) cell cultures in normal (5.5mM) or high (25mM) glucose for 24 hours. Mitochondrial respiration was measured using a NeoFox fluorescent oxygen sensor in both conventional and microfluidic chambers. Glucose oxidase bound MNs were used for calibration of the microfluidic device.

Results : The conventional NeoFox oxygen sensor requires mitochondrial isolation from ~700mg of tissue or about 0.05mg of total protein to achieve the detection limit. The microfluidic device reduced tissue requirements to ~7mg or about 0.0027mg of total protein making it possible to analyze mouse retinal mitochondrial metabolism from a single animal. We have demonstrated that MNs can be used to concentrate mitochondria in the microfluidic chamber. Using this approach we found a 3.6 ± 0.2 fold increase in mitochondrial outer membrane permeability in RPE cells cultured in 25mM glucose compared to control. The increase was corrected by ASM inhibitor desipramine.

Conclusions : MN-based mitochondria isolation coupled with a microfluidic sensor for respiratory and electrochemical activity shows potential for identification of complex-specific changes in the mechanism of ceramide-induced mitochondrial dysfunction in the diabetic retina.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×