title: Quantification of Oxygen Consumption in Retina Ex Vivo Demonstrates Limited Reserve Capacity of Photoreceptor Mitochondria.
abstract: OBJECTIVE
  Cell death in neurodegeneration occurs at the convergence of diverse metabolic pathways. In the retina, a common underlying mechanism involves mitochondrial dysfunction since photoreceptor homeostasis and survival are highly susceptible to altered aerobic energy metabolism. We sought to develop an assay to directly measure oxygen consumption in intact retina with the goal of identifying alterations in respiration during photoreceptor dysfunction and degeneration.
  
  
  METHODS
  Circular punches of freshly isolated mouse retina, adjacent to the optic nerve head, were used in the microplate-based Seahorse Extracellular Flux Analyzer to measure oxygen consumption. Tissue integrity was evaluated by propidium iodide staining and live imaging. Different substrates were tested for mitochondrial respiration. Basal and maximal respiration were expressed as oxygen consumption rate (OCR) and respectively measured in Ames' medium before and after the addition of mitochondrial uncoupler, BAM15.
  
  
  RESULTS
  We show that glucose is an essential substrate for retinal mitochondria. At baseline, mitochondria respiration in the intact wild-type retina was close to maximal, with limited reserve capacity. Similar OCR and limited mitochondrial reserve capacity was also observed in cone-only Nrl-/- retina. However, the retina of Pde6brd1/rd1, Cep290rd16/rd16 and Rpgrip1-/- mice, all with dysfunctional or no photoreceptors, had reduced OCR and higher mitochondrial reserve capacity.
  
  
  CONCLUSIONS
  We have optimized a method to directly measure oxygen consumption in acutely isolated, ex vivo mouse retina and demonstrate that photoreceptors have low mitochondrial reserve capacity. Our data provide a plausible explanation for the high vulnerability of photoreceptors to altered energy homeostasis caused by mutations or metabolic challenges.
authors.affiliation: Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Keshav
authors.initials: K
authors.lastname: Kooragayala
authors.affiliation: Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Norimoto
authors.initials: N
authors.lastname: Gotoh
authors.affiliation: Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Cogliati
authors.affiliation: Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Jacob
authors.initials: J
authors.lastname: Nellissery
authors.affiliation: Retinal Neurobiology Section, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Talia R
authors.initials: TR
authors.lastname: Kaden
authors.affiliation: Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Stephanie
authors.initials: S
authors.lastname: French
authors.affiliation: Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Balaban
authors.affiliation: Retinal Neurobiology Section, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Raul
authors.initials: R
authors.lastname: Covian
authors.affiliation: Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Anand
authors.initials: A
authors.lastname: Swaroop
mesh_terms: D000818:Animals; D016923:Cell Death; D004734:Energy Metabolism / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism*; D018384:Oxidative Stress; D010101:Oxygen Consumption / Q000502:physiology*; D017949:Retinal Cone Photoreceptor Cells / Q000166:cytology / Q000378:metabolism*