Kooragayala Keshav, Gotoh Norimoto, Cogliati Tiziana, Nellissery Jacob, Kaden Talia R, French Stephanie, Balaban Robert, Li Wei, Covian Raul, Swaroop Anand
Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
Retinal Neurobiology Section, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
Invest Ophthalmol Vis Sci. 2015 Dec;56(13):8428-36. doi: 10.1167/iovs.15-17901.
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.
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.
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.
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.
神经退行性变中的细胞死亡发生在多种代谢途径的交汇点。在视网膜中，一个常见的潜在机制涉及线粒体功能障碍，因为光感受器的内环境稳定和存活对有氧能量代谢的改变高度敏感。我们试图开发一种检测方法来直接测量完整视网膜中的氧气消耗，目的是识别光感受器功能障碍和退化过程中呼吸作用的改变。
使用紧邻视神经乳头的新鲜分离的小鼠视网膜圆形切片，在基于微孔板的海马细胞外通量分析仪中测量氧气消耗。通过碘化丙啶染色和实时成像评估组织完整性。测试了不同的底物对线粒体呼吸的作用。基础呼吸和最大呼吸分别表示为氧消耗率（OCR），并分别在添加线粒体解偶联剂BAM15之前和之后在艾姆斯培养基中进行测量。
我们表明葡萄糖是视网膜线粒体的必需底物。在基线时，完整野生型视网膜中的线粒体呼吸接近最大值，储备能力有限。在仅含视锥细胞的Nrl-/-视网膜中也观察到类似的氧消耗率和有限的线粒体储备能力。然而，Pde6brd1/rd1、Cep290rd16/rd16和Rpgrip1-/-小鼠的视网膜，所有这些小鼠的光感受器功能失调或没有光感受器，其氧消耗率降低，线粒体储备能力更高。
我们优化了一种直接测量急性分离的离体小鼠视网膜中氧气消耗的方法，并证明光感受器的线粒体储备能力较低。我们的数据为光感受器对由突变或代谢挑战引起的能量内环境稳定改变高度敏感提供了一个合理的解释。
