Optical imaging of mitochondrial redox state in rodent model of retinitis pigmentosa

Abstract

Oxidative stress (OS) and mitochondrial dysfunction contribute to photoreceptor cell loss in retinal degenerative disorders. The metabolic state of the retina in a rodent model of retinitis pigmentosa (RP) was investigated using a cryo-fluorescence imaging technique. The mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent and can be monitored without exogenous labels using optical techniques. The cryo-fluorescence redox imaging technique provides a quantitative assessment of the metabolism. More specifically, the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), the NADH redox ratio (RR), is a marker of the metabolic state of the tissue. The NADH RR and retinal function were examined in an established rodent model of RP, the P23H rat compared to that of nondystrophic Sprague-Dawley (SD) rats. The NADH RR mean values were 1.11 ± 0.03 in the SD normal and 0.841 ± 0.01 in the P23H retina, indicating increased OS in the P23H retina. Electroretinographic data revealed a significant reduction in photoreceptor function in P23H animals compared to SD nozrmal rats. Thus, cryo-fluorescence redox imaging was used as a quantitative marker of OS in eyes from transgenic rats and demonstrated that alterations in the oxidative state of eyes occur during the early stages of RP.

Oxidative stress and its downstream signaling in aging eyes

“…The pathophysiology of some of them includes oxidative damage to the RPE, as in AMD or Stargardt disease 64,65. The mechanism of RPE injury has not been clearly described, but it may be related to A2E (a lipofuscin fluorophore) 69,70–74. AMD, a leading cause of severe visual loss, is a multifactorial disease, and thus its pathogenesis remains poorly understood.…”

Organ specific optical imaging of mitochondrial redox state in a rodent model of hereditary hemorrhagic telangiectasia‐1

“…Thus, the measurement of mitochondrial metabolic activity could potentially be an early indicator of organ dysfunction during the disease. Our present method measures fluorescence from both NADH and FAD, allowing calculations of the redox ratio, which has proven to be a powerful diagnostic marker, independent of the number of mitochondria and hemodynamics [50, 65, 76]. Furthermore, this study demonstrates the ability of cryoimaging to measure FAD RR of various organs.…”

“…Before fluorescence imaging, the extracted tissue were frozen and embedded as previously described [65]. …”

“…Details of this instrument has been previously reported [65]. For this study, we used a resolution of 50 μm for kidney and 10 μm for eyes in the z direction, which resulted in 300 and 250 z-slices per kidney and eye, respectively.…”

“…To compensate for day-to-day variation of light intensity and non-uniformity of the illumination pattern, the calibration method described previously [65] was carried out.…”