MRI of Retinal Free Radical Production With Laminar Resolution In Vivo.

Rod cell oxidative stress, the continual generation of free radicals exceeding a cell's quenching capacity, has been implicated in the pathogenesis of currently untreatable, degenerative diseases of the retina, including retinitis pigmentosa and diabetic retinopathy.1–16 However, clinical benefits from antioxidant treatment have not been realized, perhaps because a one-size-fits-all approach may be too simple and also because we cannot assess antioxidant treatment efficacy in vivo on rod cells. Conventional methods, such as optical coherence tomography (OCT) and electrophysiology (ERG), do not measure rod cell free radical production. Recent methods have examined redox-responsive probes to evaluate oxidative stress from specific reactive oxygen species in vivo, but these approaches are problematic because they (1) modify the redox environment by the presence of the reporter probes, (2) require careful consideration of the biologic time course of the probe, (3) require intravitreal injections, and (4) measure fundus oxidative stress without laminar resolution.17–20 Thus, the use of these methods for longitudinal studies and in the clinic is unlikely, limiting objective evaluation of antioxidant treatment efficacy over time within rod cells. Recently, we, and others, unmasked a contribution of continuously produced free radicals to the spin-lattice relaxation rate (1/T1) magnetic resonance imaging (MRI) signal by collecting data in the absence and presence of a quenching condition (“quench-assisted MRI”).21,22 Quench-assisted 1/T1 MRI measured outer retina oxidative stress following pharmacological manipulation.21 Furthermore, quench-assisted MRI reliably reported the in vivo suppression of free radical production in healthy outer retina in response to a light quench.21 For example, light quench-assisted MRI readily distinguished between the expected differences in the light response of rods of 129S6 wild-type mice versus cones of R91W;Nrl−/− mice.21 These results suggest that light-evoked suppression of outer retinal 1/T1 signal is linked with reduced leakage of free radicals from mitochondria.9,23 Here, we extend the above studies by testing the spatial and detection sensitivity of quench-assisted MRI to strain, age, or retinal cell layer-specific genetic manipulations. The following groups were evaluated: (1) a common wild-type mouse strain, the C57Bl/6 mouse, (2) immature mice before outer retinal photosensitivity develops, postnatal day 7 mice (P7), (3) mutant mice missing mitochondrial superoxide dismutase (SOD) from only the retinal pigment epithelium (RPE) layer (and their controls), and (4) mice lacking rod specific α-transducin (Gnat1). α-transducin is an essential signaling element for rod phototransduction.24,25 In general, because Gnat1−/− mice have inoperant rod phototransduction without retinal degeneration, they allow for the exploration of cone responses to light, as well as rhodopsin regeneration via the visual cycle.26,27 In this study, quench-assisted MRI outcomes in vivo in these groups matched results generated from previous studies using ex vivo methods. Overall, our results provide further support for the hypothesis that quench-assisted MRI has the cellular and subcellular spatial resolution, and detection sensitivity, for evaluating normal and pathologic production of retinal free radical production in vivo.