PURPOSE. To test the hypothesis that the extent of outer retina uptake of manganese, measured noninvasively with manganese-enhanced MRI (MEMRI), is a quantitative biomarker of photoreceptor ion channel regulation by visual cycle activity. METHODS. Four groups of animals were studied: control rats adapted to three different background light intensities, darkadapted control mice systemically pretreated with retinylamine, and dark-adapted mice with a nonsense mutation in exon 3 of the RPE65 gene (RPE65 rd12 ) with and without systemic 11-cis-retinal pretreatment. In all cases, rodents were anesthetized and studied with MEMRI 4 hours after manganese administration IP. Central retinal thickness and intraretinal ion channel regulation were measured from the MEMRI data. RESULT. No differences (P Ͼ 0.05) in retinal thickness were noted within any arm of this study. In rats, manganese uptake was inversely proportional to the background light intensity in the outer retina but not in the inner retina. Specific inhibition at the level of RPE65 activity, either acutely with retinylamine or chronically in RPE65 rd12 mice, similarly reduced (P Ͻ 0.05) outer retinal manganese uptake compared with that in control mice. In RPE65 rd12 mice, outer retinal manganese uptake returned to normal (P Ͼ 0.05) after 11-cis retinal treatment. Inner retinal uptake was supernormal (P Ͻ 0.05) in retinylaminetreated mice but normal in untreated or 11-cis treated RPE65 rd12 mice. CONCLUSIONS. The present data support measuring the extent of manganese uptake in the outer retina as an analytic noninvasive metric of visual cycle regulation of photoreceptor ion channel activity in vivo. (Invest Ophthalmol Vis Sci. 2009;50: 188050: -188550: ) DOI:10.116750: /iovs.08-2958 N ormal vision involves conversion of photons to electrical activity in the retina. In rod photoreceptors, this process starts when light interacts with rhodopsin (which consists of opsin and covalently bound 11-cis-retinal) to produce a cis-totrans isomerization. Through a series of signal transduction steps, this isomerization causes cyclic guanosine monophosphate (cGMP)-gated ion channels in rods, which are maximally opened in the dark, to close in a graded fashion depending on the light intensity. 1,2 Rhodopsin and 11-cis-retinal are regenerated via the visual cycle to produce fresh rhodopsin. A key step in the visual cycle involves retinal pigment epitheliumspecific protein 65 kDa (RPE65), an isomerase that converts all-trans retinol back to 11-cis-retinal. 3 Prolonged impairment of the visual cycle resulting in inhibited 11-cis-retinal production-for example, by long-term inhibition of RPE65 activitycan produce persistent build-up of unbound opsin to concentrations high enough to induce chronic channel closure, which is linked with photoreceptor degeneration. 4 New pharmaceutical and gene therapies are being developed to address abnormal visual cycle activity and associated retinal degeneration. As these treatment options enter clinical trials, there is a need for noninvasive metrics of abnormal visual cycle activity in focal retinal regions (to identify which locations are most likely to benefit from treatment intervention before overt retinal thinning is evident), and, after different dosage schedules and concentrations of treatments for visual cycle abnormalities, can prognostically measure local rescue efficacy in emerging retinopathy. 5 Currently, the electroretinogram (ERG) and optical coherence tomography (OCT) have been essential in documenting overall progression and treatment response for retinal dystrophy. Manganese-enhanced MRI (MEMRI) is a noninvasive method that allows for simultaneous measurement of regional retinal uptake of manganese ion (Mn 2ϩ , a strong MRI contrast agent and biomarker for regulation of ions such as calcium) colocalized with retinal thickness, after systemic injection of a modest and nontoxic amount of MnCl 2 . 6 -8 We have found that MEMRI is sensitive to the state of cGMP-gated channels, since significantly more manganese was taken up in the outer retina in dark-adapted rodents relative to that in light adapted rodents. In this study we tested the hypothesis that the extent of outer retina uptake of manganese is a quantitative biomarker of photoreceptor ion channel regulation by visual cycle activity. At intermediate light intensities, photoreceptors respond to background light levels with a proportionate closure of the ion cation channels. 1 It is not yet known if such a graded response is reflected in the extent of outer retinal manganese uptake in rodents. We also examined the sensitivity of MEMRI to specific inhibition of the visual cycle at the level of RPE65 that subsequently reduces 11-cis-retinal production, which is expected to result in a buildup of unbound opsin and closure of photoreceptor ion channels. 4,9,10 MEMRI was used to study darkadapted control mice and mice systemically pretreated with retinylamine. 9 A single application of retinylamine produces a long-lasting but impermanent inhibition of 11-cis-retinal production without associated retinal degeneration. 10,11 METHODS The animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. In all cases, rats or mice were housed and maintained in a normal 12 hour/12 hour light/dark cycle. The day before the MRI experiment, rodents were placed and maintained in total darkness overnight. All procedures (e.g., weighing animals, injecting MnCl 2 , anesthesia for MRI, and MRI examination) were performed in dim red light, darkness, or the light level being studied. MnCl 2 was administered as an intraperitoneal injection to awake rats (44 mg/kg) or mice (66 mg/kg), as described previously (Roberts R, et al. IOVS 2008;43:ARVO E-Abstract 4926). 7,8 Different doses were necessary, since, in a preliminary study, the 44-mg/kg dose of MnCl 2 did not produce reliable contrast changes in the mouse retina (data not shown), possibly due to the relatively higher overall metabolic rate in the mouse. Instead, it was found empirically that a somewhat higher dose of manganese (66 mg/kg) produced more robust retinal contrast changes. In all cases, rodents were maintained awake in dark conditions for another 3.5 hours, anesthetized, and imaged (MEMRI study). Experimental Arms Light/Dark. Male albino control Sprague-Dawley (SD) rats (204 -276 g; mean age, 46 days) were examined with MEMRI after exposure to the following light intensities: 1.8 Ϯ 0.7 (n ϭ 10, mean Ϯ SEM), 51.3 Ϯ 11.7 (n ϭ 5), and 250.2 Ϯ 19.3 (n ϭ 6) lux. Light meter (traceable dual-range light meter; Control Co., Friendswood, TX) values were multiplied by 0.91 to correct fluorescent light values to the tungsten light calibration values. The intermediate light levels were produced by positioning the cage different distances from a 25-W fluorescent light bulb. Every hour after manganese administration, intensity readings were obtained at the brightest and dimmest portions of the cage and averaged; mean readings over the 4-hour time course (before the MEMRI experiment) were then averaged. Retinylamine. Two groups were studied in this arm of the study: noninjected C57BL/6 mice (n ϭ 8 males; 28 -33 g; mean age, 184 days) and C57BL/6 mice (n ϭ 6 males; 27-32 g; mean age, 184 days) treated with retinylamine (kind gift of Martin Golczak and Krzysztof Palczewski, Case Western Reserve University, Cleveland, OH). 9 For each treated mouse, retinylamine (0.5 mg) was dissolved in DMSO (100 L) and injected intraperitoneally. The next day, treated mice were briefly anesthetized with ether. Their eyes were dilated with 1 drop of atropine and they were allowed to fully wake up. They were then positioned close to laboratory lights (300 lux) for 4 hours to ensure the remaining rhodopsin levels were bleached. In all cases, the mice were dark adapted overnight, during the injection, and MEMRI examination. These two groups had similar extraocular muscle signal intensities (as reported in the results section) implying similar whole body handling of manganese (i.e., no DMSO effect). RPE65 rd12 . In this arm of the study, the following groups were examined with MEMRI: noninjected C57BL/6 mice (n ϭ 4 males; 26 -29 g; mean age, 90 days) and noninjected RPE65 rd12 on a C57BL/6 background (n ϭ 4 males; 16 -20 g; mean age, 35 days; Jackson Laboratories, Bar Harbor, ME). 11 Note that the 11-cis-retinal formulation and administration used in this study killed three other RPE65 rd12 mice and impaired systemic handling of manganese in three surviving RPE65 rd12 mice that required correction (vide infra). In all cases, the mice were dark adapted overnight, during the injection, and during the MEMRI examination. Note that C57BL/6 mice have the methionine amino acid at codon 450 of the RPE65 gene. 12 Manganese-Enhanced MRI. Immediately before the MRI experiment, each animal was anesthetized with urethane (36% solution, IP 0.083 mL/20 g animal weight, prepared fresh daily; Aldrich, Milwaukee, WI). In addition, some mice received an injection of xylazine (1-8 mg/kg, IP). In mice, urethane was found to increase respiratory frequency and thus motion artifacts on MEMRI. The addition of a small amount of the muscle relaxant xylazine helped to minimize these artifacts. To maintain the core temperature, a recirculating heated water blanket was used. Rectal temperatures were continuously monitored throughout each experiment, as previously described. 13 MRI data were acquired on a 4.7-T system (Avance; Bruker AXS, Madison, WI) using a two-turn transmit/receive surface coil (1.0 cm diameter) placed over the eye. Images were acquired with an adiabatic spin-echo imaging sequence (repetition time [TR] 14 A single transverse slice through the center of the eye (based on sagittal localizer images collected using the same adiabatic pulse sequence as just described) was obtained for each animal. Data Analysis Retinal Thickness. Whole retinal thicknesses were determined from each MEMRI-generated image as the radial distance between the anterior edge and the posterior edge of the retina at distances Ϯ 0.4 to 1 mm from the optic nerve. Layer-Specific Signal Intensity. Within each group, individual linearized retinas were averaged into a composite image and used for visual comparison purposes only. For quantitative analysis, signal intensities were analyzed using the program NIH IMAGE (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/ index.html) and derived macros. Downloaded from iovs.arvojournals.org on 06/29/2019 tive of the inner and outer retina, respectively, and were analyzed as previously described. Statistical Analysis The retinal thicknesses were consistent with a normal distribution, and comparisons between groups were performed by unpaired two-tailed t-test. Comparison of retinal signal intensities and Mn 2ϩ ion enhancements were performed in a generalized estimating equation approach. RESULTS Background Light Intensit
