Effect of Methylimidazole-Induced Hypothyroidism in a Model of Low Retinal Neovascular Incidence

PURPOSE. To determine the effect of methylimidazole (MMI)-induced hypothyroidism in a newborn rat model of low retinal neovascular (NV) incidence. METHODS. Control and MMI-exposed newborn rats were raised either in room air or variable oxygen (40/15) until P14. All groups were then exposed to room air between postnatal day (P)14 and P20. Dams drank either tap water or water containing MMI. Eyes of animals in all groups were enucleated, and retinas were removed and stained with adenosine diphosphatase and analyzed for peripheral avascularity, vascular density, and NV incidence and severity. RESULTS. In the control group, MMI treatment did not promote the development of retinal NV although a linear relationship (r ϭ 0.99, P Ͻ 0.01) was found between increased MMI dose and lower peripheral retinal vascular densities. In all the 40/15 groups, peripheral retinal vascular densities were lower (P Ͻ 0.05) than normal and were not a function of MMI dose. Increased MMI dose produced increased retinal incidence of NV (r ϭ 0.99, P Ͻ 0.05). CONCLUSIONS. These data are consistent with the notions that thyroid function contributes to normal retinal vascular density and that hypothyroidism can play a permissive role in the development of retinal NV. (Invest Ophthalmol Vis Sci. 2004; 45:919 -921) DOI:10.1167/iovs.03-0914 V ery-low-birth-weight infants are at substantially higher risk for blinding complications, such as the development of retinal neovascularization (NV) associated with retinopathy of prematurity (ROP). To minimize the impact of retinal NV on vision, photocoagulation is currently used, but it is a destructive approach that is not always effective. A better understanding is needed of the pathogenic factors involved in the formation of retinal NV in ROP so that new methods of prevention and treatment can be developed. To date, studies have demonstrated an important link between insulin-like growth factor (IGF)-1 and normal and abnormal retinal vascular development. 1-3 IGF-1 is, among other functions, a downstream modulator of thyroid activity. Infants born very prematurely (Ͻ27 weeks) are more likely to have low thyroxine (T 4 ) levels, indicating an abnormal hypothalamus-pituitary-thyroid axis function. In this study, we used a clinically relevant model of ROP involving newborn rats exposed to a variable oxygen environment from postnatal day (P)0 to P14 and then to room air between P15 and P20. 6 -8 In animals exposed to an oxygen environment that alternates between 40% and 15% every other day (the 40/15 model), only a small percentage (Ͻ10%) of the rat pups exhibit 1 clock hour of NV in the peripheral retina. -14 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. MMI Between P0 and P20, rat pups were raised in room air and received MMI (0.05%, 0.1%, 0.13%, or 0.15% wt/vol; Sigma-Aldrich, St. Louis, MO), added to the drinking water of the dam. Control dams and their pups drank untreated water. In the control and 0.13% and 0.15% MMI-treated 40/15 groups, measurements of T 4 , thyroid stimulating hormone (TSH), and IGF-1 were performed (Anilytics Inc., Gaithersburg, MD) on pooled blood samples. Pooled blood samples were used because of the limited amount of blood available from individual newborn rats (Ͻ30 g). However, retinal histopathology was not investigated, because 0.13% and 0.15% MMI doses usually resulted in substantial (Ͼ90%) pup attrition (data not shown). Animal Model 7 Briefly, Sprague-Dawley dams and litters (12-15 pups per litter) were housed in modified pediatric incubators where the oxygen levels were varied between 40% and 15% (40/15) every 24 hours for the first 14 days after birth. Rats were then allowed to recover in room air (21%) during the next 6 days until P20. The drinking water in one of two cages per incubator was supplemented with MMI between P0 and P20. Holes in the sides of the pediatric incubators were purposely not sealed so that the incubators would be somewhat leaky and minimize the unwanted buildup of carbon dioxide. Although we did not directly assess whether airflow at the holes reversed during the variable oxygen exposure, it is unlikely that this happened, because the computercontrolled (Oxycycler; Biospherix, Ltd., Redfield, NY) variable oxygen procedure constantly maintain positive pressure inside the incubator by injecting the appropriate mixture of 100% oxygen or nitrogen to maintain either a 40% or 15% oxygen environment. In addition, each incubator housed one untreated and one MMI-treated 40/15 cage and so these groups experienced similar variable oxygen exposures. From th

Refractive Development in the “ROP Rat”

Although retinopathy of prematurity (ROP) is clinically characterized by abnormal retinal vessels at the posterior pole of the eye, it is also commonly characterized by vascular abnormalities in the anterior segment, visual dysfunction which is based in retinal dysfunction, and, most commonly of all, arrested eye growth and high refractive error, particularly (and paradoxically) myopia. The oxygen-induced retinopathy rat model of ROP presents neurovascular outcomes similar to the human disease, although it is not yet known if the “ROP rat” also models the small-eyed myopia characteristic of ROP. In this study, magnetic resonance images (MRIs) of albino (Sprague-Dawley) and pigmented (Long-Evans) ROP rat eyes, and age- and strain-matched room-air-reared (RAR) controls, were examined. The positions and curvatures of the various optical media were measured and the refractive state (℞) of each eye estimated based on a previously published model. Even in adulthood (postnatal day 50), Sprague-Dawley and Long-Evans ROP rats were significantly myopic compared to strain-matched controls. The myopia in the Long-Evans ROP rats was more severe than in the Sprague-Dawley ROP rats, which also had significantly shorter axial lengths. These data reveal the ROP rat to be a novel and potentially informative approach to investigating physiological mechanisms in myopia in general and the myopia peculiar to ROP in particular

Oral rivaroxaban versus standard therapy for the treatment of symptomatic venous thromboembolism : a pooled analysis of the EINSTEIN-DVT and PE randomized studies

Background: Standard treatment for venous thromboembolism (VTE) consists of a heparin combined with vitamin K antagonists. Direct oral anticoagulants have been investigated for acute and extended treatment of symptomatic VTE; their use could avoid parenteral treatment and/or laboratory monitoring of anticoagulant effects. Methods: A prespecified pooled analysis of the EINSTEIN-DVT and EINSTEIN-PE studies compared the efficacy and safety of rivaroxaban (15 mg twice-daily for 21 days, followed by 20 mg once-daily) with standard-therapy (enoxaparin 1.0 mg/kg twice-daily and warfarin or acenocoumarol). Patients were treated for 3, 6, or 12 months and followed for suspected recurrent VTE and bleeding. The prespecified noninferiority margin was 1.75. Results: 8282 patients were enrolled. 4151 received rivaroxaban and 4131 received standard-therapy. The primary efficacy outcome occurred in 86 rivaroxaban-treated patients (2.1%) compared with 95 (2.3%) standard-therapy-treated patients (hazard ratio, 0.89; 95% confidence interval [CI], 0.66-1.19; pnoninferiority<0.001). Major bleeding was observed in 40 (1.0%) and 72 (1.7%) patients in the rivaroxaban and standard-therapy groups, respectively (hazard ratio, 0.54; 95% CI, 0.37-0.79; p=0.002). In key subgroups, including fragile patients, cancer patients, patients presenting with large clots and those with a history of recurrent VTE, the efficacy and safety of rivaroxaban was similar compared with standard-therapy. Conclusion: The single-drug approach with rivaroxaban resulted in similar efficacy to standard-therapy and was associated with a significantly lower rate of major bleeding. Efficacy and safety results were consistent among key patient subgroups

Functional Changes Within the Rod Inner Segment Ellipsoid in Wildtype Mice: An Optical Coherence Tomography and Electron Microscopy Study

Purpose: To test the hypothesis that changing energy needs alter mitochondria distribution within the rod inner segment ellipsoid. Methods: In mice with relatively smaller (C57BL/6J [B6J]) or greater (129S6/ev [S6]) retina mitochondria maximum reserve capacity, the profile shape of the rod inner segment ellipsoid zone (ISez) was measured with optical coherence tomography (OCT) under higher (dark) or lower (light) energy demand conditions. ISez profile shape was characterized using an unbiased ellipse descriptor (minor/major aspect ratio). Other bioenergy indexes evaluated include the external limiting membrane–retinal pigment epithelium (ELM-RPE) thickness and the magnitude of the signal intensity of a hyporeflective band located between the photoreceptor tips and apical RPE. The spatial distribution of rod ellipsoid mitochondria were also examined with electron microscopy. Results: In B6J mice, darkness produced a greater ISez aspect ratio, thinner ELM-RPE, and a smaller hyporeflective band intensity than in light. In S6 mice, dark and light ISez aspect ratio values were not different and were greater than in light-adapted B6J mice; dark-adapted S6 mice showed smaller ELM-RPE thinning versus light, and negligible hyporeflective band intensity in the light. In B6J mice, mitochondria number in light increased in the distal inner segment ellipsoid and decreased proximally. In S6 mice, mitochondria number in the inner segment ellipsoid were not different between light and dark, and were greater than in B6J mice. Conclusions: These data raise the possibility that rod mitochondria activity in mice can be noninvasively evaluated based on the ISez profile shape, a new OCT index that complements OCT energy biomarkers measured outside of the ISez region

Quantitative mapping of ion channel regulation by visual cycle activity in rodent photoreceptors in

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

Evidence for Diffuse Central Retinal Edema In Vivo in Diabetic Male Sprague Dawley Rats

Background: Investigations into the mechanism of diffuse retinal edema in diabetic subjects have been limited by a lack of animal models and techniques that co-localized retinal thickness and hydration in vivo. In this study we test the hypothesis that a previously reported supernormal central retinal thickness on MRI measured in experimental diabetic retinopathy in vivo represents a persistent and diffuse edema. Methodology/Principal Findings: In diabetic and age-matched control rats, and in rats experiencing dilutional hyponatremia (as a positive edema control), whole central retinal thickness, intraretinal water content and apparent diffusion coefficients (ADC, ‘water mobility’) were measured in vivo using quantitative MRI methods. Glycated hemoglobin and retinal thickness ex vivo (histology) were also measured in control and diabetic groups. In the dilutional hyponatremia model, central retinal thickness and water content were supernormal by quantitative MRI, and intraretinal water mobility profiles changed in a manner consistent with intracellular edema. Groups of diabetic (2, 3, 4, 6, and 9 mo of diabetes), and age-matched controls were then investigated with MRI and all diabetic rats showed supernormal whole central retinal thickness. In a separate study in 4 mo diabetic rats (and controls), MRI retinal thickness and water content metrics were significantly greater than normal, and ADC was subnormal in the outer retina; the increase in retinal thickness was not detected histologically on sections of fixed and dehydrated retinas from these rats