Micah A. Chrenek

Exaggerated eye growth in IRBP-deficient mice in early development

PURPOSE Because interphotoreceptor retinoid-binding protein (IRBP) is expressed before being needed in its presumptive role in the visual cycle, we tested whether it controls eye growth during development. METHODS The eyes of congenic IRBP knockout (KO) and C57BL/6J wild-type (WT) mice ranging in age from postnatal day (P)2 to P440 were compared by histology, laser micrometry, cycloplegic photorefractions, and partial coherence interferometry. RESULTS The size and weight of IRBP KO mouse eyes were greater than those of the WT mouse, even before eye-opening. Excessive ocular enlargement started between P7 and P10, with KO retinal arc lengths becoming greater compared with WT from P10 through P30 (18%; P < 0.01). The outer nuclear layer (ONL) of KO retinas became 20% thinner between P12 to P25, and progressed to 38% thinner at P30. At P30, there were 30% fewer cones per vertical section in KO than in WT retinas. Bromodeoxyuridine (BrdU) labeling indicated the same number of retinal cells were born in KO and WT mice. A spike in apoptosis was observed in KO outer nuclear layer at P25. These changes in size were accompanied by a large decrease in hyperopic refractive error, which reached -4.56 ± 0.70 diopters (D) versus +9.98 ± 0.993 D (mean ± SD) in WT, by postnatal day 60 (P60). CONCLUSIONS; In addition to its role in the visual cycle, IRBP is needed for normal eye development. How IRBP mediates ocular development is unknown.

Aerobic Exercise Protects Retinal Function and Structure from Light-Induced Retinal Degeneration

Aerobic exercise is a common intervention for rehabilitation of motor, and more recently, cognitive function (Intlekofer and Cotman, 2013; Wood et al., 2012). While the underlying mechanisms are complex, BDNF may mediate much of the beneficial effects of exercise to these neurons (Ploughman et al., 2007; Griffin et al., 2011; Real et al., 2013). We studied the effects of aerobic exercise on retinal neurons undergoing degeneration. We exercised wild-type BALB/c mice on a treadmill (10 m/min for 1 h) for 5 d/week or placed control mice on static treadmills. After 2 weeks of exercise, mice were exposed to either toxic bright light (10,000 lux) for 4 h to induce photoreceptor degeneration or maintenance dim light (25 lux). Bright light caused 75% loss of both retinal function and photoreceptor numbers. However, exercised mice exposed to bright light had 2 times greater retinal function and photoreceptor nuclei than inactive mice exposed to bright light. In addition, exercise increased retinal BDNF protein levels by 20% compared with inactive mice. Systemic injections of a BDNF tropomyosin-receptor-kinase (TrkB) receptor antagonist reduced retinal function and photoreceptor nuclei counts in exercised mice to inactive levels, effectively blocking the protective effects seen with aerobic exercise. The data suggest that aerobic exercise is neuroprotective for retinal degeneration and that this effect is mediated by BDNF signaling.

In Vivo Imaging of Retinal Oxidative Stress Using a Reactive Oxygen Species-Activated Fluorescent Probe.

PURPOSE In vivo methods for detecting oxidative stress in the eye would improve screening and monitoring of the leading causes of blindness: diabetic retinopathy, glaucoma, and age-related macular degeneration. METHODS To develop an in vivo biomarker for oxidative stress in the eye, we tested the efficacy of a reactive oxygen species (ROS)-activated, near-infrared hydrocyanine-800CW (H-800CW) fluorescent probe in light-induced retinal degeneration (LIRD) mouse models. After intravitreal delivery in LIRD rats, fluorescent microscopy was used to confirm that the oxidized H-800CW appeared in the same retinal layers as an established ROS marker (dichlorofluorescein). RESULTS Dose-response curves of increasing concentrations of intravenously injected H-800CW demonstrated linear increases in both intensity and total area of fundus hyperfluorescence in LIRD mice, as detected by scanning laser ophthalmoscopy. Fundus hyperfluorescence also correlated with the duration of light damage and functional deficits in vision after LIRD. In LIRD rats with intravitreal injections of H-800CW, fluorescent labeling was localized to photoreceptor inner segments, similar to dichlorofluorescein. CONCLUSIONS Hydrocyanine-800CW detects retinal ROS in vivo and shows potential as a novel biomarker for ROS levels in ophthalmic diseases.

Analysis of the RPE sheet in the rd10 retinal degeneration model

The normal RPE sheet in the C57BL/6J mouse is subclassified into two major tiling patterns: a regular generally hexagonal array covering most of the surface and a “soft network” near the ciliary body made of irregularly shaped cells. Physics models predict these two patterns based on contractility and elasticity of the RPE cell, and strength of cellular adhesion between cells. We hypothesized and identified major changes in RPE regular hexagonal tiling pattern in rd10 compared to C57BL/6J mice. In rd10 mice, RPE sheet damage was extensive but occurred later than expected, after most retinal degeneration was complete. RPE sheet changes occur in zones with a bullseye pattern. In the posterior zone, around the optic nerve, RPE cells take on larger irregular and varied shapes to maintain an intact monolayer. In mid periphery, RPE cells have a compressed or convoluted morphology that progress into ingrown layers of RPE under the retina. Cells in the periphery maintain their shape and size until the late stages of the RPE reorganization. The number of neighboring cells varies widely depending on zone and progression. RPE morphology continues to deteriorate after the photoreceptors have degenerated. The RPE cells are bystanders to photoreceptor degeneration in the rd10 model, and the collateral damage to the RPE results in changes in morphology as early as 30 days old. Quantitative measures of the tiling patterns and histopathology detected here were scripted in a pipeline written in Perl and Cell Profiler (an open source MatLab plugin) and are directly applicable to RPE sheet images from noninvasive fundus autofluorescence (FAF), adaptive optics confocal scanning laser ophthalmoscope (AO-cSLO), and spectral domain optical coherence tomography (SD-OCT) of patients with early stage AMD or RP.

Subretinal Delivery and Electroporation in Pigmented and Nonpigmented Adult Mouse Eyes

Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, Müller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumulating to high concentrations because there is no dilution due to transport processes or diffusion. Dilution in the interphotoreceptor space (IPS) is minimal because the IPS volume is only 10-20 μl in the human eye and less than 1 μl in the mouse eye. For gene delivery purposes, we wished to transfect the cells adjacent to the IPS in adult mouse eyes. Others transfect these cells in neonatal rats to study the development of the retina. In both neonates and adults, electroporation is found to be effective. Here we describe the optimization of electroporation conditions for RPE cells in the adult mouse eye with naked plasmids. However, both techniques, subretinal injection and electroporation, present some technical challenges that require skill on the part of the surgeon to prevent untoward damage to the eye. Here we describe methods that we have used for the past 10 years (Johnson et al. Mol Vis 14: 2211-2226, 2008).

Non-contact measurement of linear external dimensions of the mouse eye

Biometric analyses of quantitative traits in eyes of mice can reveal abnormalities related to refractive or ocular development. Due to the small size of the mouse eye, highly accurate and precise measurements are needed to detect meaningful differences. We sought a non-contact measuring technique to obtain highly accurate and precise linear dimensions of the mouse eye. Laser micrometry was validated with gauge block standards. Simple procedures to measure eye dimensions on three axes were devised. Mouse eyes from C57BL/6J and rd10 on a C57BL/6J background were dissected and extraocular muscle and fat removed. External eye dimensions of axial length (anterior-posterior (A-P) axis) and equatorial diameter (superior-inferior (S-I) and nasal-temporal (N-T) axes) were obtained with a laser micrometer. Several approaches to prevent or ameliorate evaporation due to room air were employed. The resolution of the laser micrometer was less than 0.77 microm, and it provided accurate and precise non-contact measurements of eye dimensions on three axes. External dimensions of the eye strongly correlated with eye weight. The N-T and S-I dimensions of the eye correlated with each other most closely from among the 28 pair-wise combinations of the several parameters that were collected. The equatorial axis measurements correlated well from the right and left eye of each mouse. The A-P measurements did not correlate or correlated poorly in each pair of eyes. The instrument is well suited for the measurement of enucleated eyes and other structures from most commonly used species in experimental vision research and ophthalmology.

The Rpe65 rd12 Allele Exerts a Semidominant Negative Effect on Vision in Mice

PURPOSE The rd12 mouse was reported as a recessively inherited Rpe65 mutation. We asked if the rd12 mutation resides in Rpe65 and how the mutation manifests itself. METHODS A complementation test was performed by mating Rpe65(KO) (KO/KO) and rd12 mice together to determine if the rd12 mutation is in the Rpe65 gene. Visual function of wild-type (+/+), KO/+, rd12/+, KO/KO, rd12/rd12, and KO/rd12 mice was measured by optokinetic tracking (OKT) and ERG. Morphology was assessed by retinal cross section. qRT-PCR quantified Rpe65 mRNA levels. Immunoblotting measured the size and level of RPE65 protein. Rpe65 mRNA localization was visualized with RNA fluorescence in situ hybridization (FISH). Fractions of Rpe65 mRNA-bound proteins were separated by linear sucrose gradient fractionation. RESULTS The KO and rd12 alleles did not complement. The rd12 allele induced a negative semidominant effect on visual function; OKT responses became undetectable 120 days earlier in rd12/rd12 mice compared with KO/KO mice. rd12/+ mice lost approximately 21% visual acuity by P210. rd12/rd12 mice had fewer cone photoreceptor nuclei than KO/KO mice at P60. rd12/rd12 mice expressed 71% +/+ levels of Rpe65 mRNA, but protein was undetectable. Mutant mRNA was appropriately spliced, exported to the cytoplasm, trafficked, and contained no other coding mutation aside from the known nonsense mutation. Mutant mRNA was enriched on ribosome-free messenger ribonucleoproteins (mRNPs), whereas wild-type mRNA was enriched on actively translating polyribosomes. CONCLUSIONS The rd12 lesion is in Rpe65. The rd12 mutant phenotype inherits in a semidominant manner. The effects of the mutant mRNA on visual function may result from inefficient binding to ribosomes for translation.

Functional Principal Component Analysis Reveals Discriminating Categories of Retinal Pigment Epithelial Morphology in Mice

PURPOSE To determine whether multivariate, functional principal component analysis of the size and shape of retinal pigment epithelial (RPE) cell morphology allows discrimination of mouse RPE genotypes and age. METHODS Flatmounts of RPE sheets obtained from C57BL/6J (n = 50) and rd10 (n = 61) mice at postnatal days 30 to 720 were stained for zonula occludens-1 (ZO-1) and imaged with confocal microscopy. A total of 111 flatmounts were prepared. Twenty-one morphometric measurements were made on tiled, composite images of complete flatmounts, including cell location, area, and eccentricity, using automated image analysis software for quantitatively measuring cell phenotypes. RESULTS In young (≤61-day-old) C57BL/6J mice, the RPE morphology resembled a regular hexagonal array of cells of uniform size throughout the retina, except near the ciliary body, where the shapes of RPE resembled a soft network. Old (≥180-day-old) C57BL/6J eyes had a subpopulation of large cells. A clear disruption of the regular cell size and shape appeared in rd10 mutants. Aspect ratio and cell area gave rise to principal components that predictively classified mouse age and genotype. CONCLUSIONS Quantitative differences in the RPE sheet morphology allowed discrimination of rd10 from C57BL/6J strains despite the confounding effect of aging. This has implications for RPE sheet morphology as a potential early biomarker for diagnosis of eye disease and prognosis of the eye at early stages when disease is subtle. We conclude that an RPE cells area and aspect ratio are very early quantitative indicators that predict progression to advanced RPE disease as manifested in rd10.

Complementation Test of Rpe65 Knockout and Tvrm148

PURPOSE A mouse mutation, tvrm148, was previously reported as resulting in retinal degeneration. Tvrm148 and Rpe65 map between markers D3Mit147 and D3Mit19 on a genetic map, but the physical map places RPE65 outside the markers. We asked if Rpe65 or perhaps another nearby gene is mutated and if the mutant reduced 11-cis-retinal levels. We studied the impact of the tvrm148 mutation on visual function, morphology, and retinoid levels. METHODS Normal phase HPLC was used to measure retinoid levels. Rpe65(+/+), tvrm148/+ (T(+/-)), tvrm148/tvrm148 (T(-/-)), RPE65(KO/KO) (Rpe65(-/-)), and Rpe65(T/-) mice visual function was measured by optokinetic tracking (OKT) and electroretinography (ERG). Morphology was assessed by light microscopy and transmission electron microscopy (TEM). qRT-PCR was used to measure Rpe65 mRNA levels. Immunoblotting measured the size and amount of RPE65 protein. RESULTS The knockout and tvrm148 alleles did not complement. No 11-cis-retinal was detected in T(-/-) or Rpe65(-/-) mice. Visual acuity in Rpe65(+/+) and T(+/-) mouse was -0.382 c/d, but 0.037 c/d in T(-/-) mice at postnatal day 210 (P210). ERG response in T(-/-) mice was undetectable except at bright flash intensities. Outer nuclear layer (ONL) thickness in T(-/-) mice was -70% of Rpe65(+/+) by P210. Rpe65 mRNA levels in T(-/-) mice were unchanged, yet 14.5% of Rpe65(+/+) protein levels was detected. Protein size was unchanged. CONCLUSIONS A complementation test revealed the RPE65 knockout and tvrm148 alleles do not complement, proving that the tvrm148 mutation is in Rpe65. Behavioral, physiological, molecular, biochemical, and histological approaches indicate that tvrm148 is a null allele of Rpe65.

DNA Delivery in Adult Mouse Eyes: An Update with Corneal Outcomes

Ocular injection (intravitreal, subretinal, or into the anterior space) is an efficient approach to deliver many classes of drugs, cells, and other treatments to various cell types of the eye. In particular, subretinal injection is efficient since delivered agents accumulate as there is no dilution due to transport processes or diffusion and the volume of the interphotoreceptor space (IPS) is minimal (10-20 μl in the human eye, less than 1 μl in the mouse eye). We previously reported methods using subretinal injection and electroporation to deliver DNA to photoreceptor and retinal pigment epithelium cells in retinas of live mice (Johnson et al., 14:2211-2226; Nickerson et al. 884:53-69, 2012; Andrieu-Soler et al. 13:692-706, 2007). Here we detail further optimization of that approach and additionally report its use in delivering DNA expression plasmids to the corneal endothelium.