M. D. Fischer

Vision tests in the mouse: Functional phenotyping with electroretinography.

Electroretinography (ERG) is an established diagnostic technique in clinical ophthalmology and provides objective information about retinal function. This technique is also applied in basic research, where animal models of hereditary retinopathies have significantly contributed to our understanding of the composition of ERG responses in general and how retinal degenerative pathologies alter retinal function specifically. Indeed, electrophysiologic assessment of transgenic mice, which are genetically engineered to mimic human mutations that lead to retinal diseases, can be well compared with clinical data. Furthermore, limitations on examinations (e.g. length of measurement, range of light intensity) are much less of a concern when assessing mice compared to human patients. In order to measure and analyze retinal responses properly, several important aspects have to be considered. This paper focuses on these aspects, and shows exemplary ERG data which were obtained from normal wild-type mice and from transgenic mice with specific functional properties, namely Rho-/- (rod opsin knockout, cone function only), and Cnga3-/- (cone CNG channel deficient, rod function only) to illustrate rod and cone system contributions to ERG responses.

Gene expression profiling of the retina after transcorneal electrical stimulation in wild-type Brown Norway rats.

PURPOSE Transcorneal electrical stimulation (TES) has been beneficial in several neurodegenerative ocular diseases, but the exact mechanisms remain to be elucidated. This study was conducted to investigate the effects of TES on the retinas of wild-type Brown Norway (BN) rats by gene expression profiling and to assess its effects on retinal function and morphology. METHODS TES was applied to BN wild-type rat retinas in vivo for 1 hour (1-ms biphasic pulses at 20 Hz; 200 μA). RNA was isolated and processed for microarray-based profiling 4 hours after TES; differentially expressed genes from TES compared with those from sham-treated animals were validated by quantitative real-time polymerase chain reaction. Furthermore, the effect of TES was assessed at the structural and functional levels using electroretinography, confocal scanning laser ophthalmoscopy, optical coherence tomography, and immunohistochemistry. RESULTS Transcriptome changes associated with TES versus sham-stimulated BN wild-type retina were identified. Four hundred ninety genes were differentially expressed in TES and included potentially neuroprotective genes such as Bax or members of the tumor necrosis factor family (Tnfrsf11b, Tnrsf12a, Tnfsf13b, Tnfsf13). ERG recordings showed physiological retinal function after TES, and structural in vivo and ex vivo studies revealed intact retinal anatomy. CONCLUSIONS These results demonstrate that TES applied to the retina of the wild-type BN rats induces distinct transcriptome level changes and may help in the understanding of the mechanisms underlying TES. In addition, TES treatment indicates no negative effect on structure and function of the wild-type BN retina up to 35 hours after application.

Induction of STAT3-related genes in fast degenerating cone photoreceptors of cpfl1 mice

Cone dystrophies are genetic diseases characterized by loss of cone photoreceptor function and severe impairment of daylight vision. Loss of function is accompanied by a progressive degeneration of cones limiting potential therapeutic interventions. In this study we combined microarray-based gene-expression analysis with electroretinography and immunohistochemistry to characterize the pathological processes in the cone photoreceptor function loss 1 (cpfl1) mouse model. The cpfl1-mouse is a naturally arising mouse mutant with a loss-of-function mutation in the cone-specific Pde6c gene. Cpfl1-mice displayed normal rod-specific light responses while cone-specific responses were strongly diminished. Despite the lack of a general retinal degeneration, the cone-specific functional defect resulted in a marked activation of GFAP, a hallmark of Müller-cell gliosis. Microarray-based network-analysis confirmed activation of Müller-glia-specific transcripts. Unexpectedly, we found up-regulation of the cytokine LIF and the anti-apoptotic transcription factor STAT3 in cpfl1 cone photoreceptors. We postulate that STAT3-related pathways are induced in cpfl1 cone photoreceptors to counteract degeneration.

Effects on colour discrimination during long term exposure to high altitudes on Mt. Everest

Aim To investigate changes in colour discrimination as a result of chronic hypoxic exposure induced by extreme altitudes (above 8000 m) during an expedition to Mt Everest. Methods Colour discrimination thresholds for tritan, protan and deutan axes were measured extensively in two male participants (four eyes) during an expedition to Mt Everest, using a quantitative, computer controlled psychophysical colour vision test (modified version of the Cambridge Colour Test). The tests were carried out over a period of 54 days at altitudes of 1300 m, 3450 m, 4410 m, 5060 m, 5300 m, 6450 m, 7200 m and 8000 m. Results Colour vision tests 1 week before and 6 months after the expedition indicated normal colour discrimination in both participants. With increasing altitude, colour discrimination thresholds were found to rise, predominantly for the tritan (blue) axes in both observers. Deutan (green) thresholds were minimally elevated at high altitude, whereas protan (red) was altered in one observer. Tritan colour discrimination thresholds decreased as a function of time spent at a given altitude and normalised upon return to low altitude. Conclusions Chronic hypoxia induced by high altitude exposure transiently affects colour discrimination, in particular tritan axis discrimination. Decreased tritan discrimination is partly reversible upon physiological adaptation to high altitude and completely normalised upon return to low altitude.

In vivo analysis of cone survival in mice.

PURPOSE To identify individual cone photoreceptors in a transgenic mouse line in vivo based on selective expression of green fluorescent protein (GFP) using cSLO (confocal scanning laser ophthalmoscopy) and to use this approach to monitor cone cell fate in mouse models of retinal degeneration. METHODS Transgenic mice expressing GFP under the control of a red-green opsin promoter (RG-GFP mice) were analyzed in vivo with respect to GFP expression in cone cells using cSLO and functional integrity using electroretinography (ERG). Histology was performed to correlate the pattern of GFP expression with light microscopic data. Longitudinal monitoring of cone survival was evaluated in crossbreds of RG-GFP mice with cpfl1 and Rpe65(-/-) mutant mice, respectively. RESULTS The authors found that RG-GFP transgenic mice had a stable GFP expression that did not interfere with retinal function up to at least 3 months of age. Thus, a longitudinal analysis of cone degeneration in individual RG cpfl1 and RG Rpe65(-/-) cross-bred mice in vivo was successfully performed and demonstrated distinct time frames of cone survival in the particular mouse model. CONCLUSIONS Monitoring GFP expression in cone photoreceptor cells, such as in the RG-GFP mouse, is a promising in vivo approach for the analysis of cone survival in mice.

In vivo assessment of retinal vascular wall dimensions.

PURPOSE Retinal blood vessel diameter and arteriovenous ratio (AVR) are commonly used diagnostic parameters. Because vascular walls are typically not visible in funduscopy, clinical AVR estimation is based on the lumen rather than the entire vessel diameter. Here the authors used a transgenic mouse model to quantify AVR in vivo based on total vessel dimensions (wall and lumen). METHODS Confocal scanning laser ophthalmoscopy (cSLO) and indocyanine green angiography of the retinal vasculature were performed in wild-type and transgenic mice expressing green fluorescent protein (GFP) under the transcriptional control of the smooth muscle type α-actin (αSMA) promoter. Spectral-domain-OCT and ERG were performed to control for integrity of retinal structure and function in vivo and histology to demonstrate the location of GFP expression. RESULTS Native cSLO imaging and angiography yielded only inner vessel diameters similar to those observed through clinical funduscopy. In αSMA-GFP mice, autofluorescence imaging of the GFP-marked vascular walls also allowed the determination of outer vessel diameters. The mean AVR based on either inner diameter (AVR(id) = 0.72 ± 0.08) or outer diameter (AVR(od) = 0.97 ± 0.09) measurements were significantly different (P < 0.01). CONCLUSIONS Transgenic αSMA-GFP expression in murine vessel wall components allowed quantification of retinal vessel outer diameters in vivo. Although arterioles and venules differ in lumen and vessel wall width, they share a common outer diameter, leading to an AVR(od) close to unity. Because vessel walls are primary targets in common hypertensive and metabolic diseases, αSMA-GFP transgenic mice may prove valuable in the detailed assessment of such disorders in vivo.

Impact of Acute Exposure to High Altitude on Anterior Chamber Geometry

PURPOSE This study aimed to quantify the impact of exposure to high altitude on individual layers of the cornea in regard to central corneal thickness (CCT) and the geometry of the anterior chamber angle (ACA). This work is related to the Tübingen High Altitude Ophthalmology study. METHODS Anterior segment spectral domain optical coherence tomography was used to quantify changes in individual corneal layers and to study ACA and angle opening distance (AOD). Peripheral oxygen saturation, heart rate, and scores of acute mountain sickness (AMS) were assessed in 14 healthy subjects at baseline (341 m) and altitude (4559 m) for respective correlations. RESULTS Longitudinal analysis revealed a significant (P < 0.05) increase of CCT during altitude exposure (CCT(baseline) = 539.27 ± 32.30 μm; CCT(day1) = 558.87 ± 29.39 μm; CCT(day3) = 567.17 ± 33.40 μm; mean ± SD) due to stromal edema. This change was completely reversible upon descent. Geometric measures of aqueous outflow structures remained consistent with no significant changes in AOD or ACA. Incidence of AMS on day 1 was 64% followed by a decrease in AMS scores over time spent at high altitude; while AMS correlated significantly with stromal edema formation just after arrival (r = 0.71; P = 0.01), no correlation was found on day 3 (r = 0.05; P = 0.87); no correlations were found for vital parameters. CONCLUSIONS Significant stromal edema was found during exposure to high altitude in healthy subjects. This seems to occur due to decreased atmospheric pressure under hypoxia but independent of systemic acclimatization. Other measures of anterior chamber geometry remained stable during the challenge to hypoxic conditions at high altitude.

In vivo assessment of rodent retinal structure using spectral domain optical coherence tomography.

To determine the potential of a commercially available optical coherence tomography (OCT) device (Spectralis™ HRA + OCT, Heidelberg Engineering) for small animal retinal imaging, we achieved to adapt this third generation OCT system to obtain and quantify high-resolution morphological sections of rodent retina in models with acquired and inherited retinal degenerations. Genetically induced (rd1, rho−/−, RPE65) and acquired retinal degeneration (light damage) was similarly clear as in histology and could be followed in a timeline fashion. We were able to detect and analyze a wide range of retinal pathology in a variety of established animal models used in vision research. As this technique allows longitudinal study designs, it will facilitate characterization of disease dynamics while reducing the numbers of study animals needed. Use of identical outcome measures and even the same diagnostic device in animal and clinical studies bears the potential to streamline translational approaches, e.g., in the assessment of putative therapeutic interventions.

Exposure to High Altitude Alters Tear Film Osmolarity and Breakup Time

AIMS High altitude provides environmental conditions with dry air and cold temperatures that may facilitate the development of dry eye symptoms. This study investigated, for the first time, the quality of the tear film during high altitude exposure in healthy subjects. This study is related to the Tübingen High Altitude Ophthalmology (THAO) study. METHODS Tear film osmolarity (TFO), tear film breakup time (TBUT), and Schirmer I and II were used to assess tear film properties under standardized conditions in 14 healthy subjects on day 1, 2, and 4 during exposure to high altitude at the Capanna Margherita (CM; 4559 m, Italy) compared to baseline measurements in Tübingen (341 m, Germany) before (BL1) and after (BL2) exposure. RESULTS Upon arrival at CM, a significant increase in intra-individual TFO (309.1 ± 19.3, 332.2 ± 24.1, 335.5 ± 28.7, 329.7 ± 19.0, and 308.5 ± 15.3 mOsms/L at BL1, day 1, 2, 4, and BL2, respectively) and a significant decrease of TBUT (11.2 ± 5.2, 7.3 ± 5.2, 7.2 ± 11.6, 4.5 ± 2.3, and 8.7 ± 4.6 seconds at BL1, day 1, 2, 4, and BL2, respectively) were found. Schirmer test changes at high altitude remained statistically nonsignificant compared to baseline. Comparisons of parameters between BL1 and BL2 showed no statistically significant differences and recordings of right and left eyes for TBUT and Schirmer did not differ significantly on any day measured. CONCLUSION High altitude exposure leads to an altered tear film resulting in an increased TFO and a reduced TBUT. These changes were fully reversible after descent. This is of clinical importance to populations living in high altitude areas and to trekkers and mountaineers exposed to high altitude due to their ever-increasing number.

In vivo imaging reveals novel aspects of retinal disease progression in Rs1h(-/Y) mice but no therapeutic effect of carbonic anhydrase inhibition

PURPOSE X-linked juvenile retinoschisis (XLRS) is the most common juvenile maculopathy in men and is caused by mutations in the gene encoding retinoschisin (RS1). Evidence in the literature on the therapeutic effect of carboanhydrase inhibitors (CAIs) to treat schisis formation in the retina has remained equivocal. Here, we evaluate the effect of the CAI dorzolamide on the structural and functional disease progression in the mouse model for XLRS (Rs1h(-/y)). METHODS Rs1h (-/y) mice were treated unilaterally with dorzolamide eye drops (Trusopt(®) 20 mg/mL) every 12 h for 2 weeks starting on postnatal day 14 (n = 27). Changes of retinal structure were monitored by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography 12 h, 14 days, 4 weeks, 2 months, and 6 months after completion of the treatment. RESULTS Schisis formation (peak at 3 months) preceded photoreceptor degeneration and hyper-fluorescence (peak at 7 months). Structural pathology was most severe in the superior hemi-retina with previously unreported hyper-fluorescent lesions. Quantitative analysis showed no significant differences regarding the inner or outer retinal thickness of the treated vs. untreated eyes 12 h after the completion of treatment (IRT(12 h) = -1.29 ± 1.89 μm; ORT(12 h) = 0.61 ± 2.08 μm; mean ± 95%CI) or at any later time point. CONCLUSION Time line analysis after short-term treatment with CAI failed to show short-, intermediate-, or long-term evidence of structural improvement in Rs1h(-/y) mice. Schisis formation in the inner retina peaked at the age of 3 months and was followed by photoreceptor degeneration predominantly in the superior hemi-retina. Previously unreported hyper-fluorescent lesions co-register with structural retinal pathologies.