A scoring system for the evaluation of the mutated Crb1/rd8-derived retinal lesions in C57BL/6N mice

Introduction

Retinal degeneration in mice occurs in many forms, many of which can be attributed to mutations in specific genes. Some of the reported types of retinal degeneration display a similar phenotype, characterised by the presence in the fundus of white spots of different shapes and sizes^1–3. One of the causative mutations for retinal degeneration in the mouse is the spontaneous single nucleotide deletion rd8 in the Crb1 gene, situated on chromosome 1^1,4. It has been previously reported that the C57BL/6N strain, derived from the unaffected C57BL/6J strain, often presents typical retinal white spots (flecks) caused by the Crb1/rd8 mutation⁴. These have been described as dysplastic lesions affecting the retinal region between the inner and the outer nuclear layer and are mainly localised in the inferior part of the retina^5,6. The observed phenotype is considered a possible confounding factor that could mask a phenotype with a similar appearance but a different causative gene mutation (Figure 1). This is of particular importance considering that the C57BL/6N line is a widely used commercial line and is the background strain used for the generation of gene-targeted mice in several mouse mutagenesis/phenotyping programmes, including the International Knockout Mouse Consortium (IKMC) and the International Mouse Phenotyping Consortium (IMPC).

Figure 1. Picture showing the retinal fundus with an example of the flecks treated in this study.

The flecks appear in the superior hemicycle of the fundus because the image is inverted by the ophthalmoscope.

Over the last 5 years of phenotyping mice through the IMPC pipeline at MRC Harwell, we have observed the presence of fundus flecks in both the knockout lines and in the C57BL/6NTac wild type mice. The number of affected individuals for each knockout line generated has been variable, as has the number of flecks present in each individual. As a result of such variability, the probability that the flecking in the mutant line is a phenotype attributable to the gene mutations rather than the background strain effect becomes questionable. To correctly interpret similar phenotypes in the knockout lines and exclude the contribution of Crb1/rd8 -related flecks, we have created a scoring system to allow us to fully categorise the lesions present in the C57BL/6NTac mice in a systematic manner in order to provide a comprehensive background strain reference. The flecks scoring system takes into account the position of the flecks in the superior and inferior retinal hemicycle, as the retina is not uniformly affected by the phenotype⁵. As an innovative approach, we also scored the number of flecks in each hemicycle as a measure of the phenotypic penetrance. In addition, in order to determine any sexual dimorphism, we applied our scoring system to both males and females.

Methods

Fundus imaging and flecks scoring

For the fundus examination an Omega 180 ophthalmoscope (Heine Ltd, USA) and a Superfield NC lens (Volk Optical Inc., USA) were used. Each eye pupil was dilated using a drop of 1% w/v Minims Tropicamide (Bausch & Lomb Inc., USA) and the observation was performed after 2 minutes. Images of the fundus were acquired by the use of a topical endoscopy fundus imaging (TEFI) camera.

The examinations were conducted by trained technicians on both eyes and the flecks on individual eyes were evaluated according to an in-house scoring system (Figure 2), taking into account their position in the fundus with respect to the optic nerve head (superior or inferior) and their number (with respect to the retinal surface covered by the flecks) as a measure of the severity grade. Therefore, the combination of both the position and the severity grade formed a scoring category for each eye.

Figure 2. In-house flecks scoring system.

The retinal fundus has been divided into two hemicycles: inferior and superior. In each one of the hemicycles the percentage of the surface that is covered by flecks represents the severity grade within a range of 25% for each level. The combination of the position, superior (S) or inferior (I), and the severity grade (from 0 to 4) represents the flecks score.

Results

As shown in Table 1, the total percentage of affected males was higher than that of affected females (14.4% of males and 5% of females). Further categorising the flecks according to our scoring system, we observed that the males were still the most affected in the score classes ranging from I1 to I3 (Figure 3), with a symmetrical distribution of the frequencies centred in the I2 class (25–50% of inferior retina surface) in both sexes (8.2% of males and 3.0% of females). In the sample, there were no males affected in the class I4 (75 to 100% of inferior retina surface), whilst only one female (0.5% of the total) presented that severity grade. We mentioned above that the presence of this kind of flecks has already been associated with the inferior hemicycle of the fundus by other authors, a fact supported by our data that show just one male in the S3 (50–75% of superior retina surface) class of flecking.

Figure 3. Frequency distribution of the flecks in male and female C57BL/6NTac mice according to our score system.

The chart shows the percentage distribution of the flecks in the retinal fundus of males (black columns) and females (white columns) C57BL/6NTac mice. The horizontal categories represent the flecks class as previously explained in Figure 2.

Conclusions

With this study we make available both the observational data on the retinal flecks in C57BL/6NTac mice determined by the use of our scoring system, and the scoring system itself. Our findings, using a large population of wild type mice, provide a reference baseline that could significantly contribute to the further evaluation of Crb1 mutations-based eye morphology phenotypes. In addition to the flecks distribution data, the scoring system used represents a reliable quantitative method to evaluate the degree of flecking of an affected mouse retina and to make the comparison process between two or more strains (or treatment groups) more accurate and manageable.

Data availability

Dataset 1: Flecks scores raw data. A spreadsheet with the raw data related to the manual scoring of flecks made by the trained technicians, according to our in-house scoring system. The spreadsheet contains one column related to the animal ID and one column with the score class for males and females. The score class represents the combination of the position, superior (S) or inferior (I), and the severity grade (from 0 to 4) as described in Figure 2.

DOI, 10.5256/f1000research.11252.d156405⁷