Abstract
Purpose.:
A mouse mutant identified during a recessive N-ethyl-N-nitrosourea (ENU) mutagenesis screen exhibited ocular hemorrhaging resulting in a blood-filled orbit, and hence was named “redeye.” We aimed to identify the causal mutation in redeye, and evaluate it as a model for diabetic retinopathy (DR).
Methods.:
The causative gene mutation in redeye was identified by haplotype mapping followed by exome sequencing. Glucose tolerance tests, detailed histologic and immunofluorescence analyses, and vascular permeability assays were performed to determine the affect of redeye on glucose metabolism, pericyte recruitment, and the development of the retinal vasculature and blood–retinal barrier (BRB).
Results.:
A mutation was identified in the Pdgfrb gene at position +2 of intron 6. We show that this change causes partial loss of normal splicing resulting in a frameshift and premature termination, and, therefore, a substantial reduction in normal Pdgfrb transcript. The animals exhibit defective pericyte recruitment restricted to the central nervous system (CNS) causing basement membrane and vascular patterning defects, impaired vascular permeability, and aberrant BRB development, resulting in vascular leakage and retinal ganglion cell apoptosis. Despite exhibiting classic features of diabetic retinopathy, redeye glucose tolerance is normal.
Conclusions.:
The Pdgfrbredeye/redeye mice exhibit all of the features of nonproliferative DR, including retinal neurodegeneration. In addition, the perinatal onset of the CNS-specific vascular phenotype negates the need to age animals or manage diabetic complications in other organs. Therefore, they are a more useful model for diseases involving pericyte deficiencies, such as DR, than those currently being used.
Initial mapping by SNP array analysis (University of Edinburgh, Wellcome Trust Clinical Research Facility) identified an 8 Mb region of interest. All exons and splice junctions in this region were captured on a custom oligonucleotide-array, amplified, and sequenced, identifying a mutation in the Pdgfrb gene, which was confirmed by Sanger sequencing. To characterize the mutation we extracted RNA from enucleated eyes and performed quantitative real-time PCR using the Mouse Universal ProbeLibrary Set (Roche, West Sussex, UK) with probes for Pdgfrβ and TBP on a Roche Lightcycler LC480 (Roche). The Lightcycler LC480 software (Lightcycler LC480[C] 1.5.0 SP4 [1.5.0.39]; Roche) was used to normalize results to TBP controls and calculate relative expression values.
Mice were killed at the appropriate age, and whole mount retinae were prepared and stained as described.
36 We stained mutant and control retinae in the same well to control for changes in staining efficiency, and retinae were distinguished by different numbers of radial incisions.
Embryonic hindbrains were dissected as described previously,
37 and all other organs were dissected from animals of the appropriate age and fixed in 4% paraformaldehyde (PFA) overnight at 4°C. Postnatal day 5 (P5) brains were embedded in 4% agarose in PBS and 200 μm sections were cut on a Vibratome Series 1000 (Technical Products International, Inc., St. Louis, MO). Immunofluorescence for hindbrains and brain sections was performed as for retinae.
We embedded the kidneys in paraffin for sectioning on a Leica RM 2235 microtome (Leica Microsystems, Inc., Buffalo Grove, IL). Placentae and hearts were cryopreserved in 30% sucrose and embedded in OCT compound (VWR International, Leicestershire, UK) for sectioning on a Leica CM30505 cryostat (Leica Microsystems, Inc.). Citrate buffer antigen retrieval was used for paraffin sections. We blocked all the sections in 10% heat inactivated donkey serum in PBS with Tween-20 (PBST) for an hour, and performed all antibody incubations at room temperature for an hour in block (antibody details are given in
Supplementary Table S1). Hematoxylin and eosin staining was performed according to standard procedures.
Tissue from at least five mice of each genotype from at least three different litters was used for all analysis. All tissues were mounted in Vectashield (Vector Laboratories Ltd., Peterborough, UK), imaged by confocal microscopy (Nikon A1R; Nikon Instruments, Inc., Melville, NY), and maximum intensity projections of z-stacks were created using NisElements AR Version 4.0 software (Nikon UK, Kingston Upon Thames, UK). All images are representative of at least three animals.
The MetaMorph Angiogenesis Tube Formation application (Molecular Devices, Berkshire, UK) was used for quantification. Confocal images were used to determine the total area covered by vessels and pericytes to calculate percentage pericyte coverage of vessels and the total number of branchpoints/mm2. We imaged three areas of each retina; three different regions of the central retina each encompassing an artery and vein, and two images each of peripheral arteries and peripheral veins; a total of five images/retina. For pericyte quantification of the cerebral cortex capillaries, sections from the frontal, parietal, and occipital regions of the brain were used, and four images were taken from the cerebral cortex of each section. Threshold values were kept the same for analysis of samples of the same stage.
Eight-week-old animals were fasted for five hours before intraperitoneal injection of 2 g glucose (Sigma-Aldrich Company Ltd.) per kilogram of body weight. Blood was drawn from the tail vein at 0, 30, 60, 90, and 120 minutes after injection, and glucose values were monitored using an automatic glucometer (Accuchek; Roche). Numbers of mice tested were females (4 wild type [WT], 6 homozygote animals [HOM]) and males (6 WT, 5 HOM).
The authors thank David Black and animal staff for animal husbandry, Matthew Pearson and Paul Perry for imaging assistance, Allyson Ross for histology assistance, and Alexi Balmuth at the Genepool for exome sequencing. Clare Isacke, BBCRC, London, United Kingdom, provided the kind gift of the anti-Endosialin antibody.
Supported by Eumodic; the European Mouse Disease Clinic, an EU Integrated Research Programme, and MRC Core Support to the MRC Human Genetics Unit and the MRC Mammalian Genetics Unit.
Disclosure: S. Jadeja, None; R.L. Mort, None; M. Keighren, None; A.W. Hart, None; R. Joynson, None; S. Wells, None; P.K. Potter, None; I.J. Jackson, None