Abstract
Purpose :
Mutation in the podosomal adaptor protein SH3PXD2B (nee) causes anterior segment dysgenesis, elevated intraocular pressure (IOP), and congenital glaucoma, as previously described in B10.A-H2h4/(4R)SgDvEg mice. We investigated the effect of the nee mutation in C57BL/6J mice with respect to IOP, total retinal ganglion cell (RGC) death, and RGC subtype specific death in nee mice containing the Trhr-GFP transgene (selectively expresses GFP in ON-OFF direction selective RGCs).
Methods :
IOP and RGC death were measured in B6.Sh3pxd2bnee mutant (MUT) and wild type (WT) mice at post-natal days 30, 60, 75, and 90. C57BL/6J mice containing the Trhr-GFP transgene were crossed with B6.Sh3pxd2bnee to obtain nee mutant mice expressing GFP in ON-OFF direction selective RGCs. IOP was measured using a TonoLab tonometer. RGC damage was assessed by immunofluorescence of labeled retinal flat mounts using the GFP biomarker and NeuN.
Results :
Significant IOP elevation was observed in MUT mice at days 30, 60, 75, and 90 compared to WT mice (p<0.05). By 90 days of age, MUT mice had an IOP of 29.3 ± 7.6 mmHg compared to WT, 13.16±1.8 mmHg, p<0.01. MUT mice exhibited significant differences in percent cell survival of RGCs at 60 days (67.4±8.5%; n=4-6; p<0.0001), 75 days (23.7 ± 3.6%; n=4-6; p<0.0001), and 90 days (15.9 ± 8.2%; n=4; p<0.0001) compared to WT. IOP inversely correlated to RGC survival with an R2 value of 0.742. Significant differences in the percent cell survival of GFP positive RGCs was observed in MUT mice containing the Trhr-GFP transgene at 30 days (55.1±15%; n=4-6; p=0.0017), 60 days (16.5±4.6%; n=4-6; p<0.0001), 75 days (10.9±3.0%; n=4; p<0.0001), and 90 days (5.8±7.2%; n=4; p=0.013) when compared to WT. The half-life (T1/2) of GFP positive RGCs was T1/2=32 days and total RGC death T1/2=52 days. A shorter half-life suggests the ON-OFF direction selective RGCs are more susceptible to IOP elevation than the total RGC population. We have also established a protocol to visualize damage in the optic nerve and brain via tissue clearing and 3D visualization techniques.
Conclusions :
These studies characterized the nee glaucoma phenotype in C57BL/6J mice and demonstrate the specific susceptibility of ON-OFF direction selective RGCs. Future studies will identify susceptibility to additional subtypes of RGCs using this model system. These data are important to determine timing and onset of disease as well as identifying novel therapeutic targets.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.