Abstract
Purpose:
We previously described that deficiency of Vav2, 3 proteins leads to an ocular phenotype in mice similar to human angle closure glaucoma. To establish an animal model for the assessment of pressure-dependent damage to retinal ganglion cells (RGCs), we generated cyan fluorescent protein (CFP)-expressed Vav2-deficient mice that express CFP in RGCs and gradually develop the elevated intraocular pressures (IOP). We clarify this new animal model to evaluate the correlation between pressure insult and RGC survival rate.
Methods:
Vav2-deficient mice were crossed with B6.Cg-Tg (Thy1-CFP) 23Jrs/J mice to express CFP in RGCs (CFP/Vav2 mice). The daytime IOPs of CFP/Vav2 mice were measured starting at 8 week-old ages every 2 weeks up to 14 weeks using the microneedle method. To assess the magnitude of IOP elevation, the area under the curve was calculated in units of mmHg×day as a surrogate value of the pressure insult. Then the mice were sacrificed and the eyes were enucleated and immediately fixed in 4% paraformaldehyde in 0.1M PBS. Four radial relaxing incisions were made and the retina was prepared as a flattened whole mount on a glass slide (n=23). Images were obtained using a fluorescence microscope with a CFP filter set. Concerning the whole eye, the number of RGCs expressing CFP was manually counted in 12 separated areas of 40,000μm2, the central, middle, and peripheral areas measured from the optic disc of each four retinal quadrant areas.
Results:
In CFP/Vav2 mice, 20 percent of mice showed a statistically higher IOP than control WT mice. They were regarded as having pressure insult, that is, the area under curve indicated 1926±123 mmHg×day, while that of wild type (WT) mice was 1593±85 mmHg×day. After 6-week duration of pressure insult, the number of RGCs (cells/mm2) in CFP/Vav2 mice showed a decrease compared to those of WT mice (n=23, P<0.01). In particular, a decrease was clearly detected in the nasal area as 1308±181 cells/mm2 compared to that of WT mice, 1443±94 cells/mm2. In addition, in the inferior area, the number decreases to 1298±191 cells/mm2, while that of WT, 1499±161mmHg cells/mm2 (P<0.01). Our results demonstrate that specific retinal area was damaged by IOP elevation.
Conclusions:
CFP/Vav2 mice are likely to be a valuable animal model for the easy assessment of pressure-dependent damage to RGCs and evaluation of pressure-dependent RGC death.
Keywords: 568 intraocular pressure •
740 transgenics/knock-outs