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Keiko Fujikawa, Kaoru Inoue, Reiko Yamagishi, Takae Koshiyama, Makoto Aihara; Characterization of RGC death in Rho-GTPases activating proteins Vav proteins, deficient mice. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2437.
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© ARVO (1962-2015); The Authors (2016-present)
We previously described that deficiency of Rho-GTPases activating proteins, Vav2,3 and Vav2 (Vavs), lead to an ocular phenotype in mice. These mice indicated anterior segment dysgenesis, various levels of ocular hypertension (OH) and subsequent RGC death. Here we generate the CFP-expressed Vavs- deficient mice and evaluate them as animal models for glaucoma, with the comparison of RGC death observed in normal and OH mice.
Vav2,3-deficient and Vav2-deficient mice were crossed with B6.Cg-Tg (Thy1-CFP) 23Jrs/J mice to express CFP in RGCs (CFP/Vav2,3 mice, CFP/Vav2 mice). The daytime IOPs of CFP/Vavs mice were measured starting at 8 week-old ages every 2 weeks up to 14 weeks using the microneedle method. 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. Images were obtained using a fluorescence microscope with a CFP filter set. Concerning the whole eye, the number of RGCs (cells/mm2) expressing CFP was manually counted in 12 separated areas of 40,000μm2. Mice were classified into normal and OH IOP groups based on the definition of normal IOP range determined as mean±2SD from IOP of wild-type (WT) mice.
Normal IOP in this investigation is set 11.0±1.4mmHg determined from IOP of control WT mice (n=24).<br /> In CFP/Vav2,3 mice, 69 percent of eyes (11/16) showed OH IOP; 16.4±4.1mmHg, whereas 21 percent of eyes (5/16) showed normal IOP. The number of RGCs in OH IOP group was remarkably decreased to 1021 compared to 1441 of age-matched control WT mice group (P<0.001), and even in normal IOP group of CFP/Vav2,3 mice it was statistically decreased to 1214(P<0.001). In addition, in CFP/Vav2 mice, normal IOP group (13/22 eyes) also showed statistically decreased RGCs number; 1342 (P<0.01), while the number of RGCs in OH IOP group (9/22 eyes) that is 13.4±0.9 mmHg IOP, was decreased to 1293 (p<0.001).<br /> Our results seem to indicate that even in the circumstance of normal IOP Vavs-deficient mice appear to cause RGCs death
We demonstrate here CFP/Vav2,3 and CFP/Vav2 mice show not only the pressure-dependent RGCs death, but also under the normal IOP level, pressure-independent RGC death. It implies that Vav2,3 and Vav2 deficiency in mice may cause RGC death apart from that of pressure insult.
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