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Anthony Saraco, Alexander K Ball, Trevor Williams, Judith A West-Mays; Retinal Ganglion Cell Numbers In Early Post-Natal AP-2β Neural Crest Cell Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3708.
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Our lab has previously described a mouse model of conditional deletion of AP-2β transcription factor in neural crest cells (NCC) contributing to the periocular mesenchyme (POM). This model is characterized by an adherence of the iris to the cornea, accompanied by an elevated intraocular pressure (IOP) and a 35% decrease in retinal ganglion cells (RGCs) by 2 months of age. The purpose of the current study was to investigate whether the observed decrease in cells within the RGC layer in the mutant model occurs progressively following birth or if it occurred as an embryonic defect.
Using the Cre-loxP system we generated a conditional knockout of tfap2b exclusively in cranial NCC (AP-2β NCC KO). Postnatal day 4 (P4) mice eyes extracted, fixed in 10% formalin, and processed for paraffin embedding. Immunohistochemistry was performed using a goat anti-Brn3a polyclonal antibody (santa cruz biotechnology lot#: E1413) to label RGCs. Representative micrographs of the retina were taken at 40x and RGC layer cells were counted using Image-J. Statistical analysis was performed with RStudio.
At P4 there were 347 +/- 58 DAPI cells/mm in the AP-2β NCC KO mice compared to 387 +/- 74 DAPI cells in wild-type (WT) mice. Of these, 92 +/- 26 cells/mm expressed Brn3a in the KO compared to 102 +/- 16 cells/mm in the WT. An ANOVA indicated no statistical difference between the total number of cells in the RGC layer or the number of RGCs (Brn3a) in AP-2β NCC KO or WT (F=0.909, p=0.3654 and F=1.449, p=0.234 respectively). These results demonstrate that the RGC layer in KO and WT retinas contain the same proportion of RGCs (26.35% and 26.48% respectively) as early as P4.
In our previous work we had shown that the AP-2β NCC KO mutant mice exhibited significantly fewer RGCs by 2 months of age. In the current study, we examined the retinas of mutants and WT littermates at the earlier stage of P4. In this case we did not observe a statistical difference in the number of cells in the RGC cell layer, nor the proportion of cells in the mutants as compared to WT. These findings support the hypothesis that the RGC layer cell loss described in older mutants occurs progressively and did not occur due to an embryonic defect caused by the KO. These findings suggest that the AP-2β NCC KO mice may serve as a novel model for investigating RGC cell loss due to high IOP in glaucoma.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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