Abstract
Purpose :
Glaucomatous IOP elevation is due to decreased aqueous humor outflow facility at the trabecular meshwork (TM).Using mice as a glaucoma research model is challenging because measuring outflow facility in mouse eyes is very challenging. Currently, there are an in vivo model and an ex vivo model using the entire mouse globe for outflow facility measurement. Both methods require cannulation of the anterior segment with a fine needle which is technically challenging. Here, we are developing an ex vivo model using the mouse anterior segment for outflow facility studies and long-term cultures.
Methods :
A perfusion culture dish and a rigid compression ring were designed and 3D printed. A custom-made gasket was attached to the compression ring to improve sealing. Mouse eyes of C57BL/6J background were was cut at the optic nerve head region. Four cuts were made latitudinally (from posterior to anterior) ending at the equator. The retina, lens, and uveal tissue were all removed. The eye was then mounted on the perfusion dish with the cornea facing up, the equator sitting at the bottom of the dish, and the dissected posterior segment (with 4 cuts) evenly spread out like a rosette. The compression ring was placed on top of the eye so that the region between the limbus and equator was compressed. After tightening the four bolts, the anterior segment was covered with a wet pad (no direct contact with the corea), perfused with opti-MEM medium at 37 degrees in a cell culture incubator, and IOP was recorded.
Results :
We found that removing the mouse lens, ciliary body and iris did not affect the anatomic integrity of the TM. We observed mouse outflow facility at 4.917±2.277 nl/min/mmHg (mean±SD; N=6) with linear regression analysis. We also conducted power law analysis of the same raw data with a reference IOP at 8mmHg, showing outflow facility at 5.860±2.277 nl/min/mmHg. Power law analysis showed R square values from 0.9119 to 0.9959 and β values from -0.483 to 0.6506. There is no statistical difference between the outcome using the two methods (Paired t-test; p<0.05, N=6).
Conclusions :
We believe that our novel ex vivo anterior segment perfusion culture model is a suitable tool for studying mouse outflow facility and potential long-term culture.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.