Abstract
Purpose :
Glaucoma is one of the leading causes of blindness worldwide. The outflow pathway, especially fibrosing trabecular meshwork (TM), plays an essential role in this disease, and the availability of TM cells is crucial for in vitro research. To date, extracting TM cells from mice is complex and relies on injecting magnetic microbeads in the anterior chamber of living mice with subsequent isolation. Therefore, a cost-effective and animal experiment-reducing strategy for the extraction of mouse TM cells is required.
Methods :
After Enucleation, eyes were cut in half anterior-to-posteriorly. Lens and posterior segment were removed, Iris and the attached ciliary body were gently pulled backwards and disconnected from the remaining tissue to expose the TM. Cuts anterior and posterior the TM allowed for removal of the TM region. Tissue was cultured facing down in a 6-well filled with Eagle`s Minimum Essential Medium supplemented with 10% FBS. Phagocytotic properties were assessed using fluorescent microbeads, and Immunocytochemistry (ICC) analysis was performed for different markers present in TM cells.
Results :
An outgrowth of cells could be observed after 4-7 days. These cells were able to phagocytize fluorescent microbeads and were positive in ICC for collagen IV, fibronectin 1, vimentin and α-smooth muscle actin. Also, typical cross-linked actin networks (CLANs) were expressed after one-week TGFB2 exposure. As previously reported for TM cells, treatment of the isolated cells with 100 nM Dexamethasone for one week led to increased Myocilin expression.
Conclusions :
The isolated cells show phagocytotic properties and specific expression of markers reported in TM cells. Therefore, our presented dissection-based method is an inexpensive, reproducible method for isolating TM cells in mice.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.