An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes.

Investigative Ophthalmology & Visual Science

PubMedID: 27701632

Kizhatil K, Chlebowski A, Tolman NG, Freeburg NF, Ryan MM, Shaw NN, Kokini AD, Marchant JK, John SW. An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes. Invest Ophthalmol Vis Sci. 2016;57(13):5207-5215.
PURPOSE
The molecular mechanisms controlling aqueous humor (AQH) outflow and IOP need much further definition. The mouse is a powerful system for characterizing the mechanistic basis of AQH outflow. To enhance outflow studies in mice, we developed a perfusion system that is based on human anterior chamber perfusion culture systems. Our mouse system permits previously impractical experiments.

METHODS
We engineered a computer-controlled, pump-based perfusion system with a platform for mounting whole dissected mouse eyes (minus lens and iris, ~45% of drainage tissue is perfused). We tested the system's ability to monitor outflow and tested the effects of the outflow-elevating drug, Y27632, a rho-associated protein kinase (ROCK) inhibitor. Finally, we tested the system's ability to detect genetically determined decreases in outflow by determining if deficiency of the candidate genes Nos3 and Cav1 alter outflow.

RESULTS
Using our system, the outflow facility (C) of C57BL/6J mouse eyes was found to range between 7.7 and 10.4 nl/minutes/mm Hg (corrected for whole eye). Our system readily detected a 74.4% Y27632-induced increase in C. The NOS3 inhibitor L-NG-nitroarginine methyl ester (L-NAME) and a Nos3 null mutation reduced C by 28.3% and 35.8%, respectively. Similarly, in Cav1 null eyes C was reduced by 47.8%.

CONCLUSIONS
We engineered a unique perfusion system that can accurately measure changes in C. We then used the system to show that NOS3 and CAV1 are key components of mechanism(s) controlling outflow.