title: An In Vitro Perfusion System to Enhance Outflow Studies in Mouse Eyes.
abstract: OBJECTIVE
  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.
authors.affiliation: The Howard Hughes Medical Institute, and The Jackson Laboratory, Bar Harbor, Maine, United States.
authors.forename: Krishnakumar
authors.initials: K
authors.lastname: Kizhatil
authors.affiliation: The Howard Hughes Medical Institute, and The Jackson Laboratory, Bar Harbor, Maine, United States.
authors.forename: Arthur
authors.initials: A
authors.lastname: Chlebowski
authors.affiliation: The Howard Hughes Medical Institute, and The Jackson Laboratory, Bar Harbor, Maine, United States.
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Tolman
authors.affiliation: The Howard Hughes Medical Institute, and The Jackson Laboratory, Bar Harbor, Maine, United States.
authors.forename: Nelson F
authors.initials: NF
authors.lastname: Freeburg
authors.affiliation: The Howard Hughes Medical Institute, and The Jackson Laboratory, Bar Harbor, Maine, United States.
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Ryan
authors.affiliation: The Howard Hughes Medical Institute, and The Jackson Laboratory, Bar Harbor, Maine, United States.
authors.forename: Nicholas N
authors.initials: NN
authors.lastname: Shaw
authors.affiliation: The Howard Hughes Medical Institute, and The Jackson Laboratory, Bar Harbor, Maine, United States.
authors.forename: Alexander D M
authors.initials: AD
authors.lastname: Kokini
authors.affiliation: Department of Integrative Physiology and Pathobiology, Tufts University School of Medicine, Boston, Massachusetts, United States.
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Marchant
authors.affiliation: The Howard Hughes Medical Institute, and The Jackson Laboratory, Bar Harbor, Maine, United States 3Department of Ophthalmology and Sackler School of Graduate Biomedical Sciences, Tufts University of Medicine, Boston, Massachusetts, United States.
authors.forename: Simon W M
authors.initials: SW
authors.lastname: John
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology; D001082:Aqueous Humor / Q000378:metabolism*; D004867:Equipment Design; D007429:Intraocular Pressure / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010477:Perfusion / Q000295:instrumentation*; D011725:Pyridines / Q000494:pharmacology; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
chemical_list: D000577:Amides; D000959:Antihypertensive Agents; D011725:Pyridines; C108830:Y 27632