PurposeThe 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.MethodsWe 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.ResultsUsing 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%.ConclusionsWe 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.
Working with undergraduates in an
organic synthesis research laboratory
presents a unique set of challenges. An undergraduate research program
must be designed to focus on teaching students to “think like
a chemist” while advancing the principal investigator’s scientific agenda. However,
as novices in the laboratory, undergraduate students lack formal scientific
training, proficiency in required technical skills, and the chemical
safety knowledge required to work independently in a high hazard environment.
The lack of chemical knowledge and intuition poses considerable risk
in the laboratory, and mentors must develop and integrate methods
which allow undergraduate students to experiment safely. A well-designed
program will produce research assistants who recognize that good science
is a system consisting of many connected areas of knowledge that must
be codeveloped. Endeavoring to teach science as a process and create
a single method approach, the authors present “research storyboarding”.
Storyboarding combines the multiple components of research into a
single, transferrable method which can transform novice undergraduate
research students into safe, independent, competent, and productive
chemists. The method allows a scientist to break down the necessary
components of a successful experiment–the scientific
procedure, the chemical transformation, the tactile process, scientific
theory, instrument use, hazard recognition, risk assessment and minimization,
and emergency responseinto a single executable process. The
authors will present a storyboard conceptualized using a common synthetic
organic experiment.
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