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Enhua Zhou, Michael Paolucci, sam shrestha, Christopher William Wilson, Ernie Hixon, Andrew Brady, Aaron Bickel, Ted Manley, Ganesh Prasanna, Dennis S Rice, Amy Chen; Design and validation of a compact ex vivo whole eye perfusion system. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
A clinically validated, manageable risk factor for glaucoma progression is elevated Intraocular pressure (IOP). IOP is maintained by the balance between aqueous humor production and outflow resistance. To investigate the regulation of outflow resistance, ex vivo perfusion of animal eyes provides a valuable tool. However, typical whole eye perfusion systems reported in glaucoma research have limited throughput (e.g. 1-2 pairs a time). Here we report the design of a compact, scalable perfusion system with improved throughput. This is achieved by incorporating flow sensors into a gravity-driven perfusion system.
IOP was controlled by the height of the reservoir, which was adjusted by a motorized stage. Flow rate was measured by flow sensors (Sensirion, Switzerland). Custom software was developed in Matlab (Mathworks, Natick, MA) to control reservoir height and to record flow rate. We validated the system by measuring response of eyes to Y-39983 (a Rho kinase inhibitor), latrunculin B (actin disrupting agent), prostaglandin E1 (PGE1), endothelin-1 (ET-1; potent vasoactive peptide), ambrisentan (inhibitor for type A endothelin receptor), and S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide [NO] donor). All compounds were evaluated in bovine eyes and a few selected ones were also tested in rabbit and porcine eyes.
The instrument design enabled simultaneous measurements of 10 eyes with a small footprint of 4 square feet. At fixed IOP of 6 mmHg, Y-39983, Latrunculin B, or PGE1 increased outflow rate by up to 50-100%, compared to vehicle control, in a time- and dose-dependent manner. On the contrary, ET-1 decreased outflow rate by up to 50-70% with an EC50 around 7nM. ET-1’s effect was inhibited by ambrisentan as expected. Interestingly, it was also inhibited by pretreatment of SNAP (by up to 84% in bovine eyes) or Y-39983 (by 21% in porcine eyes and 100% in rabbit eyes).
We have developed a compact system that can reliably measure outflow resistance in ex vivo cultured eyes. Results from Y-39983, latrunculin B, and PGE1 recapitulated in vivo animal studies reported in the literature. This platform offers an opportunity in enabling compound prescreen for in-vivo efficacy studies for novel glaucoma therapies.Acknowledgement: We thank Tom Vollmer for technical assistance.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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