DZF-Project: C. Ross Ethier

2001

Human Anterior Segment Model for Myocilin Perfusion

Principal Investigator: Prof. C. Ross Ethier

Department of Mechanical and Industrial Engineering, University of Toronto, 5 King's College Rd., Toronto, Ontario, Canada

ethier(at)mie.utoronto.ca

Co-investigator: Dr. Ernst Tamm, University of Erlangen-Nuremburg

Keywords: glaucoma, anterior segment organ culture, myocilin, human eyes

Beginning and End of the Project: 2001-2002

Background and Aims

Background: Glaucoma describes a group of ocular conditions that involve progressive optic nerve damage causing loss of visual function. Glaucoma occurs in 0.5 to 1% of the general population in Western countries, and its prevalence increases with age, affecting up to 2.5% of those over 65 years of age. Recent estimates of world-wide prevalence by the year 2000 predict approximately 67 million people affected by glaucoma, with 6.7 million bilaterally blind. A central feature of glaucoma is elevated pressure within the eye, and there is therefore a great deal of interest in understanding why this pressure is elevated. This frequently uses experiments in animals. Here we wished to use an alternate system employing post mortem human eyes for studying the factors controlling pressure in the eye. The tissue that controls the pressure and which is therefore of interest in this research is known as the trabecular meshwork, and the pressure in the eye is generated as a fluid known as the aqueous humor flows through this tissue.

The overall goal of the research funded by the Foundation was to demonstrate the utility of an organ culture system for studying aqueous humor outflow dynamics in enucleated human eyes. The organ culture system maintains the function of the trabecular meshwork for days or weeks. An understanding of trabecular meshwork function is important in glaucoma, an ocular disease where the normal flow resistance to aqueous humor drainage becomes pathologically elevated. Use of enucleated human eyes to study trabecular meshwork function, rather than the more conventional animal models, reduces the use of larger animals in biomedical research.

Aim: The specific focus of the funded research was to determine the effects of the protein, myocilin, on aqueous outflow resistance in human eyes. We wished to:

Refine and further develop the hardware and experimental protocol for organ culture perfusions of human anterior segments.
To perfuse recombinant myocilin into human anterior segments using the organ culture model.

Methods and Results

The funded research was quite successful. More specifically, we were able to:

Improve and refine the organ culture system to obtain good trabecular meshwork morphology and structure for long-term perfusions in our hands (up to 2 weeks). Demonstration of the viability of the target tissues in this type of culture system is clearly necessary to speed acceptance of the technique among the research community.
Express and isolate the olfactomedin (C-terminal) domain of myocilin, using eukaryotic 293 EBNA cells. Most disease-causing mutations in myocilin occur in the olfactomedin domain, and therefore this domain is critical for proper myocilin function.
Perfuse the recombinant protein from step 2 into enucleated human anterior segments in the organ culture model. We showed that large quantities of this recombinant protein did not affect aqueous outflow resistance. This finding was unexpected, and suggests that both the olfactomedin and N-terminal domains must be present for myocilin to have full function.Alternatively, post-translational modifications of myocilin may have a major impact on protein function.

The above results have been documented in a refereed journal paper: A. Goldwich, C.R. Ethier, D. W.-H. Chan and E.R. Tamm, “Perfusion with the Olfactomedin Domain of Myocilin Does not Affect Outflow Facility”, Investigative Ophthalmology and Visual Science, Vol. 44, pp. 1953-1961, 2003.

In addition, Ethier carried out further work to optimize the use of the organ culture system. Specifically, his lab investigated the transport of virus within cultured anterior segments. Viral infection studies are now being used to modulate trabecular meshwork function by over- or under-expressing key target proteins. Most previous studies have been in animals, primarily rabbits and monkeys. We were able to develop a protocol to more reliably deliver virus to the trabecular meshwork in perfused human anterior segments. This has resulted in better trabecular meshwork function and higher levels of recombinant protein expression in infected trabecular meshwork cells. This work further extends the utility of the cultured human anterior segment model, and has been published: C.R. Ethier, S. Wada, D. Chan and W.D. Stamer, “Experimental and Numerical Studies of Adenovirus Delivery to Outflow Tissues of Perfused Human Anterior Segments”, Investigative Ophthalmology and Visual Science, Vol. 45, pp. 1863-1870, 2004.

Conclusions and Relevance for 3R

Support from the Foundation has resulted in two successful studies, both of which further the acceptance of the anterior segment organ culture system by the research community. This acceptance is recognized by the adoption of this system by other labs, both in the research and industrial community, and the concomitant reduction in the use of large animals in studies of trabecular meshwork function.