DOI

10.17077/etd.rnlroxyo

Document Type

Dissertation

Date of Degree

Spring 2015

Access Restrictions

.

Degree Name

PhD (Doctor of Philosophy)

Degree In

Molecular Physiology and Biophysics

First Advisor

Michael G. Anderson

First Committee Member

VAL SHEFFIELD

Second Committee Member

AMY LEE

Third Committee Member

ANDY RUSSO

Fourth Committee Member

MARK STAMNES

Fifth Committee Member

ROBERT MULLINS

Abstract

Pigment dispersion syndrome (PDS) and its potential sequela, pigmentary glaucoma (PG) are ocular diseases characterized by disruption of the iris with subsequent dispersion of pigment throughout the anterior chamber. In some cases, PDS can be accompanied by intraocular pressure (IOP) elevation and initiate conversion to PG. PG results in vision loss by the death of retinal ganglion cells (RGC). However, the pathophysiologic mechanisms that contribute to IOP elevation and conversion to PG are not known. Mice represent a powerful resource for studies of human eye disease. The DBA/2J (D2) mouse model of PG exhibit iris phenotypes that resemble PDS in humans, including pigment dispersion from the iris pigmented epithelium and a characteristic pattern of iris transillumination defects (iris-TID).

Mutations in Lyst and Dct each elicit mild iris phenotypes of PDS in C57BL/6J mice (B6) and were hypothesized to exacerbate the PDS in D2. Using a genetic-background driven approach, the Lyst and Dct mutations were transferred onto the glaucoma-susceptible D2 strain (D2.Lyst and D2.Dct). Characterization of both strains show that the iris phenotypes of PDS were rapidly intensified, resulting in the development of two new mouse models with accelerated forms of PDS. A novel technique for grading severity of iris-TID revealed that a mutation in Tyrp1 modified the Lyst-mediated iris-TID in D2.Lyst.

Using a physiologic approach to identify factors contributing to IOP elevation, PDS was experimentally induced in mice by intraocular infusion of homogenized irides from mouse donors. Induction led to characteristic features of PDS including pigment deposition along the cornea, lens, and trabecular meshwork. Induction also resulted in IOP elevation. This novel model provides a unique experimental platform for manipulating disease parameters of PDS, since the amount of pigment, frequency of infusions, and genotypes of the recipient and donor mouse strains may be modified.

Studies of PDS and conversion to PG require high-throughput methods for detecting glaucomatous death of RGCs. To complement our studies, we developed a software tool that performs automated counting (RetFM-J) and classification (RetFM-J) of cells in the inner retina of flat-mounted retinas. In testing performance, output count data from these tools was determined to be consistent with previously published results for several well-characterized mouse models of eye disease and their controls, including: D2 (glaucoma), Jun-sufficient and Jun-deficient mice receiving controlled optic nerve crush (induced retinal damage), and B6 (normal). We show that these tools are feasible and can be utilized to study RGC death in a high-throughput manner.

In conclusion, novel mouse models with severe forms of naturally occurring and experimentally-induced PDS were developed and characterized. An objective technique for measuring severity of iris-TID was developed. Automated software tools for quantitative analyses and random forest classification techniques of cellularity in the inner retina provide a new approach for measuring glaucomatous damage.

Public Abstract

Pigment dispersion syndrome (PDS) and its potential sequela, pigmentary glaucoma (PG), are ocular diseases characterized by disruption of the iris with pigment dispersion throughout the anterior chamber. In some cases, PDS can be accompanied by intraocular pressure (IOP) elevation and conversion to PG. However, the mechanisms contributing to IOP elevation and conversion to PG are not known. PG results in vision loss by the death of retinal ganglion cells (RGC).

The DBA/2J (D2) mouse model of PG exhibits an iris disease similar to human PDS. Mutations in the Lyst and Dct genes cause PDS and were hypothesized to exacerbate PDS in D2. The Lyst and Dct mutations were individually transferred onto the glaucoma-susceptible D2 strain. Assessment of each strain shows PDS was exacerbated, resulting in two new mouse models with accelerated PDS.

PDS was experimentally induced in mice by injecting iris pigment from mouse donors, leading to characteristic features of PDS and IOP elevation. This novel model provides an experimental platform for manipulating disease parameters of PDS. To complement our studies, we developed a new software tool that performs high- throughput automated counting and classification of inner retinal cells to measure loss of RGCs. Cell counts from these tools were determined to be consistent with previously published data for several well-characterized mouse models of RGC damage.

In conclusion, novel mouse models with severe forms of naturally-occurring and experimentally-induced PDS were developed and characterized. The automated counting and classification techniques for the inner retina provide a new approach for measuring glaucomatous damage.

Pages

xx, 161 pages

Bibliography

Includes bibliographical references.

Copyright

Copyright © 2015 Adam Hedberg-Buenz

Included in

Biophysics Commons

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