Date of Award

12-2011

Document Type

Thesis

Degree Name

Master of Science (MS)

Program

Biomedical Sciences

Track

Molecular Therapeutics and Cell Signaling

Research Advisor

Jena J. Steinle, Ph.D.

Committee

Edward Chaum, M.D., Ph.D. Monica Jablonski, Ph.D. Duane Miller, Pharm D., Ph.D. Charles R. Yates, Pharm D., Ph.D.

Abstract

Diabetic retinopathy is the leading cause of blindness in working Americans. While there are therapeutic regimens for the disease, more effective methods are needed. We have previously shown that a non-specific beta-adrenergic receptor agonist, isoproterenol, was effective in preventing functional and morphological changes associated with diabetic retinopathy in the rat. Isoproterenol also produced left ventricle remodeling suggesting it entered the systemic circulation. We therefore synthesized various novel beta-adrenergic receptor compounds and screened these compounds in vitro for their ability to reduce markers of inflammation and apoptosis. Of the various compounds tested, Compound 49b was able to reduce both inflammation and apoptosis in vitro in both retinal endothelial cells (REC) and Müller cells and did so at a significantly lower concentration than isoproterenol, deeming it the most effective compound.

Objective. We evaluated the effectiveness of Compound 49b to prevent and delay changes associated with diabetic retinopathy without systemic effects. We hypothesized that topical application of Compound 49b could prevent and delay the onset of functional and morphological markers of diabetic retinopathy and restore retinal cellular signaling, without affecting the heart.

Methods. Male rats were made diabetic with a streptozotocin injection (60mg/kg). Two days after the injection, glucose measurements were obtained. Two groups of rats were used for these two separate studies. In the prevention studies, a subset of the diabetic rats were started on daily 1mM Compound 49b eye drops for either 2-months or 8-months upon the verification of diabetes, while the control and diabetic only subgroups received no treatment. In the delayed treated studies, animals were placed on Compound 49b after 6-months of untreated diabetes. Once optimal dose and time course was obtained, body weight, blood glucose, intraocular pressure, blood pressure, and electrical activity of the retina were measured monthly over the duration of the prevention and delayed treatment studies. Insulin signaling, tumor necrosis factor!alpha (TNF-!) levels, and cleavage of caspase 3 were analyzed after 2- and 8-months or 2- and 6-months of treatment.

Results. Protein levels of TNF-! and cleaved caspase 3 were increased in the retinas of diabetic rats, but were returned to normal values following 1mM Compound 49b treatment. Since we have previously found that topical isoproterenol produced left ventricular remodeling, we wanted to investigate whether Compound 49b could overcome this obstacle. Left ventricular samples from diabetes+Compound 49b-treated rats were not significantly different than diabetes alone rats after statistical analyses.

Conclusion. Data showed that daily topical 1mM Compound 49b eye drops reduced TNF-! levels and cleaved caspase 3 levels, while maintaining insulin receptor signal transduction in the diabetic retina. Additionally, Compound 49b maintained retinal electrical activity, as measured by electroretinogram (ERG) analysis. Data indicates that Compound 49b does not reach the systemic circulation or alter blood pressure or intraocular pressure, suggesting that it may represent a novel treatment option for diabetic retinopathy.

DOI

10.21007/etd.cghs.2011.0349