Abstract
Purpose: :
Myocilin has been linked to the increase in intraocular pressure in juvenile– and adult–onset POAG in man. CD44, a multifunctional receptor and cell–adhesion molecule is increased in iris, ciliary body, and aqueous humor in POAG in man. The goal of this study was to compare the aqueous humor levels of myocilin and CD44 between primary glaucoma, secondary glaucoma, primary cataracts and diabetic cataracts of different breeds of dogs.
Methods: :
Aqueous humor samples (0.1–0.2 ml) were obtained from the anterior chambers of over 300 dogs of various breeds. Each dog’s aqueous humor samples were diagnosed as either primary glaucoma, secondary glaucoma, cataracts, or cataracts due to diabetes. Two [[Unsupported Character – WP Symbol :]]L aqueous samples from different dogs were loaded and run on 12% Bis–Tri gels (Invitrogen, Carlsbad CA). The separated proteins were then stained using the microwave Simply Blue Safestain protocol described in the Invitrogen 12% Bis–Tris gel manual, and imaged using a BioRad Scanner and Quantity One software for analysis. Western blot analysis was performed by running the samples on a 12% Bis–Tris Gel. The separated proteins were then transferred onto nitrocellulose membranes and the blot incubated with an anti–myocilin rabbit polyclonal antibody or anti–CD–44H mouse monoclonal antibody and stained (Supra Signal West Pico Chemiluminescent, Pierce, Rockford IL) for scanning. Myocilin levels were then computed using a known concentration of human myocilin protein (Alcon Labs) as a control. The relative amount of CD44 was expressed as a ratio of the band intensity between the primary glaucomatous, secondary glaucomatous, diabetic cataractous, and primary cataractous.
Results: :
Myocilin levels were detected in all dogs. The overall levels of myocilin were highest in the primary (21051 µg/ml) and secondary glaucoma (32247 µg/ml) dogs; there was no significant difference between the two groups. Low levels occurred in the primary cataract and the diabetic cataract eyes. CD44 was detected in the aqueous humor in most dogs, with few exceptions with the cataractous dogs. The ratios were 10.0 to 10.8 to 1.0 to 2.0 for primary glaucoma, secondary glaucoma, primary cataract, and diabetic cataract respectively. A distinct common broad immunoreactive 61 kDa band of myocilin and CD44 was detected.
Conclusions: :
Aqueous myocilin and CD44 levels were significantly increased in glaucomatous eyes of many breeds of dogs. The co–expression of the 61 kDa band of myocilin and CD44 suggests a formation of aggregates between CD44 and myocilin.
Keywords: anterior chamber • aqueous • outflow: trabecular meshwork