Investigative Ophthalmology & Visual Science, Vol 34, 2097-2104, Copyright © 1993 by Association for Research in Vision and Ophthalmology

ARTICLES AND REPORTS

Novel procedures for isolating intact retinal vascular beds from diabetic humans and animal models

NM Laver, WG Robison Jr and BA Pfeffer
National Eye Institute, NIH, Bethesda, MD 20892.

PURPOSE. To improve the 30-year-old "trypsin digestion" procedure for isolation of the complete retinal vasculature, which, in its time, was a revolutionary advance that allowed important discoveries about diabetic retinopathy; to provide a method that will yield more consistent results when applied to retinas representing a wide range of ages, species, and severity of vascular disease, such as that occurring in diabetes. METHODS. Because the Difco trypsin preparation (Difco Laboratories, Detroit, MI) is a crude pancreatic extract, containing variable amounts of chymotrypsin, elastase, amylase, lipase, ribonuclease, collagenase, and other contaminants, an attempt was made to determine which of the major enzymes alone (using purified preparations), or what combination of enzymes, might be most effective in providing consistently clean yet intact retinal vasculatures from eyes of different origins. RESULTS. Purified elastase alone (40 U/ml) in 100 mmol/l sodium phosphate buffer with 150 mmol/l sodium chloride and 5 mmol/l EDTA at pH 6.5 and 37 degrees C gave better results than various concentrations of purified trypsin or chymotrypsin alone, or mixtures of trypsin/chymotrypsin, trypsin/elastase, chymotrypsin/elastase, or the crude trypsin preparation. CONCLUSIONS. Elastase, which exhibits broad protease activity, and not trypsin, is the most important enzyme of the standard, crude trypsin digestion procedure for removal of the nonvascular tissues of the retina.

This article has been cited by other articles:

				G. R. Barile, S. I. Pachydaki, S. R. Tari, S. E. Lee, C. M. Donmoyer, W. Ma, L. L. Rong, L. G. Buciarelli, T. Wendt, H. Horig, et al. The RAGE Axis in Early Diabetic Retinopathy Invest. Ophthalmol. Vis. Sci., August 1, 2005; 46(8): 2916 - 2924. [Abstract] [Full Text] [PDF]

				S. Y. Kim, M. A. Johnson, D. S. McLeod, T. Alexander, T. Otsuji, S. M. Steidl, B. C. Hansen, and G. A. Lutty Retinopathy in Monkeys with Spontaneous Type 2 Diabetes Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4543 - 4553. [Abstract] [Full Text] [PDF]

				M. B. Grant, P. E. Spoerri, D. W. Player, D. M. Bush, E. A. Ellis, S. Caballero, and W. G. Robison Plasminogen Activator Inhibitor (PAI)-1 Overexpression in Retinal Microvessels of PAI-1 Transgenic Mice Invest. Ophthalmol. Vis. Sci., July 1, 2000; 41(8): 2296 - 2302. [Abstract] [Full Text]

				D. F. Lazarous, M. Scheinowitz, M. Shou, E. Hodge, M. A. S. Rajanayagam, S. Hunsberger, W. G. Robison Jr, J. A. Stiber, R. Correa, S. E. Epstein, et al. Effects of Chronic Systemic Administration of Basic Fibroblast Growth Factor on Collateral Development in the Canine Heart Circulation, January 1, 1995; 91(1): 145 - 153. [Abstract] [Full Text]