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J.D. Lindsey, A.J. Tran, J.G. Crowston, R.N. Weinreb; Transscleral Delivery of Macromolecules to the Retina Enhanced by MMPs . Invest. Ophthalmol. Vis. Sci. 2006;47(13):418.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have identified increased transscleral permeability following exposure following matrix metalloproteinase (MMP) production. The present study was undertaken to quantify delivery of extraocular macromolecules to the retina following exposure of intact eyes to MMPs.
Freshly enucleated mouse eyes were incubated with 1–µg/ml each of human MMP–1, MMP–2, and MMP–14 for 4 hours at 37°C. The eyes were then rinsed in phosphate buffered saline (PBS) and incubated in 2.5 mg/ml tetramethylrhodamine conjugated to 10–kDa dextran (TMRD) for 30 minutes. The retinas were isolated and homogenized with 60 µl of PBS. The homogenate was centrifuged at 14000 x g for 10 minutes. Fluorescence in 2 µl samples of supernatant was measured directly using a microspectrofluorometer. Calibration of the measurements was achieved by measuring fluorescence in serial dilutions of TMRD standards. Parallel experiments were conducted except that at the end of the incubation in TMRD, the eyes were fixed with 4% formaldehyde and frozen sections were evaluated by fluorescence microscopy.
The mean concentration of fluorescent dextran was 5.14 ± 1.61 µg/ml (SD, n = 33) in retinal homogenate supernatants from the control eyes and 6.37 ± 2.67 µg/ml (n = 40) in retinal homogenates of the eyes that had been incubated with MMPs. This 24% increase was statistically significant (P<0.02) by the two–tailed Students t test. The structural organization of the retina and other ocular tissues was maintained in both the control and MMP–treated eyes.
Incubation of mouse eyes with MMPs 1, 2, and 14 significantly enhances 10 kDa dextran access to the retina.
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