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Kelsey Sadlek, Catalina Ardila, Jonathan Pieter Vande Geest; Decellularization of the porcine lamina cribosa. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3170.
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The current literature in primary-open-angle-glaucoma (POAG) has shown damaged retina ganglion cell axons at the level of the lamina cribosa (LC) in the optic nerve head. LC cells and astrocytes play a major role in the remodeling of the LC ECM and the release of growth factors and other cellular signals that can affect how axons respond to changes in intraocular pressure (IOP). The aim of this study is to develop a technique to decellularize a porcine lamina cribosa to use as a biological scaffold to study changes in LC cells and astrocytes under varying IOP.
Twenty-four pig eyes were obtained from Thoma’s meat market (Saxonburg, PA) 4-5h post mortem. The eyes were dissected and the posterior poles including the LC and peripapillary sclera were decellularized using four different treatments. Treatment one included dry and wet freeze-thaw cycles, followed by immersion in hypotonic buffer (HB) and incubation with SDS and MgCl2. Treatment two used an incubation in HB and 3% Triton x-1000. In treatment three, incubations in HB and 0.5% Trypsin containing 0.2% EDTA were performed. Treatment four included sequential incubations in HB, 0.5% Trypsin containing 0.2% EDTA, 3% Triton x-1000, 4% deoxycholic acid, and 3% H2O2 solution. All of the treatments involved at least one incubation in 10KIU/ml aprotinin and 0.2 µg/mL deoxyriribonuclease I. DNA and cell content was studied using Qubit Fluorometric Quantitation (ThermoFisher) and histology (H&E and Masson’s trichrome (MTC) stains) respectively (n=3). Untreated pig eyes were the control. An one-way ANOVA was used for statistical analysis and p≤0.05 was considered significant.
DNA content was significantly decreased in treatments one (1.74 ± 0.8 ng/µl) and four (0.59 ± 0.4 ng/µl) compared to the control (25.33 ± 8.7 ng/µl). By a qualitative histological analysis (Fig 1), it is possible to observe a reduction in the cell number in samples from treatment 1 and 4, nevertheless the collagen and axonal structures are more preserved in treatment 1.
Using decellularization treatment one, it is possible to obtain a LC biological scaffold that allows for better analysis of biological changes of cells seeded on it in response to increases in IOP. Since changes in the LC ECM are important modulators of glaucoma, in future work we will measure the biomechanical properties of the decellularized scaffolds.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Fig 1. Histological sections of the treated eyes.
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