Abstract
Increasing evidence suggests that unknown collagen remodeling mechanisms in the sclera underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantify collagen remodeling at the tissue- and lamella-level. Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; gluing the eye to a washer using (iii) 50 μL and (iv) 200 μL of cyanoacrylate adhesive; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in our new bioreactor. Two scleral shells of normal juvenile tree shrews were fluorescently labeled using a collagen specific protein and cultured in our bioreactor. Using two-photon microscopy, grid patterns were photobleached into and across multiple scleral lamellae. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. No significant reduction in cell viability was observed under conditions (i) and (v). Compared to condition (i), cell viability was significantly reduced starting at day 0 (condition (ii)) and day 3 (conditions (iii, iv, vi)). Tissue-level strain and intralamellar shear angel increased significantly during the culture period. Some scleral lamellae elongated while others shortened. Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are sensitive to tissue manipulations and tissue gluing. However, Ham's F-12 Nutrient Mixture has a protective effect on cell viability and can offset the cytotoxic effect of cyanoacrylate adhesive. This is the first study to quantify collagen micro-deformations over a prolonged period in organ culture providing a new methodology to study scleral remodeling in myopia.
Highlights
Myopia, or nearsightedness, is the most common type of refractive error affecting over 40% of adults in the United States as of 2004 [1] and over 80% of some Asian populations [2, 3]
The aim of this paper is to present a new organ culture system and imaging strategy based on two-photon fluorescence microscopy (2PFM) to gain insight into the remodeling mechanism that underlie scleral elongation in myopia
Because serum contains agents and growth factors known to stimulate the biosynthesis of matrix metalloproteinase (MMP) [38], which may alter scleral remodeling in situ, we aimed to optimize our culturing conditions towards maximum cell viability using serum-free culture media
Summary
Nearsightedness, is the most common type of refractive error affecting over 40% of adults in the United States as of 2004 [1] and over 80% of some Asian populations [2, 3]. There is increasing evidence from animal studies in support of an active but unknown remodeling mechanism that elongates the posterior scleral shell to match the eye’s axial length to its optical power, producing eyes with retinal focus (emmetropia) [10,11,12,13,14]. The sclera is an avascular tissue [15], which surrounds the posterior eye It serves as the principal load bearing tissue of the eye, as well as an important regulator of refractive error through a remodeling mechanism that alters the eye’s axial length. The scleral extracellular matrix (ECM) is produced and remodeled by resident fibroblasts This scleral ECM is primarily composed of collagen type I fibrils, and contains proteoglycans, such as aggrecan, decorin, and biglycan [10, 16]. Collagen fibrils aggregate to form interwoven layers of lamellae, which make up the bulk of the scleral structure [10]
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