A Novel Organ Culture Model to Quantify Collagen Remodeling in Tree Shrew Sclera

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.

A Novel Tree Shrew (Tupaia belangeri) Model of Glaucoma

“…Genetic analysis does show that the tree shrew is in proximity to primates, 38 and they have been used widely as an alternative model to higher primates for studies of psychosocial stress, 39 hepatitis, 40 and myopia. 33 , 34 However, tree shrews do not appear to share any derived characteristics. Thus, they are now classified independently in the order Scandentia.…”

“…In contrast, the tree shrew ( Tupaia belangeri ) is a small mammal closely related to primates 32 that currently is used in vision research as an inducible myopia model. 33 , 34 Tree shrews are accessible and, most importantly, have a robust connective tissue lamina with horizontally-oriented laminar beams that insert into the peripapillary sclera and, thus, are load-bearing. 35 We validated a novel animal model of glaucoma in tree shrews by demonstrating a reliable method to chronically elevate IOP with resultant selective gradual loss of the RGC and retinal nerve fiber (RNFL) layers, histopathologic evidence of glaucomatous optic neuropathy, and remodeling of the load-bearing connective tissues of the ONHs consistent with human glaucoma.…”

Thin Lamina Cribrosa Beams Have Different Collagen Microstructure Than Thick Beams

“…Elsewhere, arguments have been made about the connective tissues of the eye remodeling in response to IOP load changes, 43 with age 29 , 44 and disease. 45 How the collagen of LC beams changes in these conditions remains unknown and should be investigated. Herein we have demonstrated that this can be done using PLM.…”

The characterization of auditory brainstem response (ABR) waveforms: A study in tree shrews (Tupaia belangeri)

“…The tree shrew has been proposed to be a viable and alternative animal model to primates in disease studies and drug safety testing. Many researchers have recently used the tree shrew to create animal models for studying hepatitis B or C virus infections and myopia as well as social stress and depression (Baldivia et al., 2016; Fuchs, 2005; Norton et al., 2006; Wang et al., 2012; Zhao et al., 2002). Nevertheless, there have been few studies on the otology of tree shrews.…”