Altered Structure and Function of Murine Sclera in Form-Deprivation Myopia

Abstract

Purpose The sclera is believed to biomechanically influence eye size, facilitating the excessive axial elongation that occurs during myopigenesis. Here, we test the hypothesis that the sclera will be remodeled and exhibit altered biomechanics in the mouse model of form-deprivation (FD) myopia, accompanied by altered retinoid concentrations, a potential signaling molecule involved in the process. Methods Male C57 Bl/6J mice were subjected to unilateral FD ( n = 44 eyes), leaving the contralateral eye untreated (contra; n = 44). Refractive error and ocular biometry were measured in vivo prior to and after 1 or 3 weeks of FD. Ex vivo measurements were made of scleral biomechanical properties (unconfined compression: n = 24), scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 18, and immunohistochemistry: n = 22), and ocular all-trans retinoic acid (atRA) concentrations (retina and RPE + choroid + sclera, n = 24). Age-matched naïve controls were included for some outcomes ( n = 32 eyes). Results Significant myopia developed after 1 (−2.4 ± 1.1 diopters [D], P < 0.001) and 3 weeks of FD (−4.1 ± 0.7 D, P = 0.025; mean ± standard deviation). Scleral tensile stiffness and permeability were significantly altered during myopigenesis (stiffness = −31.4 ± 12.7%, P < 0.001, and permeability = 224.4 ± 205.5%, P < 0.001). Total scleral sGAG content was not measurably altered; however, immunohistochemistry indicated a sustained decrease in chondroitin-4-sulfate and a slower decline in dermatan sulfate. The atRA increased in the retinas of eyes form-deprived for 1 week. Conclusions We report that biomechanics and GAG content of the mouse sclera are altered during myopigenesis. All scleral outcomes generally follow the trends found in other species and support a retina-to-sclera signaling cascade underlying mouse myopigenesis.

Exogenous All-TransRetinoic Acid Induces Myopia and Alters Scleral Biomechanics in Mice

“…We have also established that the myopic phenotype induced by atRA is accompanied by altered scleral biomechanics, with the sclera becoming more extensible and permeable, an effect consistent with that seen in visually mediated myopia in other species, 10 , 46 – 48 including our previous report in the mouse. 28 The overall impact of atRA on the eye appeared to be limited entirely to the posterior eye, except for a minor effect on anterior chamber depth that was also observed to occur in chickens. 24 We did not identify changes in scleral sGAGs or obvious signs of systemic or ocular toxicity.…”

“…Characterization of retinoids in the ocular tissues was performed as previously described. 28 In brief, the same tissues from left and right eyes of each animal were combined and weighed prior to homogenizing in 0.9% NaCl (normal saline). A two-step acid base extraction was performed, including internal standards (RA: 4,4-dimethyl-RA; retinol, total retinyl esters: retinyl acetate).…”

“…Total scleral sGAG and DNA content were quantified via spectrophotometric assays, the DMMB assay and PicoGreen assay, respectively. 28 In brief, samples were thawed and digested in 200 µL papain solution (0.1 M sodium acetate [anhydrous], 10 mM cysteine hydrochloride, 50 mM EDTA, pH 6, 2% v/v papain), distributing the digestate between the two assays. For the DMMB assay, absorbance was read at 525 nm immediately following the addition of the DMMB dye mixture (8 mg DMMB, 2.5 mL ethanol, 1 g sodium formate, 1 mL formic acid, and 496.5 mL deionized water).…”

Effects of exogenous retinoic acid on ocular parameters in Guinea pigs with form deprivation myopia

“…Citral is a competitive inhibitor of the key dehydrogenase required for RA generation. Studies have shown that the retinal RA content of myopia is significantly higher than that of non-myopia (Brown et al, 2022a;Brown et al, 2022b). Also, Wang et al found that the level of RA and the expression level of Zonula occludens-1 (ZO-1) and occludin in the retinal pigment epithelium (RPE) -choroid complex of lens induced myopia (LIM) guinea pigs were significantly increased, whereas the expression of RA and the proteins of ZO-1and occludin in LIM guinea pigs treated with RA antagonists were inhibited (Wang et al, 2014).…”

“…Retinoic acid (RA) is an acid derivative of vitamin A, which is the one-way oxidation product of photosensitive cells in the eye ( Brown et al, 2022a ; Brown et al, 2022b ), and RA has an important role in eye development. Citral is a competitive inhibitor of the key dehydrogenase required for RA generation.…”