Regulation of scleral cell contraction by transforming growth factor-β and stress competing roles in myopic growth

Reduced extracellular matrix accumulation in the sclera of myopic eyes leads to increased ocular extensibility and is related to reduced levels of scleral transforming growth factor-β (TGF-β). The current study investigated the impact of this extracellular environment on scleral cell phenotype and cellular biomechanical characteristics. Scleral cell phenotype was investigated in vivo in a mammalian model of myopia using the myofibroblast marker, α-smooth muscle actin (α-SMA). In eyes developing myopia α-SMA levels were increased, suggesting increased numbers of contractile myofibroblasts, and decreased in eyes recovering from myopia. To understand the factors regulating this change in scleral phenotype, the competing roles of TGF-β and mechanical stress were investigated in scleral cells cultured in three-dimensional collagen gels. All three mammalian isoforms of TGF-β altered scleral cell phenotype to produce highly contractile, α-SMA-expressing myofibroblasts (TGF-β3 > TGF-β2 > TGF-β1). Exposure of cells to the reduced levels of TGF-β found in the sclera in myopia produced decreased cell-mediated contraction and reduced α-SMA expression. These findings are contrary to the in vivo gene expression data. However, when cells were exposed to both the increased stress and the reduced levels of TGF-β found in myopia, increased α-SMA expression was observed, replicating in vivo findings. These results show that although reduced scleral TGF-β is a major contributor to the extracellular matrix remodeling in the myopic eye, it is the resulting increase in scleral stress that dominates the competing TGF-β effect, inducing increased α-SMA expression and, hence, producing a larger population of contractile cells in the myopic eye.

Metrics of the normal cornea : anterior segment imaging with the Visante OCT

Purpose:  The purpose of the study was to obtain anterior segment biometry for 40 normal eyes and to measure variables that may be useful to design large diameter gas permeable contact lenses that sit outside the region normally viewed by corneal topographers. Also, the distribution of these variables in the normal eye and how well they correlated to each other were determined.

Methods:  This is a cross-sectional study, in which data were collected at a single study visit. Corneal topography and imaging of the anterior segment of the eye were performed using the Orbscan II and Visante OCT. The variables that were collected were horizontal K reading, central corneal/scleral sagittal depth at 15 mm chord, and nasal and temporal angles at the 15 mm chord using the built-in software measurement tools.

Results:  The central horizontal K readings for the 40 eyes were 43 ± 1.73 D (7.85 ± 0.31 mm), with ± 95% confidence interval (CI) of 38.7 (8.7 mm) and 46.6 D (7.24 mm). The mean corneal/scleral sagittal depth at the 15 mm chord was 3.74 ± 0.19 mm and the range was 3.14 to 4.04 mm. The average nasal angle (which was not different from the temporal angle) at the 15 mm chord was 39.32 ± 3.07 degrees and the ± 95%CI was 33.7 and 45.5 degrees. The correlation coefficient comparing the K reading and the corneal/scleral sagittal depth showed the best correlation (0.58, p < 0.001). The corneal/scleral sagittal depth at 15 mm correlated less with the nasal angle (0.44, p = 0.004) and the weakest correlation was for the nasal angle at 15 mm with the horizontal readings (0.32, p = 0.046).

Conclusion:  The Visante OCT is a valuable tool for imaging the anterior segment of the eye. The Visante OCT is especially effective in providing the biometry of the peripheral cornea and sclera and may help in fitting GP lenses with a higher percentage of initial lens success, when the corneal sag and lens sag are better matched.

Expression of muscarinic receptor subtypes in tree shrew ocular tissues and their regulation during the development of myopia

Muscarinic receptors are known to regulate several important physiologic processes in the eye. Antagonists to these receptors such as atropine and pirenzepine are effective at stopping the excessive ocular growth that results in myopia. However, their site of action is unknown. This study details ocular muscarinic subtype expression within a well documented model of eye growth and investigates their expression during early stages of myopia induction. Total RNA was isolated from tree shrew corneal, iris/ciliary body, retinal, choroidal, and scleral tissue samples and was reverse transcribed. Using tree shrew-specific primers to the five muscarinic acetylcholine receptor subtypes (CHRM1-CHRM5), products were amplified using polymerase chain reaction (PCR) and their identity confirmed using automated sequencing. The expression of the receptor proteins (M1-M5) were also explored in the retina, choroid, and sclera using immunohistochemistry. Myopia was induced in the tree shrew for one or five days using monocular deprivation of pattern vision, and the expression of the receptor subtypes was assessed in the retina, choroid, and sclera using real-time PCR. All five muscarinic receptor subtypes were expressed in the iris/ciliary body, retina, choroid, and sclera while gene products corresponding to CHRM1, CHRM3, CHRM4, and CHRM5 were present in the corneal samples. The gene expression data were confirmed by immunohistochemistry with the M1-M5 proteins detected in the retina, choroid, and sclera. After one or five days of myopia development, muscarinic receptor gene expression remained unaltered in the retinal, choroidal, and scleral tissue samples. This study provides a comprehensive profile of muscarinic receptor gene and protein expression in tree shrew ocular tissues with all receptor subtypes found in tissues implicated in the control of eye growth. Despite the efficacy of muscarinic antagonists at inhibiting myopia development, the genes of the muscarinic receptor subtypes are neither regulated early in myopia (before measurable axial elongation) nor after significant structural change.

Biomechanics of the sclera in myopia: Extracellular and cellular factors

Excessive axial elongation of the eye is the principal structural cause of myopia. The increase in eye size results from active remodelling of the sclera, producing a weakened scleral matrix. The present study will detail the biomechanics of the sclera and highlight the matrix and cellular factors important in the control of eye size. Scleral elasticity (load vs. tissue extension) and creep rate (tissue extension vs. time) have been measured postmortem in human eyes. Animal models of myopia have allowed the direct relevance of scleral biomechanics to be investigated during myopia development. Recently, data on tissue matrices incorporating scleral fibroblasts have highlighted the role of cellular contraction in scleral biomechanics. Scleral elasticity is increased in eyes developing myopia, with a reduction in the failure load of the tissue. Scleral creep rate is increased in the sclera from eyes developing myopia, and reduced in eyes recovering from myopia. These changes in biomechanical properties of the sclera occur early in the development of myopia (within 24 h). Alterations in scleral biomechanics during myopia development have been attributed to changes in matrix constituents, principally reduced collagen content. Although the biochemical structure of the sclera plays a critical role in defining the mechanical properties, recent studies investigating the cellular mechanics of the sclera, implicate myofibroblasts in scleral biomechanics. Scleral myofibroblasts have the capacity to contract the matrix and are regulated by tissue stress and growth factors such as transforming growth factor-ß. Changes in these regulatory factors have been observed during myopia development, implicating cellular factors in the resultant weakened sclera. Changes in the biomechanical properties of the sclera are important in facilitating the increase in axial length that results in myopia. Understanding the matrix and cellular factors contributing to the weakened sclera may aid in the development of a clinically appropriate treatment for myopia.

Inhibition of matrix metalloproteinase activity in the chick sclera and its effect on myopia development

To investigate the contribution of matrix degradation in the two-layer avian sclera to the development of myopia. Tissue inhibitor of metalloproteinase-2 (TIMP-2) was used to inhibit chick scleral collagen degradation with (3)H-proline, a marker for this effect. Ex vivo scleral culture experiments confirmed TIMP-2 doses for in vivo experimentation. Ocular growth and refractive response to exogenous TIMP-2 (11.25, 2.25, and 0.45 picomoles, plus vehicle only) were monitored in 7-day-old chicks during the induction of myopia over 4 days with a translucent occluder. Collagen degradation was assessed, in whole sclera and in separated scleral layers by using the same paradigm (11.25 picomoles TIMP-2; vehicle only).Approximately 60% of collagen degradation was inhibited with low (2 nM) doses of TIMP-2 in the ex vivo sclera. Degradative activity in the in vivo chick sclera increased significantly (46%) during myopia development, with all the altered activity confined to the fibrous layer. Addition of TIMP-2 significantly reduced (by 46%) this accelerated scleral collagen degradation, also by acting in the fibrous layer. TIMP-2 had no significant effect on (3)H-proline incorporated in the cartilaginous scleral layer and cornea. Despite inhibiting collagen degradation TIMP-2 had no significant effect on myopia development. Increased collagen degradation is a feature of scleral remodeling in chick myopia development, but is confined to the fibrous scleral layer. Significant inhibition of this collagenolytic activity with TIMP-2 has little effect on refractive error development, suggesting that collagen degradation in the sclera contributes little to the development of myopia in the chick.