Alex Gentle

Role of the sclera in the development and pathological complications of myopia

Myopia is one of the most prevalent ocular conditions and is the result of a mismatch between the power of the eye and axial length of the eye. As a result images of distant objects are brought to a focus in front of the retina resulting in blurred vision. In the vast majority of cases the structural cause of myopia is an excessive axial length of the eye, or more specifically the vitreous chamber depth. In about 2% of the general population, the degree of myopia is above 6 dioptres (D) and is termed high myopia. The prevalence of sight-threatening ocular pathology is markedly increased in eyes with high degrees of myopia ( > -6 D). This results from the excessive axial elongation of the eye which, by necessity, must involve the outer coat of the eye, the sclera. Consequently, high myopia is reported as a leading cause of registered blindness and partial sight. Current theories of refractive development acknowledge the pivotal role of the sclera in the control of eye size and the development of myopia. This review considers the major biochemical mechanisms that underlie the normal development of the mammalian sclera and how the scleral structure influences the rate of eye growth during development. The review will characterise the aberrant mechanisms of scleral remodelling which underlie the development of myopia. In describing these mechanisms we highlight how certain critical events in both the early and later stages of myopia development lead to scleral thinning, the loss of scleral tissue, the weakening of the scleral mechanical properties and, ultimately, to the development of posterior staphyloma. This review aims to build on existing models to illustrate that the prevention of aberrant scleral remodelling must be the goal of any long-term therapy for the amelioration of the permanent vision loss associated with high myopia.

Biomechanics of the Sclera in Myopia: Extracellular and Cellular Factors

Purpose. 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. Methods. 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. Results. 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. Conclusions. 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.

Optical correction of induced axial myopia in the tree shrew: implications for emmetropization.

PURPOSE To determine whether an active emmetropization mechanism is involved in the recovery from axial myopia through the use of a mammalian model of refractive development. Specifically, we sought to establish whether the emmetropization mechanism is visually guided by the level of clarity of the image falling on the retina, or if recovery is driven by a mechanism sensitive to abnormal eye shape. METHODS Young tree shrews had axial myopia induced by monocular deprivation (MD) of pattern vision and then the myopic eye was either: (1) accurately corrected with a negative lens or (2) had a zero-powered lens placed in front of it. Their emmetropization response was monitored, both through the use of ocular refractive and biometric measures, as well as through the assessment of scleral dry weight and glycosaminoglycan synthesis, as indicators of scleral metabolism. RESULTS Corrective lenses prevented recovery from induced myopia (-6.8 +/- 0.7 D after 5 days MD vs. -6.6 +/- 0.6 D after 5 days of lens wear), whereas animals fitted with zero-powered lenses displayed near full recovery from the induced myopia (-6.6 +/- 0.6 D vs. -1.7 +/- 0.3 D). Significant reductions in scleral dry weight (-4.6 +/- 1.3%) and glycosaminoglycan synthesis (-28.6 +/- 7.3%) were found in the posterior sclera of animals wearing corrective lenses. Conversely, animals wearing zero-powered lenses displayed elevated levels of glycosaminoglycan synthesis (+62.3 +/- 11.1%) in conjunction with scleral dry weights that did not differ significantly between treated and fellow control eyes (-1.5 +/- 2.6%). CONCLUSIONS Accurate correction of induced axial myopia prevents the refractive, biometric and scleral metabolic responses that are normally observed in tree shrew eyes recovering from induced myopia. These findings support the hypothesis that recovery is driven by an active emmetropization response dependent on the clarity of image falling on the retina and not by a mechanism that is sensitive to abnormal eye shape.

Retinal and choroidal TGF-β in the tree shrew model of myopia: Isoform expression, activation and effects on function

A visually evoked signalling cascade, which begins in the retina, transverses the choroid, and mediates scleral remodelling, is considered to control eye growth. The ubiquitous cytokine TGF-beta has been associated with alterations in ocular growth, where alterations in scleral TGF-beta isoforms mediate the scleral remodelling that results in myopia. However, while the TGF-beta isoforms have been implicated in the scleral change during myopia development, it is unclear whether alterations in retinal and choroidal isoforms constitute part of the retinoscleral cascade. This study characterised the retinal and choroidal TGF-beta isoform profiles and TGF-beta2 activation during different stages of myopia development, as induced by form deprivation, in a mammalian model of eye growth. Using quantitative real-time PCR, the mRNA for all three mammalian isoforms of TGF-beta was detected in tree shrew retina and choroid. Distinct tissue-specific isoform profiles were observed for the retina (TGF-beta1:TGF-beta2:TGF-beta3=20:2085:1) and choroid (TGF-beta1:TGF-beta2:TGF-beta3=16:23:1), which remained constant over the development period under investigation. The active and latent pools of retinal TGF-beta2 were quantified using ELISA with the majority (>94%) of total TGF-beta2 found in the latent form. Unlike previous scleral data showing early and continuous decreases in TGF-beta isoform expression during myopia development, the levels of the three isoforms remained within normal ranges for retinal (TGF-beta1, -14 to +14%; TGF-beta2, -2 to +20%; TGF-beta3, -10 to +26%) and choroidal (TGF-beta1, -19 to +21%; TGF-beta2, -26 to +8%; TGF-beta3, -11 to +28%) tissues during myopia development (induction times of 3h, 7h, 11h, 24h, and 5 days). A 40% decrease in retinal TGF-beta2 activation was observed after 5 days of myopia development, however, there was no functional correlate of altered TGF-beta2 activity, as assessed by the retinal ERG response. Overall, these data highlight the specific nature of TGF-beta isoform expression, which reflects the differences in tissue structure and function. While TGF-beta isoforms are involved in scleral regulation during myopia development in mammals, they do not have a primary role in the retinal and choroidal signals. Thus, the regulation of eye growth via the retinoscleral cascade involves more than one factor, which is likely to be tissue-specific in nature.

The role of visual information in the control of scleral matrix biology in myopia

Changes in eye size during the development of refractive error are accompanied by alterations in scleral biochemistry in both humans and animal models of myopia. This review discusses more recent data on scleral changes in mammalian models of myopia and considers the role of visual information in the control of scleral matrix biology. These visually-driven changes in scleral biochemistry are placed in the context of both the emmetropisation process and the abnormal enlargement of the eye that is characteristic of human high myopia.

The M4 muscarinic antagonist MT-3 inhibits myopia in chick: evidence for site of action

Citation information: McBrien NA, Arumugam B, Gentle A, Chow A & Sahebjada S. The M4 muscarinic antagonist MT‐3 inhibits myopia in chick: evidence for site of action. Ophthalmic Physiol Opt 2011, 31, 529–539. doi:10.1111/j.1475‐1313.2011.00841.x

Regulation of Scleral Cell Contraction by Transforming Growth Factor-β and Stress : COMPETING ROLES IN MYOPIC EYE 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-beta (TGF-beta). 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, alpha-smooth muscle actin (alpha-SMA). In eyes developing myopia alpha-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-beta and mechanical stress were investigated in scleral cells cultured in three-dimensional collagen gels. All three mammalian isoforms of TGF-beta altered scleral cell phenotype to produce highly contractile, alpha-SMA-expressing myofibroblasts (TGF-beta3>TGF-beta2>TGF-beta1). Exposure of cells to the reduced levels of TGF-beta found in the sclera in myopia produced decreased cell-mediated contraction and reduced alpha-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-beta found in myopia, increased alpha-SMA expression was observed, replicating in vivo findings. These results show that although reduced scleral TGF-beta 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-beta effect, inducing increased alpha-SMA expression and, hence, producing a larger population of contractile cells in the myopic eye.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.

Glycosaminoglycan synthesis in the separate layers of the chick sclera during myopic eye growth: Comparison with mammals

Purpose. Studies in animal models of refractive development have shown that the development of and recovery from induced myopia is associated with visually-guided changes in scleral glycosaminoglycan synthesis. The present study sought to determine whether differential patterns of scleral glycosaminoglycan synthesis are present in the fibrous scleral layer of the chick during myopia development or recovery, as has previously been reported in the mammalian sclera. Methods. Myopia was induced in young chicks by monocular deprivation of pattern vision over 5 days. Other animals underwent monocular deprivation, then had the occluder removed and were allowed 2 days of recovery. A group of age-matched normal animals served as a control. Newly synthesised glycosaminoglycans in the scleral layers were labelled in vivo, using a [ 35 S]-labelled precursor delivered intraperitoneally on the final experimental day. Incorporation of this sulphate into glycosaminoglycans of the fibrous and cartilaginous scleral layers was assessed in proteinase K digests by selective precipitation with alcian blue. Results. Glycosaminoglycan synthesis in the fibrous scleral layers of myopic and recovering eyes was not significantly different to contralateral control eyes (+14 ± 7%, p = 0.09 and -2 ± 4%, p =0.64 respectively). In contrast, glycosaminoglycan synthesis was significantly elevated, relative to controls, in the cartilaginous scleral layer of eyes developing myopia (+63 ± 18%, p < 0.02), whereas in recovering eyes there was found to be a significant decrease in synthesis in the cartilaginous layer (-40 ± 6%, p < 0.001). Conclusions. The results of the current study demonstrate that the fibrous scleral layer of the chick does not display the characteristic differential patterns of glycosaminoglycan synthesis that are found in the mammalian sclera during myopia development and recovery. However, as has previously been reported, the cartilaginous layer of the chick sclera does display differential glycosaminoglycan expression, although the direction of regulation is opposite to that found in the fibrous sclera of mammals.

Inhibition of Matrix Metalloproteinase Activity in the Chick Sclera and Its Effect on Myopia Development

PURPOSE To investigate the contribution of matrix degradation in the two-layer avian sclera to the development of myopia. METHODS 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). RESULTS 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. CONCLUSIONS 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.

Expression and cDNA Sequence of Matrix Metalloproteinase-2 (MMP-2) in a Mammalian Model of Human Disease Processes: Tupaia belangeri

Matrix metalloproteinase-2 (MMP-2) is one of a family of proteolytic enzymes that are involved in the remodelling of tissue during normal growth processes and is capable of degrading structural components of the extracellular matrix. Increases in MMP-2 expression and activity have been reported in diseases that involve degradation of the extracellular matrix. Reported here for the first time are the relative levels of expression of MMP-2 in tissues of the tree shrew along with 2587 bases of the mRNA sequence. Translation of this sequence predicts a protein 660 amino acids in length, containing all of the features expected of mammalian MMP-2. The tree shrew is a species close to the primate line and is an emerging animal model for a variety of human diseases, including hepatitis and myopia that feature MMP-2 mediated remodelling of the extracellular matrix.