Jody A. Rada

Reduced extracellular matrix in mammalian sclera with induced myopia

The purpose of this study was to learn whether visual form deprivation, which produces myopia in the deprived eye, alters the scleral extracellular matrix in tree shrew, a mammal closely related to primates. Axial myopia was induced in 10 tree shrews by monocular deprivation imposed with a translucent diffuser. The other eye in each animal was an untreated control. After 21 days of deprivation the refractive state and axial component dimensions were measured and the eyes were assayed for levels of DNA, hydroxyproline, and sulfated glycosaminoglycans (GAGs) in samples of the sclera and the cornea. In comparison to the open control eye, the deprived eyes became myopic and elongated. In the sclera, DNA levels were not significantly changed from the control eye. Sulfated GAG levels were significantly lower in the deprived eyes, as compared to the control eyes, at the posterior pole (-15.6%), at the nasal equatorial region (-18.1%), and in the rest of the sclera (-11.6%). The hydroxyproline level was significantly lower only at the posterior pole (-11.8%). Levels of sulfated GAGs were significantly reduced relative to DNA and relative to hydroxyproline in the total sclera. No significant changes were found in the cornea. The lower level of sulfated GAGs throughout the sclera of the deprived eyes, as compared with the control eyes, suggests that the deprived sclera contained less proteoglycan, or that the proteoglycans were less glycosylated or less sulfated. In contrast, the regional reduction of hydroxyproline suggests that collagen accumulation was specifically reduced only at the posterior pole of deprived eyes. These results suggest that form deprivation slows or reverses the normal process of extracellular matrix accumulation in the sclera of this mammal. This may allow the sclera to be more distensible, permitting the vitreous chamber elongation and resultant myopia.

Increased aggrecan (cartilage proteoglycan) production in the sclera of myopic chicks

A previously characterized chick model of myopia was used to evaluate biochemical changes in the sclera which are associated with ocular enlargement and myopia. Chicks were monocularly occluded for 10 days and the DNA, hydroxyproline, and glycosaminoglycan contents of the sclera were compared between the normal and the myopic eyes. No significant differences could be detected in total DNA or hydroxyproline content. There was, however, a 34% increase in glycosaminoglycans and a 20.7% decrease in cell density within the posterior sclera of myopic eyes. The biosynthesis of scleral proteoglycans was determined by measuring 35SO4 incorporation in the sclera of chicks visually occluded for 5, 10, and 15 days. No differences could be detected in 35SO4 incorporation into the cornea or the anterior sclera. However, 35SO4 incorporation was significantly increased in the posterior sclera of myopic eyes by 64% at Day 5, 39% at Day 10, and 49% at Day 15. When fractionated on Sepharose CL-4B, scleral proteoglycans were resolved into two peaks which were identified by Western blot analysis as aggrecan (cartilage proteoglycan) and decorin. Furthermore, Western blot and dot blot analyses indicated that significantly more aggrecan core protein was present in the sclera of myopic eyes compared with equivalent amounts of sclera from control eyes. These results indicate that increased synthesis and accumulation of aggrecan, which increases the volume of extracellular matrix in the posterior sclera, are responsible for the ocular enlargement observed in this model of myopia.

Proteoglycan synthesis by scleral chondrocytes is modulated by a vision dependent mechanism

Proteoglycan synthesis was measured in chick sclera at the onset of form-deprivation myopia, as well as in the period immediately following removal of the occluder. Two day-old chicks were monocularly form vision deprived for periods from one to ten days and proteoglycan synthesis was determined after placing posterior scleral buttons in organ culture and measuring 35SO4 incorporation into glycosaminoglycans. Following 24 hrs of form-deprivation, proteoglycan synthesis was 33% higher in myopic eyes as compared with paired control eyes. The rate of proteoglycan synthesis further increased to levels 83% higher than controls after four days of form-deprivation and remained elevated throughout the ten day period of deprivation. Removal of the occluder after 10 days of form-deprivation resulted in a rapid drop in the rate of proteoglycan synthesis to control levels within 24 hrs. Proteoglycan synthesis was also measured in scleral chondrocytes isolated from control and myopic eyes after 10 days of form-deprivation. Proteoglycan synthesis by chondrocytes from myopic eyes did not return to control levels until 48 hrs after plating. Since the rate of proteoglycan synthesis returns to control levels more quickly during the recovery period ex vivo than when scleral chondrocytes from myopic eyes are placed in cell culture, we suggest that a mechanism is present within the eye which rapidly lowers the rate of proteoglycan synthesis in response to form vision.

Identification of choroidal ovotransferrin as a potential ocular growth regulator

Purpose. In an effort to identify choroidal factors potentially involved in the regulation of ocular growth, proteins released into culture medium of organ-cultured choroids were compared between control eyes and eyes recovering from form deprivation myopia. Methods. The choroids were obtained from the posterior poles of control and recovering chick eyes, and placed into organ culture containing 35 S-methionine/ 35 S-cysteine. Culture medium was collected after 24 hours and proteins were separated and identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), fluorography, immunoprecipitation, western blot analysis and by amino acid sequencing. Choroidal proteins were tested for their effect on scleral proteoglycan synthesis by measuring 35 SO 4 incorporation into scleral glycosaminoglycans (GAG) in vitro. Choroidal thickness and axial elongation were measured in control and recovering eyes using high frequency A-scan ultrasound. Results. The synthesis of an 80 kD protein was greatly increased in the choroids of recovering eyes compared with those of control eyes. Amino acid sequencing and immunoprecipitation indicated that the newly synthesized 80 kD protein was ovotransferrin (transferrin, conalbumin). Ovotransferrin release into the culture medium by isolated recovering choroids was asssociated with a decrease in the rate of axial elongation in recovering eyes. When tested in vitro, ovotransferrin (500 ng/µl) inhibited scleral proteoglycan synthesis in the sclera by 62% in a dose-dependent manner. Conclusions. Chick choroids of recovering eyes synthesize and release ovotransferrin during the recovery from form deprivation myopia. Ovotransferrin significantly inhibited proteoglycan synthesis by the sclera, indicating that ovotransferrin may play a role in slowing the rate of vitreous chamber elongation and facilitating the recovery from induced myopia.

Proteoglycan Gene Families

Publisher Summary Molecular biology technology has radically changed the perception of proteoglycans. cDNAclones to over 16 different proteoglycans has revealed a kaleidoscope of core protein domains. Almost all of the proteoglycans have one or more domains that have binding activity for either matrix components or cell surfaces. This would suggest that the principal function of core proteins is to target a certain number of GAG chains to a specific location in the extracellular matrix or on the cell surface. cDNA clones to core proteins are now just starting to be used in expressing vector systems. This approach has already illustrated the dramatic effect of syndecan expression on cell shape. The expressions of recombinant core proteins will likely lead to a greater understanding of domain functions.

In vivo depth estimation of features in the chick eye by digital signal processing of ultrasonic pulse echoes

An existing biological instrumentation system which measures the depth of tissue boundaries in the chick eye has been modified to use National Instruments LabVIEW as a graphical interface for the operator. This system is used in a study to assess the effectiveness of pharmaceuticals designed to slow the progression of myopia, in which a high-speed data acquisition card digitizes the output of an ultrasound sensor to provide in vivo depth measurements of the eyeball structure. LabVIEW not only provides a real-time visualization of the ultrasonic scan, but it also allows for the interactive assessment of axial length as well as the creation of archival databases. A LabVIEW implementation of a matched filter with an echo-shaped impulse response applied to a simulated noisy ultrasound signal produces distinct peaks corresponding to the structural interfaces, suggesting a possible approach to automated peak detection.