early Þbroblasts contain many free ribosomes and polyribosomes, as well as immature roughsurfaced endoplasmic reticulum and Golgi complex. The intercellular space is Þlled with patches of immature collagen with an average diameter of 27Ð29 nm (range, 22Ð35 nm) without banding. There are no elastin deposits.

1.2.1.7 Seventh Week

By week 7.2, the cells of the anterior portion have more developed rough-surfaced endoplasmic reticulum and Golgi complex and have fewer ribosomes and polyribosomes. The intercellular space in the outer portion is wider than in the inner portion; the number and average diameter (30Ð40 nm; range, 24Ð46 nm) of collagen Þbrils have increased in both locations in comparison with week 6.4. Immature elastin deposits consisting of microÞbrillar components can be recognized for the Þrst time. The cells of the posterior portion have more ribosomes and polyribosomes and less mature rough-surfaced endoplasmic reticulum and Golgi complex than do the cells of the anterior portion. The intercellular space in the posterior portion reveals fewer collagen Þbrils than does the intercellular space in the anterior portion. There are no elastin deposits.

1.2.1.8 Ninth Week

By week 9.7, the cells of the inner anterior portion exhibit more glycogen granules and more elastin deposits than do the cells of the outer anterior portion. The number and average diameter (50Ð58 nm; range, 42Ð68 nm) of collagen Þbrils have increased in both locations in comparison with week 7.2. The cells of the posterior portion have more cytoplasmic processes and rough-surfaced endoplasmic reticulum, and the intercellular spaces have more collagen Þbrils and elastin deposits in comparison with week 7.2.

1.2.1.9 Tenth Week

By week 10.9, the cells of the anterior portion have more developed rough-surfaced endoplasmic reticulum and Golgi complex and fewer

glycogen granules than they had in week 9.7. The intercellular space has more collagen Þbrils and elastin deposits in comparison with week 9.7. By this stage, the only difference between anterior and posterior portions is that the posterior area has fewer collagen Þbrils than the anterior area.

1.2.1.10 Thirteenth Week

By week 13, the cells again exhibit more developed rough-surfaced endoplasmic reticulum and Golgi complex and fewer glycogen granules than do cells in week 10.9. Because collagen and elastin have increased in comparison with week 10.9, the volume ratio between cells and intercellular material is about 1:1. The average diameter of collagen Þbrils in this stage is 62Ð74 nm (range, 50Ð84 nm).

1.2.1.11 Sixteenth Week

By week 16, the diameter of the collagen Þbrils has increased in comparison with week 13. Our own transmission electron microscopy studies on human fetal and adult sclera showed that there are still differences between week 16 (Fig. 1.4a) and adult sclera (Fig. 1.4b); fetal sclera did not show the packed and dense, intermingled arrangements of collagen bundles with few Þbroblasts of the adult sclera. Other characteristics of this stage are further enlargement of the rough-surfaced endoplasmic reticulum, complete loss of glycogen granules, and more elastin deposits.

1.2.1.12 Twenty-Fourth Week

By week 24, the sclera has the same ultrastructural characteristics as the adult sclera. The average diameter of the collagen Þbrils is 94Ð102 nm (range, 84Ð102 nm), which is the average diameter of adult sclera. Mature elastin deposits exhibiting electron-translucent central cores also can be found.

Defects in synthesis of extracellular matrix components during scleral development at any of these stages may account for scleral abnormalities, including Marfan syndrome, osteogenesis imperfecta, pseudoxanthoma elasticum, EhlersÐDanlos syndrome, congenital myopia, and nanophthalmos.

Fig. 1.4 (a and b) Transmission electron microscopy photomicrographs (×4,000) of human fetal sclera, week 16 of development. Note the highly cellular nature of the fetal

sclera (a) compared to the adult sclera (b) and the densely packed, intermingled arrangement of collagen bundles in the adult sclera (b) compared to the 16-week fetal sclera (a)

Fig. 1.5 Immunoßuorescence microscopy (using anticollagen type IV antibody) of week 13 fetal sclera. The relatively abundant amount of collagen type IV seen in this

13-week fetal specimen steadily decreased throughout fetal development and was nearly absent in adult sclera, with the exception of its presence in the vascular walls

Our own studies on immunolocalization of extracellular matrix components in human fetal and adult scleral specimens contribute to the understanding of scleral developmental events. The indirect immunoßuorescence technique was performed on anterior and posterior portions of human fetal (13Ð22 weeks of gestation) and adult (52Ð73 years of age) sclera using monoclonal antibodies against the collagens (types IÐVII), the proteoglycans (heparan sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronic acid), and the basement membrane glycoproteins (Þbronectin, vitronectin, and laminin). No marked differences in extracellular matrix component staining were found between the anterior and posterior portions of the sclera by week 16 and onward.

1.2.2.1 Collagens

Immunoßuorescence studies localized collagen types I, III, IV, V, and VI in human fetal and adult scleral specimens; collagen types II and VII were not present. Collagen type I staining increased steadily from fetus to adult; the diffuse pattern

week 19, and an extensive network of positive Þbrils was evident by week 22, culminating in a diffuse presence in adult sclera. Collagen type III was moderately abundant, diffuse, and showed little change from 13 weeks to 73 years. Collagen type IV showed a pattern of granular positivity following individual Þbrils at 13 weeks, decreasing steadily through 16, 19, and 22 weeks (Fig. 1.5). Adult sclera revealed only subtle type IV positivity, except for its dramatic presence in the blood vessels. Collagen type V staining was present in diffuse, moderate amounts in fetal scleral tissue; adult sclera showed a Þne granular pattern along the edges of the collagen bundles. The amount of collagen type VI increased from fetal to adult scleral tissue, and the pattern became more striated in appearance.

Studies on tissue distribution of collagen type VIII in anterior and posterior human fetal (16Ð27 weeks) sclera showed an abundance in posterior fetal sclera; it gradually decreased and eventually disappeared in equatorial fetal sclera. Collagen type VIII showed a linear or Þbrous pattern in anterior and posterior human adult sclera [11].

Studies on tissue distribution of collagen type XII at the corneoscleral angle of the embryonic avian eye showed positive staining around the scleral ossicles and the scleral cartilages [12].

1.2.2.2 Proteoglycans

Proteoglycans are macromolecular core proteins covalently attached to at least one sulfated glycosaminoglycan (GAG) side chain. The GAG chain consists of a hexosamine (D-galactosamine or D-glucosamine) and galactose units (keratan sulfate) or a hexuronic acid (L-iduronic acid or D-glucuronic acid). Chondroitin sulfate and dermatan sulfate are galactosaminoglycans and heparan sulfate is a GAG. GAGs are not homogeneous even within a given GAG because of the variability in sulfate substitutions and chain lengths.

We analyzed the GAG dermatan sulfate, chondroitin sulfate, hyaluronic acid, and heparan sulfate in human fetal and adult scleral specimens. Dermatan sulfate was present in moderate amounts through the different gestational periods, and chondroitin sulfate always stained intensely without much difference between 13-week and adult sclera. In contrast, staining of hyaluronic acid changed from moderate at 13 weeks to mild at 22 weeks; this then subsided,

and adult sclera showed only a subtle positivity. Heparan sulfate was identiÞed in human fetal and adult sclera in small amounts.

1.2.2.3 Glycoproteins

Fibronectin, vitronectin, and laminin were identiÞed in human fetal and adult scleral specimens. Fibronectin staining changed from an intense reticular pattern at 13 weeks to a dramatic Þbrillar pattern at 16 weeks; staining then decreased at 22 weeks and was subtle in adult sclera (Fig. 1.6). These Þndings indicate that, as in other organs during embryogenesis, Þbronectin changes may play a major role in directing developmental events. Like Þbronectin, vitronectin staining also decreased from fetal to adult scleral tissue. Laminin staining was subtle at 13 weeks and disappeared at 16 weeks and onward, except for its dramatic presence in the blood vessels.

1.2.3Postnatal Development and Age-Related Changes

In infancy and childhood, scleral tissue is more translucent than in an adult, and a bluish color is evident, owing to visibility of the underlying