title: Surface ultrastructure of collagen fibrils and their association with proteoglycans in human cornea and sclera by atomic force microscopy and energy-filtering transmission electron microscopy.
abstract: OBJECTIVE
  We aimed to investigate the possible association of proteoglycans with D-periodic collagen fibrils in the human cornea and sclera, using energy-filtering transmission electron microscopy (EF-TEM) and atomic force microscopy (AFM).
  
  
  METHODS
  Human cornea and sclera were digested with keratanase to eliminate keratan sulfate proteoglycans (KSPGs). For EF-TEM observation, surface proteoglycans were detected by cupromeronic blue (CB) staining. For AFM observation, cornea and sclera were treated with sodium hydroxide before and after keratanase digestion, and the surface topology of collagen fibrils was analyzed.
  
  
  RESULTS
  With CB staining, numerous CB-positive short filaments of surface proteoglycans (proteoglycan filaments) were observed in the interfibrillar spaces of cornea and sclera associated with collagen fibrils. AFM imaging showed that the depth and periodicity of D-periodic collagen fibrils in keratanase-treated corneal collagens were deeper and more regular than in untreated ones. Moreover, the depth and periodicity of keratanase-untreated corneal collagens were shallow and irregular in comparison with keratanase-untreated scleral collagens. On the other hand, there was no difference in depth or regularity between keratanase-treated and -untreated scleral collagen fibrils. Using AFM imaging, additional thin grooves sub-bands were detected on the surface of keratanase-treated corneal collagen fibrils. The grooves were not detected in keratanase-untreated collagen fibrils nor in scleral collagen fibrils with or without keratanase digestion. Comparing densitometry waves, the grooves of D-periodic corneal collagen sub-bands corresponded to a and c bands.
  
  
  CONCLUSIONS
  Using AFM and EF-TEM to study corneal and scleral collagen fibrils and their association with proteoglycans, we conclude that KSPG is found in ample amounts in the human cornea in comparison with sclera. Moreover, we topologically detected KSPG attached to a and c bands of collagen fibrils.
authors.affiliation: Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan. amiyagawa@hotmail.com
authors.forename: A
authors.initials: A
authors.lastname: Miyagawa
authors.forename: M
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Y
authors.initials: Y
authors.lastname: Fujita
authors.forename: O
authors.initials: O
authors.lastname: Hamdy
authors.forename: K
authors.initials: K
authors.lastname: Hirano
authors.forename: M
authors.initials: M
authors.lastname: Nakamura
authors.forename: Y
authors.initials: Y
authors.lastname: Miyake
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D011508:Chondroitin Sulfate Proteoglycans / Q000648:ultrastructure*; D003094:Collagen / Q000648:ultrastructure*; D003315:Cornea / Q000648:ultrastructure*; D006801:Humans; D007202:Indicators and Reagents; D007211:Indoles; D007632:Keratan Sulfate / Q000648:ultrastructure*; D000071234:Lumican; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D008875:Middle Aged; D009942:Organometallic Compounds; D012590:Sclera / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D011508:Chondroitin Sulfate Proteoglycans; D007202:Indicators and Reagents; D007211:Indoles; C000604306:LUM protein, human; D000071234:Lumican; D009942:Organometallic Compounds; C015445:copper phthalocyanine; D003094:Collagen; D007632:Keratan Sulfate