September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
The distribution of elastic fibres in keratoconic corneas using en bloc tannic acid and orcein staining
Author Affiliations & Notes
  • Tomas L White
    Optometry and Vision Science, Cardiff Univeristy, CARDIFF, United Kingdom
  • Philip N Lewis
    Optometry and Vision Science, Cardiff Univeristy, CARDIFF, United Kingdom
  • Koji Kitazawa
    Opthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Tsutomu Inatomi
    Opthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Shigeru Kinoshita
    Opthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Keith M Meek
    Optometry and Vision Science, Cardiff Univeristy, CARDIFF, United Kingdom
  • Footnotes
    Commercial Relationships   Tomas White, None; Philip Lewis, None; Koji Kitazawa, None; Tsutomu Inatomi, None; Shigeru Kinoshita, None; Keith Meek, None
  • Footnotes
    Support  MRC
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 2360. doi:
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      Tomas L White, Philip N Lewis, Koji Kitazawa, Tsutomu Inatomi, Shigeru Kinoshita, Keith M Meek; The distribution of elastic fibres in keratoconic corneas using en bloc tannic acid and orcein staining. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2360.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We have recently demonstrated the presence and distribution of elastic fibres in normal human cornea using serial block face scanning electron microscopy (SBF-SEM). These fibres formed sheets at the limbus before becoming long fibres that run transversely and longitudinally, along the plane of the cornea, concentrated in the ‘pre-Descemet’s layer’ (PDL). The aim of the current study was to use SBF-SEM and transmission electron microscopy (TEM) to determine if these findings are consistent, or differ, in human keratoconic corneas.

Methods : Keratoconic corneal buttons were fixed in 4% paraformaldehyde (PFA) following penetrating keratoplasty. Buttons were then dissected, fixed in modified Karnovsky’s fixative and en bloc stained using 2 different protocols. The first involved using multiple staining solutions including osmium tetroxide, tannic acid, uranyl acetate and lead acetate, prior to dehydration and embedding in CY212 resin. The second protocol substituted tannic acid with orcein, with normal cornea and sclera samples being stained as controls. Polymerised blocks were attached to Gatan specimen pins and transferred to a Zeiss Sigma FEG equipped with a Gatan 3View system. Images of the block face surface were acquired every 50 nm by automated serial sectioning. 100nm gold sections were obtained from the same specimen pins and visualised using a Joel1010 TEM.

Results : No fibres could be clearly identified using SEM due to a lack of contrast. However, fibres were clearly visible above Descemet’s membrane in the orcein stained control samples. TEM confirmed the presence of elastic fibres in the control samples and also revealed that there were very few fibres in the PDL region of both central and peripheral keratoconic samples. Many dark stained fibres were seen below the epithelium, in the anterior stroma of the thinned cone region in keratoconus tissue. Furthermore, the central scarred region of cornea contained a distinct mass of staining where numerous amounts of fibres were evident.

Conclusions : Elastic fibre specific en bloc staining using tannic acid and orcein protocol has revealed a lack of fibres in the PDL region in keratoconic corneal buttons and an increased presence of fibres in the anterior stroma, below the epithelium. These findings are directly opposite to those that we have reported in normal cornea and may influence keratoconus morphology and/or pathogenesis.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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