Clinically normal corneas were collected from two adult female sheep immediately after slaughter at an abattoir in Fielding, New Zealand. Also, corneas normal on ophthalmological examination were obtained from an adult male cat and a female cat presented for necropsy at the Pathology Department of Massy University School of Veterinary Medicine, Palmerston North, New Zealand. The cats had been humanely euthanized for reasons unrelated to the current study. The central areas of the cats’ and sheep corneas where there is the most uniform collagen fibril arrangement(2, 22) were divided to provide samples for the following treatments which were carried out in duplicate. As freezing has no effect on X-ray scattering patterns(15), the duplicate normal control samples consisted of two cat and two sheep samples which were immediately wrapped in cling wrap and frozen at -80°C. The remaining duplicate samples were immersed in either 2 mL of 5% glutaraldehyde, 10% formalin, Triton X or 0.9% saline. Four days later the corneal samples were tested for transparency (below), analysed by SAXS (below), and fixed in Karnovsy’s fixative for ultrastructural evaluation by TEM as described below.
5% Glutaraldehyde was formulated from 40mL 25% glutaraldehyde, 50mL 0.2M cacodylate buffer and 80 mL distilled water.
10% Formalin was formulated by mixing 100mL 37-40% formaldehyde, 900 mL distilled water, monosodium phosphate (4.0 g) and anhydrous disodium phosphate (6.5 g).
Triton X was formulated from 20 mM tris-aminomethane with 1 mM ethylenediaminetetraacetic acid, 1.25 mL 10% Triton X and 1.25 mL sodium deoxycholate.
0.9% saline was prepared by dissolving 4.5 g sodium chloride in 500 mL deionized water before heat sterilization.
Karnovsky’s fixative was made up using 2.0 g of paraformaldehyde, 5.0 mL of 50% glutaraldehyde, and 20.0 mL of 0.2M cacodylate buffer before the pH was adjusted to 7.4 with 1 M sodium hydroxide.
The effect of the preservatives on corneal transparency was evaluated subjectively by observing a 4 mm by 4 mm cross (1-point black line) through the corneal samples (Figure 1).
Analyses were performed using Minitab 17 Statistical Software (2010) (State College, PA: Minitab, Inc,; www.minitab.com) for the 2-sample t-test for unequal variance. A P < 0.05 was regarded as significant.
Small Angle X-ray Scattering
The SAXS/WAXS beamline at the Australian Synchrotron in Melbourne, Australia was used to examine the collagen structure of the corneas. The samples were mounted flat-on to the X-ray beam (along their optical axis from anterior to posterior) to perform surface diffraction measurements in which nine diffraction patterns were recorded using a three by three grid with 0.25 mm spacing between points. A high-intensity undulator source was utilized with an energy resolution of 10-4 from a cryo-cooled Si (111) double-crystal monochrometer. The beam size full width half maximum (FWHM) focused at the sample was 250 x 80 µm with a total photon flux of approximately 2 x 1012 photons s-1. All diffraction patterns were calibrated with silver behenate and recorded with an X-ray energy of 12 keV using a Pilatus 1M detector with an active area of 170 x 170 mm and a sample detector distance of 3337 mm. The exposure time for diffraction patterns was in the range of 1 – 5 seconds. Data processing was carried out using the ScatterBrain software to determine collagen fibril diameters and D-spacing.
After background subtraction, Gaussian approximations were used to determine the peak position of the maxima of the fifth order diffraction peak from the intensity versus q plot (Figure 2) where q is defined as the magnitude of the scattering vector. The D-spacing was calculated by comparing diffraction peak positions with the calibrant to determine q-values.
Fibril Diameter and Fibril Diameter Distribution
Fibril diameters were determined from the intensity versus q plot over the full q-range (0.01 Å-1 - 0.1 Å-1) (Figure 2) by expressing the scattered intensities in terms of volume fraction distribution of scatters. Applying the total non-negative least squares model and the ‘cylinder AR’ model using “Irena” , a macro developed for analysing SAXS data(23) and more specifically particle size distributions in SAXS data running in a data analysis tool (Igor Pro, Wavemetrics, Lake Oswego, OR, USA), the fibril diameter spread within a sample was determined from the scatter intensity patterns, under the assumption that fibrils have a cylindrical shape.
Transmission Electron Microscopy
The corneas in Karnovsky’s fixative were trimmed, washed three times in phosphate buffered saline (0.1 M, pH 7.2) and post-fixed in osmium tetroxide (0.1 M) for one hour. After two additional washes in water, the samples were dehydrated through a graded ethanol series (from 50% to 100% in increments of 10) and embedded in epoxy resin (TAAB 812 resin kit, England). Ultra-thin sections (70 to 90 nm) were cut with a Leica EM UC7 ultra-microtome (Leica EM UC7, Leica, Germany) and mounted on copper grids. Sections were stained with uranyl acetate and lead citrate before being viewed in a Philips CM10 transmission electron microscope (Philips Electron Optical Division, Eindhoven, The Netherlands) operated at 80kV. Images were digitally recorded using the Olympus iTEM Soft Imaging System.
Transmission Electron Microscope Image Processing
A Graphical User Interface (GUI), written in C++ using the Qt framework and the Open Computer Vision library, was used to detect and measure the collagen fibrils in end-on TEM images of cat and sheep corneas fixated in various preservatives. The GUI allowed the user to compute an image’s pixel-to-nanometre scale factor and use an algorithm to detect the fibrils’ contours. Delaunay triangulation was used on the detected contours to generate a Voronoi diagram, which distinguished one fibril from another. The GUI computed each fibril’s diameter and distance between its nearest neighbours.