All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Nine adult mice, 6 to 8 weeks of age (three 129/SVJ, three C57BL/6 and three BALB/c) were euthanatized, and a few drops of 2% glutaralde-hyde in 80 mM sodium cacodylate buffer, 330 mOsm/kg fixative11 (link) were immediately applied to the cornea after resection of the eye lids. The whole eyes were carefully enucleated and immersed in fixative for 4 to 6 hours, to ensure proper cross-linking and preservation of the tissue.
After fixation, corneal diameter measurements were obtained on one eye per animal under a dissecting microscope (SZ60; Olympus) for increased magnification. A pair of digital calipers with sensitivity down to 0.1 mm was used to obtain measurements from all eyes. The corneal diameter was measured from limbus to limbus by using the easily visualized (at 25× magnification) corneoscleral junction to define the limit of the cornea. The corneal limbus-to-sclera transitional zone is very narrow, at approximately 0.1 mm and was for this reason not considered separately. The mouse cornea was found to be circular, not oval like the human cornea, and, therefore, the measurements do not differ in the horizontal and vertical meridians. All three measurements on the same cornea were obtained along the same meridian, and the corneal diameter measurements were performed on fixed eyes for consistency with the other measurements. The tissue-processing protocol used in the present study has shown that a fixative maintaining near physiological osmolality produces minimal tissue shrinkage, with the result that the undistorted, natural contour of the tissue is preserved.11 (link) The average corneal radius (diameter/2) was used to identify the location where the central corneal measurements were made.
The fixed corneas were bisected, and small pieces (1 × 1.5 mm) were cut from the central region. The corneal pieces were washed three times in sodium cacodylate buffer (pH 7.4) at room temperature and left for 10 minutes in each wash. Subsequently, the samples were immersed in a freshly prepared 1% solution of osmium tetroxide in 100 mM sodium cacodylate buffer for 1 hour under dim light. The samples were once again washed several times in sodium cacodylate buffer and left 10 minutes in each wash. A tissue processor (EM TP; Leica; Wetzlar, Germany) was used for the following steps: dehydration, transition, infiltration, and embedding. First, the tissue samples were dehydrated through a graded alcohol series (30%–100% in six steps) at room temperature. Next the tissue samples were infiltrated with propylene oxide. Embedding with agitation was achieved through an initial mixture of propylene oxide and Araldite resin 2:1 for 3 hours, followed by overnight immersion in a 1:1 mixture of propylene oxide and Araldite resin. Thereafter, the tissue samples were immersed in propylene oxide and Araldite resin 1:3 for 4 to 8 hours before final transfer to 100% Araldite resin overnight. The tissue samples were then oriented in embedding molds and left 12 hours for polymerization in an oven at 60°C.
An ultramicrotome (MT-7000; Research Manufacturing Co. Inc., Tucson, AZ) was used to cut thick transverse sections (0.5–1 μm). These sections were stained with 1% toluidine blue for examination with a light microscope (BX51; Olympus). For morphologic analysis, ultrathin sections were obtained and mounted on parallel bar copper grids (200MP, cat no. G200P; Electron Microscopy Sciences, Fort Washington, PA). The sections were double stained, first, in 3.5% uranyl acetate for 20 minutes at 60°C, followed by Reynold's lead citrate for 10 minutes at room temperature. The grids were examined in a transmission electron microscope (Tecnai G2 Bio Twin Spirit; FEI Co., Eindhoven, The Netherlands) and the images captured digitally.
Digital images were captured at 40× and 200× of two thick toluidine-stained corneal sections, cut from two levels of the same block separated by approximately 200 μm. Peripheral measurements were taken at the extremity of the cornea, defined histologically as immediately central (anterior) to limbal capillaries and central to the anterior edge of the trabecular meshes (Fig. 1A ).
Measurements for the central cornea were taken at a distance, half the corneal diameter ±14% from the peripheral measurements where the cornea had its natural contour, had taken up stain in a uniform manner, and was free of artifacts. All measurements were made with NIH Image Software (Wayne Rasband, National Institutes of Health, Bethesda, MD; available athttp://rsb.info.nih.gov/ij/index.html ) in trip-licate, at the same location centrally and peripherally.
The layers of cells forming the epithelium were counted in electron micrographs taken from both the central and peripheral regions. The cells were counted in a straight line from the basement membrane to the corneal surface and did not have to display a nucleus in the plane of the section.
An unpaired Student's t-test was used to compare the two corneal sections cut from two levels of the same block but separated by approximately 200 μm, to ensure no difference throughout the tissue sample. An unpaired Student's t-test was also used to compare the average central with the average peripheral corneal measurements in three mice within the same strain. The statistical significance was set at P < 0.05.
After fixation, corneal diameter measurements were obtained on one eye per animal under a dissecting microscope (SZ60; Olympus) for increased magnification. A pair of digital calipers with sensitivity down to 0.1 mm was used to obtain measurements from all eyes. The corneal diameter was measured from limbus to limbus by using the easily visualized (at 25× magnification) corneoscleral junction to define the limit of the cornea. The corneal limbus-to-sclera transitional zone is very narrow, at approximately 0.1 mm and was for this reason not considered separately. The mouse cornea was found to be circular, not oval like the human cornea, and, therefore, the measurements do not differ in the horizontal and vertical meridians. All three measurements on the same cornea were obtained along the same meridian, and the corneal diameter measurements were performed on fixed eyes for consistency with the other measurements. The tissue-processing protocol used in the present study has shown that a fixative maintaining near physiological osmolality produces minimal tissue shrinkage, with the result that the undistorted, natural contour of the tissue is preserved.11 (link) The average corneal radius (diameter/2) was used to identify the location where the central corneal measurements were made.
The fixed corneas were bisected, and small pieces (1 × 1.5 mm) were cut from the central region. The corneal pieces were washed three times in sodium cacodylate buffer (pH 7.4) at room temperature and left for 10 minutes in each wash. Subsequently, the samples were immersed in a freshly prepared 1% solution of osmium tetroxide in 100 mM sodium cacodylate buffer for 1 hour under dim light. The samples were once again washed several times in sodium cacodylate buffer and left 10 minutes in each wash. A tissue processor (EM TP; Leica; Wetzlar, Germany) was used for the following steps: dehydration, transition, infiltration, and embedding. First, the tissue samples were dehydrated through a graded alcohol series (30%–100% in six steps) at room temperature. Next the tissue samples were infiltrated with propylene oxide. Embedding with agitation was achieved through an initial mixture of propylene oxide and Araldite resin 2:1 for 3 hours, followed by overnight immersion in a 1:1 mixture of propylene oxide and Araldite resin. Thereafter, the tissue samples were immersed in propylene oxide and Araldite resin 1:3 for 4 to 8 hours before final transfer to 100% Araldite resin overnight. The tissue samples were then oriented in embedding molds and left 12 hours for polymerization in an oven at 60°C.
An ultramicrotome (MT-7000; Research Manufacturing Co. Inc., Tucson, AZ) was used to cut thick transverse sections (0.5–1 μm). These sections were stained with 1% toluidine blue for examination with a light microscope (BX51; Olympus). For morphologic analysis, ultrathin sections were obtained and mounted on parallel bar copper grids (200MP, cat no. G200P; Electron Microscopy Sciences, Fort Washington, PA). The sections were double stained, first, in 3.5% uranyl acetate for 20 minutes at 60°C, followed by Reynold's lead citrate for 10 minutes at room temperature. The grids were examined in a transmission electron microscope (Tecnai G2 Bio Twin Spirit; FEI Co., Eindhoven, The Netherlands) and the images captured digitally.
Digital images were captured at 40× and 200× of two thick toluidine-stained corneal sections, cut from two levels of the same block separated by approximately 200 μm. Peripheral measurements were taken at the extremity of the cornea, defined histologically as immediately central (anterior) to limbal capillaries and central to the anterior edge of the trabecular meshes (
Measurements for the central cornea were taken at a distance, half the corneal diameter ±14% from the peripheral measurements where the cornea had its natural contour, had taken up stain in a uniform manner, and was free of artifacts. All measurements were made with NIH Image Software (Wayne Rasband, National Institutes of Health, Bethesda, MD; available at
The layers of cells forming the epithelium were counted in electron micrographs taken from both the central and peripheral regions. The cells were counted in a straight line from the basement membrane to the corneal surface and did not have to display a nucleus in the plane of the section.
An unpaired Student's t-test was used to compare the two corneal sections cut from two levels of the same block but separated by approximately 200 μm, to ensure no difference throughout the tissue sample. An unpaired Student's t-test was also used to compare the average central with the average peripheral corneal measurements in three mice within the same strain. The statistical significance was set at P < 0.05.
