Yonago Acta medica 2004;47:29–35

A Scanning Electron Microscopic Study of the Basal Surface of the Corneal Endothelium and the Stromal and Endothelial Surfaces of Descemet’s Membrane in Rats
Keiko Sasaki, Akihiko Tamai* and Takao Inoué†
Division of Ophthalmology and Visual Science, Department of Medicine of Sensory and Motor
Organs and †Division of Morphological Analysis, Department of Functional, Morphological andRegulatory Science, School of Medicine, Tottori University Faculty of Medicine, Yonago 6838504 and *Clinic of Ophthalmology, Hino Hospital Association Hino Hospital, Hino 689-4504
JapanThe basal surface of the corneal endothelium and the stromal and endothelial surfaces ofDescemet’s membrane in rats were studied by scanning electron microscopy. We compared
the fine structures of the two surfaces of Descemet’s membrane both after sputter-coatingwith platinum and without sputter-coating. Fine structures were made clearly visible
without metal coating by heating specimens to 300˚C during observation. After sputtercoating, both surfaces of Descemet’s membrane were composed of granular substances,
but their sizes were larger on the stromal side than on the endothelial side. Both surfacesof Descemet’s membrane observed without sputter-coating were composed of fine fibrous
structures showing a felt-like appearance, but their diameters were thicker on the stromal
surface. These results may reflect a difference in collagen types between the two surfacesof Descemet’s membrane.
Key words: corneal endothelium; Descemet’s membrane; non-coating observation method; sputtercoating with platinum

Scanning electron microscopy (SEM) is a useful method for studying three-dimensional structures of tissues and cells. Since Blümcke and Morgenroth(1967) observed the three-dimensional structure of
the corneal endothelium of normal adult rabbits,
many researchers have studied the three-dimensionalstructure in normal or pathological corneas by SEM(Svedbregh and Bill, 1972; Sugita, 1976; Doughmanet al., 1976; Melamed et al., 1980; MacCallum et al.,
1983; Yamasaki and Inoué, 2001). However, theseobservations were restricted to the free surface ofthe endothelium. Descemet’s membrane is locatedbetween the endothelium and stroma in the cornea.
Since the membrane is firmly connected to theendothelium, it is difficult to observe the basal surface of the endothelium and the endothelial surfaceof the membrane by SEM. Thus, up until now, thethree-dimensional architecture of the cornea could
only be speculated from thin section images bytransmission electron microscopy (TEM) (Hogan etal, 1971; Komai et al., 1990).
In this study, we tried to demonstrate the corneal surfaces in rats by the mechanical separation/
SEM method instead of the previous cell-maceration/SEM method (Komai et al., 1990). In addition, we observed three-dimensional ultrastructures of both the endothelial and stromal sides ofDescemet’s membrane by the exfoliating method

Abbreviations: SEM, scanning electron microscopy; TEM, transmission electron microscopy

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using surface tension (Inoué et al., 1984). Furthermore, we compared the ultrastructures ofDescemet’s membrane with or without metal coating (Osatake and Inoué, 1998). We report heresome new findings in the ultrastructures of the basal
surface of the endothelium as well as the stromaland endothelial surfaces of Descemet’s membrane.

SEM preparation for observing the basal
surface of the corneal endothelium
After washing in 0.1 M phosphate buffer (pH 7.2,210 mOsm/kg), fixed corneas were separated
mechanically using a 26-gauge needle; the endothelial surface in each material was picked andscratched prudently by the fine needle. When theendothelial surface was finally detached from
Descemet’s membrane and turned over, we could

Materials and Methods

Animals
Adult Wistar rats of both sexes, weighing 170 to270 g, were used in this study. All experiments
conformed to the Association for Research in Vision and Ophthalmology Resolution on the HumanUse of Animals in Research, and the Guidelines for
Animal Experiments in the Tottori University Faculty of Medicine. Under sodium pentobarbital (40to 50 mg/kg body weight) anesthesia administeredintraperitoneally, the animals were perfused with amixture of 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2, 160mOsm/kg) through the left ventricle. The eyeballs
were then enucleated, and each cornea was cut outat the limbus. The corneas were further immersedin the same fixative for additional 3 to 5 days.

Transmission electron microscopy (TEM)
The corneas were cut into small pieces and rinsed
overnight in a buffer containing 0.2 M sucrose (pH7.2, 430 mOsm/kg) followed by postfixation with
1% osmium tetroxide for 1 h. After block-staining
in 1% uranyl acetate, they were dehydrated in a
graded ethanol series and embedded in Epon
through propylene oxide. Thin sections were obtained using a diamond knife and an ultramicrotome (Utracut UCT, Leica, Wien, Austria), andwere examined with a transmission electron microscope (100 CX II, JEDL Ltd., Tokyo, Japan) operated at 80 kV after staining for connective tissues(Kajikawa et al., 1975).

Fig. 1. TEM image of the cross-section of the rat corneal
endothelium (En), Descemet’s membrane (DM) andstroma (ST). The endothelial surface of Descemet’smembrane has an irregular profile (arrowheads), reflecting the basal contour of the endothelium. Microfibrils(small arrow) are seen on the stromal side of Descemet’smembrane, while they are not visible on the endothelialside. Pinocytotic vesicles (large arrow) are visible beneath the basal cell membrane of the endothelium. Bar =1 µm. AC, anterior chamber. Inset: Higher magnification of the basal surface of the endothelium. Note pinocytic vesicles (arrows). Bar = 0.5 µm.

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SEM of corneal endothelium and Descemet’s membraneobserve the basal surface of the endothelium. Thesespecimens were then thoroughly washed in the buffer, postfixed in 1% osmium tetroxide for 1 h andconductive-stained with 2% tannic acid and 1% osmium tetroxide (Murakami, 1974). After dehydration through a graded ethanol series, they were
finally freeze-dried by t-butyl alcohol (Inoué andOsatake, 1988). The dried specimens were observed with a scanning electron microscope (HFS-2ST,Hitachi Ltd., Tokyo) operated at 25 kV after sputter-coating with platinum.

phate buffer for 12 h at room temperature. After abrief rinse in distilled water, they were dehydrated
in a graded ethanol series up to a concentration of100%. The dehydrated specimens were then throwninto distilled water. The specimens moved about on
the surface and eventually sank. At this point, someparts of the endothelium and Descemet’s membrane
were separated by the surface tension produced
between the water and the ethanol. The specimens
were osmicated again with 1% osmium tetroxide in
the same buffer for 20 min, rinsed with the buffer,and conductive-stained with a 2% tannic acid and
1% osmium tetroxide. After dehydration through a
graded ethanol series again, they were finallyfreeze-dried by t-butyl alcohol. The dried specimens were observed with a Hitachi scanning electron microscope (HFS-2ST) operated at 25 kV aftersputter-coating with platinum. Some specimens
were observed without sputter-coating using a heating stage to 300˚C of a high resolution scanningmicroscope (UHS-T1, Hitachi Ltd.) operated at 25
kV (Osatake and Inoué, 1998).

SEM preparation for observing Descemet’s
membrane
The two surfaces of Descemet’s membrane wereexposed by the exfoliating method using surface
tension (Inoué et al., 1984). The corneas were fixedby immersion with 1% osmium tetroxide in 0.1 M
phosphate buffer (pH 7.2, 210 mOsm/kg) for 2 h atroom temperature. The specimens were further
treated with 0.1% osmium tetroxide in 0.1 M phos-Fig. 2. SEM image of the mechanically-fractured cornea showing surfaces of the endothelium (En), Descemet’smembrane (DM) and stroma (ST). Bar = 10 µm.

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