Yonago Acta medica 2001;44:17–24

Ultrastructural Changes in the Rat Corneal Endothelium Preserved atLow Temperature
Kaori Yamasaki*† and Takao Inoué**Second Department of Anatomy and †Department of Ophthalmology, Faculty of Medicine,
Tottori University, Yonago 683-0826, JapanThe ultrastructural differences in rat corneal endothelia preserved at 0˚C and 4˚C wereexamined by scanning electron microscopy (SEM) and transmission electron microscopy(TEM). Some corneas were preserved as whole globes in an eye preservation medium(EP-II) either at 4˚C or at 0˚C for 1, 2 and 7 days. The specimens were prepared for
SEM and TEM according to routine methods. For all storage times, the endothelium
preserved at 0˚C showed less irregularity of the cell surface, swollen mitochondria orvacuoles than that at 4˚C. A significant difference in structural changes between theendothelium preserved at 4˚C and 0˚C was seen after 7 days’ storage. The former
showed destruction of the surface cell membrane and degenerated cell organella, while
the latter maintained the structure of the surface cell membrane and cell organella.
These findings suggest that the lifetime of a rat endothelium preserved at 0˚C is longer
than that at 4˚C.
Key words: corneal endothelium; low temperature preservation (0˚C); scanning electron microscopy;transmission electron microscopy

freezing temperature of other organs (Wicomband Cooper, 1984; Storey and Storey, 1990;
Yoshida et al., 1999), it is obvious that the cornea does not freeze at 0˚C. Accordingly, we
tried to preserve rat corneas at 0˚C and examined the ultrastructural changes in the endothelium.
Today, most eye banks in the United States
store isolated corneas at 4˚C (Bourne, 1991),
but in Japan there are still many eye banks
which preserve corneas as whole globes in astorage medium (EP-II) (Manabe et al., 1984)
at 4˚C and their storage periods are limited towithin 48 h.
In this study, we tried to preserve rat corneas
as whole globes with storage medium at 0˚C toprolong the storage period. We compared theultrastructural preservation between corneal
endothelia stored at 4˚C and those stored at 0˚Cusing scanning electron microscopy (SEM) andtransmission electron microscopy (TEM).

Since Filatov (1935) succeeded in transplanting stored human corneas, corneal preservation techniques have markedly improved(Doughman, 1988; Wilson and Bourne, 1989).
From the 1930s to the 1970s, the corneas fromwhole eye balls were stored in moist chambersfor 24 h at 4˚C (Bourne, 1991).
Although a lot of effort was made to develop
an effective storage medium, less attention has
been paid to the temperature at which the corneas are kept. Cryopreservation techniques, bywhich the corneas are preserved at –196˚C,
were introduced for prolonged storage (Capellaet al., 1965), but they are not widely used today
because of the requirement of special expensive
equipment and trained technicians (McCareyand Kaufman, 1974).
It is well known that distilled water freezes
at 0˚C. However, the freezing-points of cells
and tissues are below 0˚C because they containproteins, sugars and lipids. Judging from theAbbreviations: SEM, scanning electron microscopy; TEM, transmission electron microscopy

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K.Yamasaki and T. Inoué

Materials and Methods
Results
Fourteen adult female rats, weighing 145 to 330g, were used in this study. All experiments
conformed to the Association for Research in
Vision and Ophthalmology Resolution on theHumane Use of Animals in Research. After theanimals were anesthetized by an intraperitoneal
injection with sodium pentobarbital (50 mg/kg
body weight), they were sacrificed by removalof blood from the right auricle. The eye globes
were immediately enucleated and were stored
in glass vials containing 15 mL of a storage
medium (EP-II, Kaken Pharmaceutical Co.,Tokyo, Japan). The vials were stored either at4˚C or 0˚C in a special refrigerator regulated bythermomodules (NH-60, Ninomiya Co., Ltd.,
Chiba, Japan). After preservation for 1, 2 and 7days, the corneas were excised with a 1- to 2mm scleral rim to make corneal buttons. Afterfixation with 2.5% glutaraldehyde in a 0.1 M
phosphate buffer (pH 7.4) for 2 h, the cornea
was cut into 2 pieces: one for SEM and the otherfor TEM. A freshly excised cornea was used asa control.
For SEM, the specimens were washed in abuffer and postfixed in buffered 1% osmium
tetroxide for 2 h. After washing again in the buffer, they were conductive-stained with 1% tannic acid and 1% osmium tetroxide (Murakami,1974). They were then dehydrated in a graded
series of ethanol, substituted with t-butyl alcohol, and finally freeze-dried (Inoué and Osatake,1988). The dried specimens were sputtercoated with platinum and observed with a scanning electron microscope (HFS-2ST, HitachiLtd., Tokyo) operated at 8 to 25 kV.
For TEM, the specimens were rinsed overnight in a buffer containing 7.5% sucrose, andpostfixed in buffered 1% osmium tetroxide for
2 h. They were washed again in the buffer, dehydrated in a graded series of ethanol, and finally embedded in epoxy resin. Ultrathin sections
were cut using an ultramicrotome (UltracutUCT, Leica, Wien, Austria), stained with tannic
acid, uranyl acetate and lead citrate (Kajikawaet al., 1975), and examined in a transmission
electron microscope (100 CX II, JEOL Ltd.,Tokyo) at 100 kV.

Normal corneal endothelium
The corneal endothelium, comprising a single
layer of hexagonal cells of 15 to 20 µm wideand 2 to 3 µm thick, was lined with a dense
basal lamina, referred to as Descemet’s membrane, of approximately 4 µm thick (Figs. 1aand b). The free surface was generally smooth
except for cell boundaries, scattered microvilli
and single cilia. The cell boundaries wereslightly elevated forming hexagonal ridges.
The cells included the attenuated nucleus andspherical mitochondria (Fig. 1c). On the freesurface of the cell membrane, pinocytotic vesicles were frequently visible. The intercellularspace was deeply invaginated between adjacentendothelial cells, and the zonula occludens wasusually visible at the apical portion.

Corneal endothelium preserved at 4˚CAfter 1 day’s storage, the cell boundaries of thecorneal endothelium slightly protruded and became somewhat indistinct (Fig. 2a). Most of
the mitochondria were swollen and their cristae
were indistinct. The zonula occludens was intact. The density of the cytoplasmic matrix wasalmost the same as normal endothelial cells, but
had slightly decreased in the basal region of thecells (Fig. 2b).
After 2 days’ storage, the endothelial surface showed prominent irregularity and the cellboundaries became indistinguishable (Fig. 3a).
The mitochondria showed marked swelling
accompanied by complete disruption of thecristae. The zonula occludens was intact. The
cytoplasmic matrix showed a low-density
compared with normal endothelial cells (Fig.
3b).
After 7 days’ storage, the corneal endothelium showed marked structural damage. The
cell surface became very irregular and destruction of the cell membrane was evident (Fig. 4a).
Severe damage to intracellular structures wasclarified by TEM (Fig. 4b): intracellular structures were unidentifiable except for the nucleus.
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Corneal preservation at low temperature

The surface cell membrane was disrupted andfragments of clumped chromatin and someother cell organellae protruded out of the cell.

Corneal endothelium preserved at 0˚CAfter 1 day’s storage, the surface of the endothelium was almost the same as that stored at
4˚C. The cell boundaries appeared morenatural than those preserved at 4˚C (Fig. 5a).
Although the mitochondria showed slightswelling, other intracellular organellae wererelatively well preserved. The cytoplasmic
density was almost the same as the normalendothelium (Fig. 5b). The zonula
occuludens was intact.
After 2 days’ storage, the surface of theendothelium became irregular, as observed in
the specimen preserved at 4˚C. The cell boundaries became less distinct than those preservedfor 1 day. Some microvilli were elongated (Fig.
6a). The mitochondria became swollen, but
their cristae were partly preserved. The roughsurfaced endoplasmic reticulum was relatively well-preserved and the zonula occludens
was also intact. The cytoplasmic density wasalmost the same as the normal endothelium(Fig. 6b).
After 7 days’ storage, the surface cell
membrane of the endothelium was almost
intact, but the microvilli on the peripheral
margin of the endothelium were markedly
elongated (Fig. 7a). The mitochondria werevacuolated and no cristae were visible. The
rough-surfaced endoplasmic reticulum showed swelling. The density of the cytoplasmic
matrix decreased in the basal region, but nosignificant changes were noted in the apicalregion of the cells (Fig. 7b). The zonula occludens was well preserved.

1aEDS

1bNMM

1cFig. 1. a: Scanning electron micrograph showing thesurface of a normal rat corneal endothelium. The cellsurface is generally smooth, except for elevation of thehexagonal cell boundaries. Arrows: single cilia (bar =10 µm). b: Low-magnification transmission electron
micrograph of a normal rat corneal endothelium. D,Descemet’s membrane; E, corneal endothelium; S,stroma (bar = 5 µm). c: High-magnificationtransmission electron micrograph of a normal rat
corneal endothelium. Note the slightly elevated cellboundaries and zonula occludens at the apical side of
the intercellular space (arrow). M, mitochondria; N,nucleus; arrowhead, pinocytotic vesicle (bar = 2 µm).

19Discussion
Since Filatov (1935) preserved whole eyeballs in an ice chest, hypothermic preservationof corneas at 4˚C has provided the basis oftissue storage for clinical use. Preservation at
reduced temperatures prolongs in vitro survival by slowing metabolism, reducing the demand for oxygen and other metabolites, andby conserving chemical energy (Taylor andHunt, 1989a). Although cryopreservation atK.Yamasaki and T. Inoué

*M

*M

2b2aM

M

3b3a*

*

4b4aFig. 2. Scanning (a) and transmission (b) electron micrographs of a rat cornea preserved at 4˚C for 1 day.
a: The cell boundaries protrude slightly (*) and become somewhat indistinct (bar = 10 µm).
b: Most of the mitochondria (M) are swollen and their cristae become indistinct. The zonula occludens(arrow) is intact. The density of the cytoplasmic matrix slightly decreases in the basal region of thecells (bar = 1 µm).
Fig. 3. Scanning (a) and transmission (b) electron micrographs of a rat cornea preserved at 4˚C for 2 days.
a: The cell surface shows prominent irregularity and the cell boundaries cannot be distinguished (bar =10 µm).
b: Mitochondria (M) are markedly swollen and the cristae are completely disrupted (bar = 2 µm).
Fig. 4. Scanning (a) and transmission (b) electron micrographs of a rat cornea preserved at 4˚C for 7 days.
a: The cell surface shows high irregularity and the surface cell membrane is destroyed (*) (bar = 10 µm).
b: The surface cell membrane is broken and some cell organellae protrude out of the cell (arrow). Notethe marked degeneration of the nucleus and cell organella (bar = 1 µm).

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