Corneal preservation at low temperature*M*5b5aMEM6b6aMEE7b7aFig. 5. Scanning (a) and transmission (b) electron micrographs of a rat cornea preserved at 0˚C for 1 day.
a: The cell surface protrudes slightly (*) and the cell boundaries are clearly visible (bar = 10 µm).
b: Mitochondria show slight swelling (M) and other intracellular organellae are relatively well preserved(bar = 1 µm).
Fig. 6. Scanning (a) and transmission (b) electron micrographs of a rat cornea preserved at 0˚C for 2 days.
a: The cell surface is irregular and the cell boundaries are just recognizable. Some elongated microvilli(arrows) are observed (bar = 10 µm).
b: The mitochondria (M) become swollen, but their cristae are partly preserved. The rough-surfaced
endoplasmic reticulum (E) is relatively well preserved (bar = 2 µm).
Fig. 7. Scanning (a) and transmission (b) electron micrographs of a rat cornea preserved at 0˚C for 7 days.
a: The cell surface is almost intact. The microvilli on the peripheral margin of the endothelium aremarkedly elongated (arrows) (bar = 10 µm).
b: The mitochondria are vacuolated and no cristae are visible (M). The rough-surfaced endoplasmic
reticulum is swollen (E). The density of the cytoplasmic matrix decreases in the basal region of thecells (bar = 1 µm).

21

K.Yamasaki and T. Inoué–196˚C made it possible to prolong storage
periods for up to a year or longer, freezing andthawing cause structural damage to the endothelium (Capella et al., 1965; Van Horn et al.,
1972; Doughman, 1988). Thus, it is logicallyassumed that organs should be stored at thelowest temperature possible without freezing(Yoshida et al., 1999).
All organic matter has its own specific subzero freezing temperature: –1.0˚C in the rat
liver (Yoshida et al., 1999), –0.6˚C in the rat
heart (Wicomb and Cooper, 1984) and –0.8˚Cin human plasma (Storey and Storey, 1990).
Since the rat corneal endothelium is very thin, it
is impossible to measure its freezing point witha thermometer. In this study, we tried to preserve corneas at 0˚C, assuming that corneas do
not freeze at this temperature.
Previously, Taylor et al. (1989a,1989b) storedrabbit isolated corneas at 0˚C. They reported
that the endothelial ultrastructure was maintained during storage for 3 and 5 days in a hyperkalemic solution, CPTES*. According to theirtransmission electron microscopic findings, themitochondria showed moderate swelling with adiffuse pallid matrix, as well as condensed andbeaded forms. The smooth endoplasmic reticulum was swollen and vesiculated. Their ultrastructural findings were almost identical to thepresent study. According to their method, polypropylene vials containing a preservation medium were placed in an evacuated Dewar flask
containing ice in a 4˚C refrigerator. In this study,
we used a special refrigerator that can precisely
control the temperature from room temperature
to –5.0˚C at 0.1˚C intervals (Yoshida et al.,1999).
Nowadays, Optisol (Steinemann et al., 1993)
and Dexsol (Skelnik et al., 1988; Lass et al., 1990)(Chiron Co., Irvine, CA) are used by most eyebanks in the United States as solutions for preserving isolated corneas at 4˚C. These solutions
have enabled their preservation for up to 1 week(Bourne, 1991). Although the corneoscleralpreservation has increased in Japan, it has notbeen widely used all over the world (Shimazakiet al., 1993). In this study, we preserved rat

whole globes at 0˚C in EP-II, which has been
generally used for whole globe preservation inJapan.
Fluid movement in the corneal endotheliumis thought to be related to a pump-leak transportmechanism in which fluid from the anteriorchamber leaks into the stroma across a leaky
apical junction, while fluid is actively pumped
from the stroma into the anterior chamber(Barry et al., 1995). The Na+-K+ ATPase pumpof the corneal endothelium has been describedby many investigators: the pump is located in
the lateral membrane and actively transports
sodium and bicarbonate ions into the anteriorchamber; along this osmotic gradient, water
moves from the stroma to anterior chamber(Klyce and Beuerman, 1988; Dohlman, 1994;
Edelhauser et al., 1994; Barry et al., 1995).
Hodson (1971) demonstrated that perfusion
with a bicarbonate free medium caused reversible stromal swelling. Thus, it was presumed
that the pump was partly inactivated by the removal of bicarbonate (Hodson, 1971). Since
EP-II does not contain bicarbonate ions, it
extends the lifetime of the corneal endotheliumby controlling pump function consumption.
The present scanning electron microscopic
study showed no significant ultrastructural differences in the endothelial surfaces of corneas
preserved at either 4˚C or 0˚C for up to 2 days.
After 7 days’ storage at 4˚C, the cells showedmarked destruction (Fig. 4a), but were fairlywell preserved at 0˚C. Furthermore, transmission electron microscopic findings indicatedthat the corneal endothelium preserved at 4˚Cshowed more distinct mitochondrial swellingthan that preserved at 0˚C. These changes became evident after 7 days’ storage (Figs. 4b and7b). Ultrastructural changes were also noted in
the corneas preserved at 0˚C: the presence ofvacuoles, condensation of the mitochondrial
matrix, a swollen rough-surfaced endoplasmic
reticulum and a decrease in the cytoplasmic
density in the basal region. However, most of
the surface cell membrane and cell organellae
were retained (Fig. 7b). The density of thecytoplasmic matrix decreased in the basal re-*CPTES: corneal-potassium-TES, a potassium-rich balanced salt solution containing the impermeantbiological buffer compound N-Tris(hydroxymethyl)methyl-2-amino ethane sulphonate.

22Corneal preservation at low temperature

1965;74:669–673.
5 Dohlman CH. Corneal edema. In: Albert DM,
Jakobiec FA, eds. Principles and practice of ophthalmology. vol 1. Philadelphia: WB Saunders;
1994. p. 245–256.
6 Doughman DJ. Corneal tissue preservation. IntOphthalmol Clin 1988;28:50–56.
7 Edelhauser HF, Geroski DH, Ubels JL. Physiology. In: Smolin G, Thoft RA, eds. The Cornea.
3rd ed. Boston: Little Brown; 1994. p. 25–46.
8 Filatov VP. Transplantation of the cornea. ArchOphthalmol 1935;13:321–347.
9 Hodson S. Evidence for a bicarbonate-dependent
sodium pump in corneal endothelium. Exp Eye
Res 1971;11:20–29.
10 Inoué T, Osatake H. A new drying method ofbiological specimens for scanning electron microscopy: The t-butyl alcohol freeze-drying
method. Arch Histol Cytol 1988;51:53–59.
11 Kajikawa K, Yamaguchi T, Katsuda S, Miwa A.
An improved electron stain for elastic fibers usingtannic acid. J Electron Microsc 1975;24:287–289.
12 Klyce SD, Beuerman RW. Structure and function of the cornea. In: Kaufman HE, Barron BA,
McDonald MB, Waltman SR, eds. The Cornea.
New York: Churchill Livingstone; 1988. p. 3–54.
13 Lass JH, Reinhart WJ, Skelnik DL, Bruner WE,Shockley RP, Park JY, et al. An in vitro and clinical comparison of corneal storage with chondroitin sulfate corneal storage medium with and without dextran. Ophthalmology 1990;97:96–103.
14 Manabe R, Kinoshita S, Itoi M, Kodama Y,Tanishima T. Clinical trials of new eye preservation medium (EP-II). Folia Ophthalmol Jpn
1984;35:1263–1265.
15 McCarey BE, Kaufman HE. Improved cornealstorage. Invest Ophthalmol 1974;13:165–173.
16 Murakami T. A revised tannin-osmium method
for non-coated scanning electron microscope
specimens. Arch Histol Jpn 1974;36:189–193.
17 Shimazaki J, Yamada M, Tsubota K. Corneal
transplantation with donor corneas stored inmoist chamber and chondroitin sulfate-containing medium. Cornea 1993;12:104–108.
18 Skelnik DL, Pearlstein CS, Mindrup EA,Lindstrom RL. Corneal preservation at 4˚C withchondroitin sulfate containing medium supplemented with dextran and epidermal growth factor(EGF). Invest Ophthalmol Vis Sci 1988;29
Suppl:111.
19 Steinemann TL, Kaufman HE, Lindstrom RL,
Beuerman RW, Varnell ED. Intermediate-term
storage media (K-Sol, Dexsol, Optisol). In:
Brightbill FS, ed. Corneal surgery. Theory, technique and tissue. 2nd ed. St.Louis: Mosby; 1993.
p. 609–613.
20 Storey KB, Storey JM. Frozen and alive. Sci Am1990;December:62–67.

gion of the endothelia preserved for 1 and 2days at 4˚C or 7 days at 0˚C (Figs. 2b, 3b and7b). This may have been due to the leakage offluid into the paracellular space across the leaky
apical junction.
There were fewer ultrastructural changes in
the corneal endothelia preserved at 0˚C thanthose at 4˚C, which are thought to be reversible.
Since the endothelial metabolism below 0˚C isreduced in comparison with that at 4˚C, consumption of ATP and activity of the Na+-K+
ATPase pump are minimized in an endothelium
preserved at 0˚C. Recently, Yoshida (1999) reported that the concentration of ATP in the rat
liver preserved at –0.8˚C was higher than that at
4˚C. Thus, the lifetime of the endothelial cells
is extended if they are preserved at 0˚C.
The utilization of corneas preserved aswhole globes at 4˚C is restricted to within 48 h,
because the endothelium is exposed to stagnant
aqueous humor which has metabolic waste
products and tissue necrosis (Bito and Salvador,
1970; McCarey and Kaufman, 1974). The present findings indicate the possibility for longer
preservation of whole globes if they are storedat 0˚C.

Acknowledgments: The authors wish to thank Professor Akihiko Tamai and Professor Shiro Ikawa, Departments of Ophthalmology and Clinical Laboratory
Medicine, Faculty of Medicine, Tottori University,
for their valuable suggestions and critical reading of
the manuscript.

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23

K.Yamasaki and T. Inoué

21 Taylor MJ, Hunt CJ. Hypothermic preservationof corneas in a hyperkalaemic solution (CPTES):
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24 Wicomb WN, Cooper DKC. Donor heart storage. In: Cooper DKC, Lanza RP, eds. Hearttransplantation. Lancaster: MTP Press Limited;
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Received December 19, 2000; accepted December 25, 2000
Corresponding author: Dr. Kaori Yamasaki

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