|Year : 1996 | Volume
| Issue : 1 | Page : 15-18
Changes in the corneal Na-K ATPase levels in eyes stored in moist chamber at 4°C
B Devi, SM Hasany, PK Basu
Department of Ophthalmology, University of Toronto, 1 Spadina Crescent, Toronto, Ontario M5S 2K6, Canada, USA
Department of Ophthalmology, University of Toronto, 1 Spadina Crescent, Toronto, Ontario M5S 2K6, Canada
Source of Support: None, Conflict of Interest: None
This report deals with a chronological measurement of Na-K ATPase enzyme activity in human and bovine corneas stored in a moist chamber at 4°C. Paired human and bovine eyes were sterilized by the standard eye bank procedure and stored up to 6 days. At the desired time, the corneal endothelium was assayed for Na-K ATPase activity. The protein content of each tissue sample was also determined. In a parallel set of experiments, the viability of identical stored corneas was determined by trypan blue and alizarin red staining technique, and morphometric analysis was done to quantify the extent of the corneal endothelial damage. The human corneas showed that there was a significant progressive decrease in the Na-K ATPase activity as the storage time increased. The decrease was related to morphological endothelial damage.
Keywords: Cornea, endothelium, epithelium, storage, moist chamber, eye bank, Na-K ATPase activity.
|How to cite this article:|
Devi B, Hasany S M, Basu P K. Changes in the corneal Na-K ATPase levels in eyes stored in moist chamber at 4°C. Indian J Ophthalmol 1996;44:15-8
One important role of the corneal endothelium is to maintain the proper hydration of the cornea which is essential for keeping its transparency. The endothelial cell layer has an Na-K dependent cation pump which regulates the flow of electrolytes and water from the anterior chamber into the corneal stroma. The corneal endothelial pump is largely mediated by an active transport of sodium as well as bicarbonate ions across the cell layer.
Both Na-K and bicarbonate dependent ATPase enzymes have been demonstrated biochemically, and histochemically in the corneal endothelial cells. The exact location of the bicarbonate dependent enzyme is not completely known but there is substantial evidence that this enzyme is of mitochondria] origin. On the other hand, the Na-K dependent ATPase, which plays a major role in controlling the corneal swelling, is located on the plasma membrane.,
The cornea shows a rapid increase in hydration, and a prompt loss of glycogen and ATP immediately after the death of the donor and during storage of the cornea. A decrease of the Na-K dependent ATPase enzyme in a dysfunctional endothelium has also been related to some diseases of the cornea and to aging.
We hypothesized that the level of the Na-K dependent ATPase in the endothelium of the stored cornea would be related to the functional status of the endothelium. To our knowledge, the level of the Na-K dependent ATPase has not been studied chronologically in the endothelium of corneas stored in mosit chamber. We, therefore, wished to determine the level of the Na-K dependent ATPase in the corneal endothelium of stored human eyes, and relatively fresh bovine eyes. We also tried to correlate the intensity of the endothelial enzyme activity with the viability and integrity of the endothelium.
| MATERIALS AND METHODS|
Human Donor Eyes
Paired human eyes were obtained from the Eye Bank of Canada (Ontario Division). On the average, the eyes were enucleated within 4 hours of donor's death. Each pair of eyes (n = 3) was subjected to the routine eye bank procedure and stored for 1, 2, 4, and 5 days at 4°C in the moist chamber.
| Bovine Eyes|| |
A certain amount of unavoidable delay inevitably had occurred before experiments could be started on the human donor eyes. This was due to the variation in the time lapsing between the death of the donor and the enucleation of eyes, and between the enucleation and the processing of the eyes in the laboratory. In order to reduce the effect of the delay as much as possible, we used fresh paired bovine eyes. This gave us an opportunity to compare the enzyme levels in relatively fresh bovine corneas with that in the stored human corneas.
Paired bovine eyes were collected from the slaughter house immediately after the death of the animal. Eyes were kept on ice and were brought to the laboratory at 4°C within 2 hours. Each pair of eyes was washed with normal saline and then treated by the method used for human eyes. One eye (control) of each animal was processed immediately, and the other eye (experimental) was stored in the moist chamber for 2, 4, 5 or 6 days at 4°C. Four pairs of eyes were used for each day.
| Preparation of the Tissue Sample|| |
At the desired time, the epithelium and endothelium of the cornea, which was kept on ice, were scraped separately with a surgical blade. Because of the small quantity of endothelium obtainable from a single human cornea, the tissues from both eyes were pooled. The endothelium of the bovine eyes was not pooled and was kept individually.
The endothelial cell preparations contained pieces of Descemet's membrane. As many pieces as possible of the Descemet's membrane were removed with fine forceps from the cellular suspension.
The endothelial sample was centrifuged twice at 3000 rpm for 5 minutes in cold Tris buffer pH 7.5. (Sorvall GLC -1 Newton, CT). The sediment (tissue sample) was then resuspended in the cold double distilled water and recentrifuged. The pellet was suspended in cold water, and sonicated at 4°C for one minute with a sonicator (Kontes, Vineland, NJ). Each sonicate was then lypholized, and the dry weight of the sample was determined.
| Procedure for the Na-K ATPase Assay|| |
For ATPase assay, the sample was reconstituted with ice-cold double distilled water. The reconstituent was adjusted to 8 mg (dry weight) tissue/ml. The Na-K dependent ATPase activity of each sample was determined by using the colorimetric assay technique of Bonting and co-workers as modified by Neville et al. The protein content of each sample was determined by the Lowry's method using bovine serum albumin as the reference standard. The Na-K ATPase activity was calculated as the difference between the ATPase activity determined in the presence and that in the absence of 10[-4]M ouabain. The ATPase activity of the tissue homogenate was expressed in terms of phosphate level (nmol per mg protein per hour). The data was expressed as mean or percent of control and were analysed statistically by analysis of variance.
| Morphometric Analysis of the Corneal Endothelial Damage|| |
In another set of parallel experiments, paired human donor eyes and bovine eyes were stored for 1, 2, 4, and 7 days. Three pairs of eyes were used for each day of storage. After each storage period, the cellular viability and integrity of the endothelium was tested by staining the tissue with trypan blue and alizarin red.
The central region (5 mm in diameter) of the endothelium of the experimental ard control corneas was photographed using a Zeiss photomicroscope at a constant magnification. The four different areas of each photomicrograph of the endothelial surface of paired cornea were traced individually on a clear plastic sheet, and then were analysed by morphometry.
Human Corneal Endothelium
[Figure - 1] shows that in the case of human corneal endothelium there was a significant decrease in the enzyme level (60%) after 2 days of storage. A progressive decrease of enzyme activity and of endothelial damage was evident from the 2nd day onward. When the daily data of endothelial ATPase activity were compared, it was found that datum of day 1 was significantly higher than that seen on days 2, 4 and 5 (P<0.01). It was seen that the enzyme activity progressively decreased as the storage time increased. Morphologically, the corneas showed progressively greater endothelial damage per unit area as the storage time was increased (P<0.05) (Table).
| Bovine Corneal Endothelium|| |
The ATPase activity of the control and experimental bovine corneal endothelia were compared. When the results of the experimental samples were expressed as the percentage of the control values, the data [Figure - 2] indicated a pattern similar to what we saw in the case of the human endothelium. The enzyme activity in the bovine endothelium had a significantly sharp fall on the 4th day. It then rose slightly on the 5th day of storage. However, this rise was not statistically significant when analysed by analysis of variance. Morphologically, the endothelial damage progressively increased with the storage time (P<0.01). Thus the endothelium on the 4th day of storage were more damaged as compared with those stored for 1 to 3 days (Table).
| Human and Bovine Endothelium|| |
There was a quantitative difference in the ATPase levels in the corneal layer between the two species. The bovine epithelium as compared to the human epithelium showed a greater amount of enzyme per unit weight of the tissue. However, neither of the species showed any significant change in the enzyme level at any time during 1 to 6 days of storage.
| Discussion|| |
The moist chamber technique of storage of whole eye balls is still widely in use in places (e.g. Third World countries) where the storage of excised cornea in tissue culture medium is difficult for technical and economic reasons. Some Canadian eye banks appear to use moist chamber stored eyes, according to the quarterly reports of the Canadian Eye Banks. In emergency and unusual situations, for example, during long distance transportation to our eye bank laboratories from far away places, this method is still used by the Eye Bank of Canada, Ontario Division.
During storage of the donor eyes in the moist chamber at 4°C, there appeared to be two phases in the activity of the ouabain sensitive Na-K dependent ATPase in the corneal endothelium. The ATPase activity in the endothelium of the stored human cornea decreased after 1 to 2 days of eye storage (first phase). During the first phase, the cellular membrane appeared to be intact morphologically. In the second phase (after 2 days) the level of ATPase activity continued to drop. In this phase, this phenomenon was probably related to the breakdown of the cellular membrane transport system. It may be mentioned here that in another of our studies (unpublished data) we have seen electronmicroscopically evidence of mitochondrial swelling and membrane disorganization in the endothelium of the human cornea stored for 2 to 4 days. Similar phenomena have been noticed by others. Recent studies on ouabain binding or unstored and stored cornea in tissue culture media did not show a change in Na-K ATPase pump site activity after four days of storage. However, after 7 days of storage, thee was an elevated ATPase pump site activity of cornea stored in MK medium. This increase in activity was interpreted as the cornea was under stressed condition.
Our present study indicated a decrease of the level of endothelial ATPase in stored human as well as relatively fresh bovine corneas after 2 days of storage in moist chamber at 4°C. Morphologically, the stored endothelium showed extensive cellular damage after 2 days of storage. We therefore recommend that corneas from human donor eyes should not be used for keratoplasty after 2 days of storage in moist chamber at 4°C.
We now plan to compare the patterns of the enzyme activity in the endothelium of the excised cornea stored in vitro for different periods in various storage media containing different agents.
In contrast to the ATPase activity level in the endothelium, that in the corneal epithelium did not change significantly during any of the storage periods used in this study. We have no explanation of this phenomenon at present.
We are thankful to Mr. Terry Riopka of the Department of Mechanical Engineering, University of Toronto, for help in our morphometric analysis, and to Professor A.K. Sen from the Department of Pharmacology, University of Toronto, for his suggestions.
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[Figure - 1], [Figure - 2]
[Table - 1]