T H E RESPIRATION OF T H E STORED CORNEA*

T. D. DUANE, M.D. Iowa City, Iowa

INTRODUCTION

A better understanding of certain physi­ologic processes that take place in corneas which are "stored" under various conditions may be gained by measurements of the respiration of the tissue. This method may aid in determining the optimum conditions for preservation of, the donor cornea. A number of methods for preservation have been described but most reliable and thorough reports are based on four different techniques for storage, namely: (1) over Ringer's solution in air at 4°C. ,·1 '2 (2) under oil;3'4 (3) in formalin;5·6 (4) at subzero centigrade temperatures.8*9 A study of the respiratory metabolism changes under these conditions is obviously important.

METHODS

Bovine eyes were used exclusively in this experiment since they were readily avail­able.1 The Q 0 2 of the beef cornea is ap­proximately the same as the human cornea10

and other mammalian corneas.11

On enucleation at the packing plant en­tire eyes were stored as follows: (1) at 4°C. suspended over Ringer's solution con­tained in a large sealed thermos jug; (2) under mineral oil at 1S°C. The corneas from these eyes were removed at the time of the metabolism studies.

Isolated corneas from freshly enucleated eyes were stored as follows: (1) in com­mercial formalin (5 percent and 35 per­cent) ; (2) in the deep freeze at — 40°C. ; (3) at — 40°C. after quick freezing on the expansion chamber of the carbon dioxide

* From the Departments of Physiology and Ophthalmology, Medical Laboratories, State Uni­versity of Iowa.

t Eyes obtained through courtesy of Gay's Locker, Iowa City, Iowa, and Wilson Company, Cedar Rapids, Iowa.

frozen section apparatus. The latter two groups were permitted to thaw immediately prior to being placed in the Warburg flasks.

All investigations of respiratory metabol­ism were made according to the standard Barcroft-Warburg technique. Each cornea was divided into four segments of approxi­mately equal size before being placed in the Warburg flask containing a Ringer-phos­phate buffer with 0- to 2-percent glucose.12

Manometric readings were made at frequent intervals during 2- to 6-hour periods. The oxygen consumption for each interval was computed in terms of dry tissue weights. From this data a curve was plotted to show the oxygen consumption of each cornea and individual Q 0 2 values were determined. From these values the average Q 0 2 for an experimental group was calculated.

RESULTS

1. + 4°C. storage. Over 100 intact eyes were stored by this method. It was found

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Fig. 1 (Duane). Respiration of 10 corneas from intact eyes stored for three days over Ringer's solution at +4°C. Ordinate: mm.* O» per mgm. dry weight. Abscissa: minutes.

1400

RESPIRATION OF STORED CORNEA 1401

that if they were removed from the low temperature during the first seven days of storage the corneas respired normally. Fig­ure 1 is a sample curve which shows the data obtained from corneas stored for three-

are similar to those found for corneas stored at + 4 ° C . In these experiments also bac­terial contamination prevented longer stor­age periods.

3. Formalin storage. The 11 corneas in-

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AIR STORAGE (4*C.) EACH POINT REPRESENTS 10 CORNEAS

Fig. 2 (Duane). Average QO: of corneas from intact eyes stored over Ringer's solution at +4°C. Each point represents 10 corneas.

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Fig. 3 (Duane). Average QOa of corneas from intact eyes stored under oil at +lf>°C.

12

days demonstrating that the normal rate of respiration is still present. After seven days the respiration began to fall off (fig. 2 ) . By the tenth day bacterial contamination precluded further investigations.

2. Storage under oil. Sixteen corneas were studied after a storage period from 3 to 12 days. The results shown in Figure 3

eluded in this group did not respire when placed in the manometric flasks. Merely dipping the cornea in either 5-percent or 35-percent formalin completely inhibited the respiration. No respiration appeared after longer storage in these fluids when followed up to four days.

4. Deep freeze, —40°C. Results ob-

1402 T. D. DUANE

tained from 22 corneas in this group are plotted in Figure 4. The Q 0 2 dropped to one-half normal in 24 hours and within four days had fallen to one-fifth normal where

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Fig. 4 (Duane). Average corneal Q02 of iso­lated corneas stored in the deep freeze at — 40 °C.

the cornea. However, the reproducibility of the curves presented in this report is evi­dence of their reliability. Even when the corneal respiration is depressed by low tem­peratures, readings are obtained which can be plotted upon a straight line. A typical curve, Figure 6, is offered as evidence of the feasibility of this technique in these studies.

Clinical experience has demonstrated that the storage of donor corneas at low non-freezing temperatures is only satisfactory for periods of 48 to 72 hours. Longer stor­age periods almost invariably result in opaque transplants. However, the Q 0 2 of these corneas remains normal for a consid­erably longer time than 72 hours.

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AVERAGE CORNEAL QO* QUICK FREEZE AND-40*C STORAGE

Fig. 5 (Duane). Average corneal Q02 of isolated corneas stored at —40°C. after quick freezing.

it remained. The absence of bacterial con­tamination permitted study over a much longer period. At 40 days the Q 0 2 was still one-fifth normal.

5. At —40°C. after quick freeze. The results obtained on 40 eyes which were in­cluded in this group are plotted in Figure 5. Quick freezing reduced the respiration to one-fifth normal and at 60 days it was still at this level.

DISCUSSION

Most mammalian tissues studied with the Barcroft-Warburg technique demonstrate a respiratory rate many times greater than

It was hoped that these experiments might explain the unsatisfactory surgical re­sults on the basis of a change in the activities of respiratory enzyme systems. There ap­pears to be little correlation between the maintenance of a normal Q 0 2 by a cornea and its suitability for corneal transplanta­tion. However, one should be cautious in transferring the results from bovine corneas to the conditions which exist in human eyes. Furthermore, the present studies do not in­clude measurements of glycolytic activities which may be important for the survival of tissues.

There are certain reports in the literature

RESPIRATION OF STORED CORNEA 1403

that corneas treated with formalin are suit­able as donor material for corneal trans­plants.5· 7 There are many reasons to ques­tion these reports13·14 and the results of the studies reported in this paper cast further doubt on the use of formalin as a storage medium.

The literature on cold death of tissues

corneas stored in 5-percent and 35-percent formalin, (4) in the deep freeze at —40°C, and (5) in the deep freeze after quick freez­ing.

2. The Q 0 2 of the cornea remains normal when stored over Ringer's solution or under oil at low nonfreezing temperatures for 7 days. The respiratory rate falls to one-

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12 DAY QUICK FREEZE STORAGE- 3 CORNEAS

Fig. 6 (Duane). Respiration of three corneas stored for 12 days at —40°C. after quick freezing.

is well summarized by Heilbrunn,15 but the actual effect of low temperatures on respira­tory enzyme systems · is unknown. It is hoped that further investigations may pro­vide answers to some of these questions.

SUMMARY AND CONCLUSIONS

1. Respiration studies have been made on bovine corneas stored from 1 ^ hours to 60 days under five different conditions : ( 1 ) in­tact eyes stored above Ringer's solution at 4°C, (2) under oil at 15°C, (3) isolated

fifth normal in 10 to 12 days under these conditions.

3. Formalin inhibits respiration of the cornea immediately and this is apparently irreversible.

4. The corneal Q 0 2 is reduced immedi­ately to one-fifth normal by quick freezing and drops to this level within 4 days when kept at —40°C. There is no further respira­tory depression up to 60 days storage.

College of Medicine.

R E F E R E N C E S

1. Filatov, V. P. : Transplantation of the cornea from preserved cadavers' eyes. Lancet, 232:1395 (June) 1937.

2. Castroviejo, R.: Present status of keratoplasty. Arch. Ophth., 22:114 (July) 1939. 3. Thomas, J. W. T.: Transplantation of cornea. Tr. Am. Ophth. Soc. 50:127-141, 1930. 4. . : Experimental transplantation of cornea. Tr. Am. Ophth. Soc, 51:88-95, 1931. 5. Salzer, F. : Dauhereinheilung von konservierten Hornhautsteuchchen in die Hornhaut des Kanin­

chens. 36, Heidelberg, Bericht, p. 312, 1910. 6. Scherschewskaya, O. J.: Keratoplasty: Experiments with formol transplants. Ophthalmologica,

99:4-15 (Feb.) 1940.

1404 JOHN H. DUNNINGTON

7. Wiener, M., and Rosenbaum, H. D. : Formalinized heterogenous and homogeneous corneal trans­plantation: Experimental. Am. J. Ophth., 24:1384-1391 (Dec.) 1941.

8. Bazhenova, M. A. : Experimental transplantation of dried and frozen cornea. Med. Zhur., 9 : 1447-1452,1940.

9. Leopold, I. H., and Adler, F. H. : Use of frozen-dried cornea as transplant material. Arch. Ophth., 37 .-268-276 (Mar.) 1947.

10. Duane, T. D. : Respiratory metabolism of cornea. In preparation. 11. Robbie, W., Leinfelder, P. J., and Duane, T. D. : Cyanide inhibition of corneal respiration. Am.

J. Ophth., 30:1381-1387 (Nov.) 1947. 12. Deckens, F., and Greville, G. : The metabolism of normal and tumor tissue : XIII. Neutral salt

effects. Biochem. J. 29:1468,1935. 13. Deutman, A. F. : Formalinized cornea and keratoplasty in rabbit's eyes. Ophthalmologica, 99 :

418-422, 1940. 14. Katzin, H. M., and Kuo, P. H. : Histologie study of experimental cornea transplantation. Am. J.

Ophth., 31:171-191, 1948. 15. Heilbrunn, L. V. : An Outline of General Physiology. Philadelphia, W. B. Saunders Company,

Ed. 2,1943, pp. 428-432.

SOME FACTORS IN T H E SURGICAL TREATMENT OF VERTICAL DEVIATIONS*

JOHN H. DUNNINGTON, M.D. New York

The uncertainty of postoperative results has deterred many ophthalmologists from attempting the surgical correction of vertical deviations. It is true that such muscle sur­gery has its disappointments and that there is still much to learn about it, but by taking full advantage of certain known facts, the number of failures can be reduced and more uniform results obtained.

For example, knowledge that most verti­cal deviations are noncomitant was the first step toward the solution of this problem. Then we began to appreciate the importance of preoperative information on the behavior of the eyes in the six cardinal directions of gaze. The value of accurate measurement of the amount of deviation in the different fields was drilled into us by Duane,1 White,2

and others who also showed us that the screen and parallax test was the test par

* From the Institute of Ophthalmology, Presby­terian Hospital, and the Department of Ophthal­mology, College of Physicians and Surgeons, Co­lumbia University, New York City. Read at the III Pan-American Congress of Ophthalmology, Havana, Cuba, January 7, 1948.

excellence for obtaining such information. We became increasingly aware of the

futility of the old teaching of "tenotomy of the superior rectus is the operation of choice in hyperphoria," for we began to diagnose vertical deviations not as hyperphorias, but as paralyses with or without secondary over-actions. The proper surgical treatment is therefore based not only upon the correct preoperative diagnosis of the muscle or muscles involved, but also upon which eye is used for fixation in the different directions of gaze. Failure to consider the presence or absence of a shifting fixation in the pre­operative study has done much to increase the variability of the surgical results.

Since most operations upon the vertically acting muscles are designed to give a func­tional result, they must be more exact. The preoperative studies to be complete should include information on the eye used for fixation in the primary position and in the various directions of gaze. Accepting the general principle that most vertical devia­tions are of paralytic nature, we can now