Effects of Freezing on Ocular Tissues*

Effects of Freezing on Ocular Tissues*

JEAN DUMAS AND CHARLES L. SCHEPENS 630 4. Fison, L. : Recent trends in treatment of de­ tachment of the retina. Trans. Ophth. Soc. U. Kingdom, 80:51...

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4. Fison, L. : Recent trends in treatment of de­ tachment of the retina. Trans. Ophth. Soc. U. Kingdom, 80:517, 1960. 5. Colyear, B. H. and Pischel, D. K.: Photocoagulation as an adjunct in retinal detachment surgery. Am. J. Ophth. 52:474, 1961. 6. Meyer-Schwickerath, G. : Indications and limitations of light coagulation of the retina. Tr. Am. Acad. Ophth. Otolaryng., 63:725, 1959. 7. Hruby, K. : Clinical observations of vitreous changes. In Schepens, C. L. (editor) : Importance of the Vitreous Body in Retina Surgery with Special Emphasis on Reoperations. St. Louis, Mosby, 1960, pp. 94-111. 8. Schepens, C. L., Tolentino, P., and McMeel, J. W. : Diagnostic and prognostic factors as found in preoperative examination. Tr. Am. Acad. Ophthal. Otolaryng., In press. 9. Vogt, A. : Die operative Therapie und die Pathogenese der Netzhautablösung. Stuttgart, Ferdinand Enke, Verlag, 1936. 10. Arruga, H. : Detachment of the Retina. R. Castroviejo (trans.), New York, Westermann, 1936. 11. Schepens, C. L. : Subclinical retinal detach­ ment. Arch. Ophth. 47 :593, 1952. 12. Michaelson, I. C. : Retinal detachment : Clinical evidence of the role of the choroid. Acta XVII Cone. Ophth., 1:392, 1954. 13. Michaelson, I. C. : Role of a distinctive choroido-retinal lesion in pathogenesis of retinal hole: A clinical and pathological report. Brit. J. Ophth. 40:527, 1956. 14. Pau, H. : On the etiology, pathology and

surgical treatment of retinal detachment. Am. J. Ophth., 47 :565, 1959. 15. Meyer-Schwickerath, G. : Light Coagula­ tion. (S. Drance, trans.) St. Louis, Mosby, 1960. 16. Okun, E. : Gross and microscopic pathology in autopsy eyes : Part III. Retinal breaks without detachment. Am. J. Ophth., 51:369, 1961. 17. Lincofï, H. A. : The prophylactic treatment of retinal detachment. Arch. Ophth., 66:48, 1961. 18. Straatsma, B. R., and Allen, R. A.: Lattice degeneration of the retina. Tr. Am. Acad. Ophthal. Otolaryng., 66:600, 1962. 19. Graether, J. M. : Retinal changes in degen­ erative myopia. Inter. Ophth. Clin. 1:109, 1962. 20. Lister, W. : Holes in the retina and their clin­ ical significance, (see Plate I) Brit. J. Ophth. 8:1, 1924. 21. Teng, C. C, and Katzin, Ή. M. : An ana­ tomic study of the periphery of the retina: Part I : Nonpigmented epithelial cell proliferation and hole formation. Am. J. Ophth., 34: 1237, 1951. 22. Rutnin, U., and Schepens, C. L. : Normal fundus appearances : II. The peripheral fundus. Am. J. Ophth., In press. 23. Okun, E. : Gross and microscopic pathology in autopsy eyes : Part I. Introduction and long posterior nerves. Am. J. Ophth, 50:424, 1960. 24. : Gross and microscopic pathology in autopsy eyes : Part II. Peripheral chorioretinal atrophy. Am. J. Ophth., 50:574, 1960. 25. O'Malley, P., Allen, R. A., Strattsma, B. R., and O'Malley, C. C. : Paving-stone degenera­ tion of the retina. Arch. Ophth., 73: 169, 1965.

EFFECTS OF FREEZING ON OCULAR TISSUES* I.

C L I N I C A L AND HISTOLOGIC STUDY OF CORNEAL HELEN

H.

C H I , M.D.,

A N D C H A R L E S D.

ENDOTHELIUM

KELMAN,

M.D.

New York I n view of the importance of the endothelium to corneal transparency, it is sur­ prising how little is known concerning its changes after exposure to freezing tempera­ tures. A few articles on the effects of freez­ ing on the cornea dealt mainly with wound healing in the corneal stroma after freezing, 1 " 3 with little mention of the changes in the corneal endothelium, perhaps because it is very difficult to detect the de* From the Cryosurgery Research Laboratory, Manhattan Eye, Ear and Throat Hospital. This study was supported by a grant from the John A. Hartford Foundation, Inc.

tailed cellular changes in the endothelium using the conventional methods of section­ ing. W e have found that flat preparations of the corneal endothelium examined under phase-contrast microscopy show the earliest changes of necrosis and regeneration. U s i n g these methods one can examine a greater area of endothelium at one time. Previous investigators reported that the cornea regained its normal transparency after damage to the cells by freezing. 1 - 2 · 4 These reports, however, were based on the use of applicators of 2.0, 5.5 and 6.0-mm

EFFECTS OF FREEZING ON OCULAR TISSUES

631

diameter with temperatures of — 78°C and — 72°C for three to five and 60 seconds. Since the severity of the trauma depends upon the temperature, size and duration of the application, in our investigation various low temperatures and different sizes of ap­ plicators were applied to the central part of the cornea. The time factor was standard­ ized at 20 seconds for all applications. The purposes of this investigation were: (1) to determine whether a relatively large area of destruction of corneal cells at var­ ious low temperatures will eventually pro­ duce permanent corneal opacity; (2) to find which temperatures will cause minimum or Fig. 1 (Chi and Kelman). The cannula with a specially constructed 8-mm brass applicator. no damage to the corneal endothelium and (3) to study the histologie changes in the corneal endothelium after freezing. All eyes were stained with fluorescein be­ fore and immediately after freezing. MATERIALS AND METHODS The eyes were fixed with forceps so that Fifty-eight female Dutch rabbits weigh­ an accurate application could be made to the ing from five to six pounds were used in central part of the cornea with temperatures these experiments. The rabbits were pre- of - 1 0 ° C , - 2 0 ° C and ~80°C applied for medicated with an intramuscular injection 20 seconds. Care was taken so that the tip of thorazine (4.0 mg/lb) and probanthine of the applicator was firmly in contact with (0.3 mg/lb) and then anesthetized with in­ the epithelial surface of the cornea without travenous sodium nembutal and local tetra- exerting undue pressure. No eye received caine, 0.5%. more than a single application. Operations Thé Cooper cryosurgery system which were conducted with sterile precautions and was used in these experiments feeds liquid erythromycin ophthalmic ointment was ap­ nitrogen into a stainless steel cannula. The plied locally immediately after operation. cannula is vacuum insulated except at the During the applications the cold applica­ tip where a microthermocouple is placed to tor usually became adherent to the corneal maintain the desired temperature. surface so that it was necessary to wait sev­ To produce a relative large frozen area it eral seconds for thawing to occur before re­ was necessary to cap the largest cannula moving the applicator from the cornea. (4.76 mm in diameter) with a specially con­ Both eyes of 14 rabbits were used for structed brass applicator measuring 8.0 mm each group of temperatures: — 10°C, in outside diameter, 11-mm long and 1-mm - 2 0 ° C and - 8 0 ° C , using the 8-mm appli­ in thickness (fig. 1). The end of the brass cator. Four corneas were removed for his­ applicator cap was concave to conform to tologie study at the end of each time inter­ the curvature of the rabbit cornea. The val: 5 minutes, 4, 17, 24 and 72 hours and alcohol used for sterilization was removed one and four weeks after freezing. Another from the cannula and brass applicator by four corneas were studied eight months washing it with distilled water and drying after freezing with the 8-mm applicator at with sterile swabs and a towel before use. -80°C. The eyes were fully exposed by sutures Another group of animals received appli­ through the lids and nictitating membrane. cations of — 80°C with a 4-mm applicator

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HELEN H. CHI AND CHARLES D. KELMAN

for each of the same short-term intervals already mentioned. The eyes of all animals were examined grossly and with the slitlamp biomicroscope daily and stained with fluorescein daily dur­ ing the first week, at two-day intervals for the next three weeks and at weekly intervals thereafter. Some of them were examined hourly for the first few hours. Before the animals were killed, in order to avoid post-mortem changes of the corneal endothelium, 10% neutral formolsaline was injected into the anterior chamber of the anesthetized rabbit using a 27-gauge needle. Immediately after this injection, the cornea was removed and placed in a large volume of the same fixative. The animals were then killed with intravenous injection of air and the remainder of the eye was enucleated. The flat preparations of the endothelium, involving the endothelium, Descemet's mem­ brane and as little as possible of the stroma were stained with hematoxylin only and mounted with the endothelial surface upper­ most. The slides were examined with both ordinary light and phase-contrast micro­ scopes. The rest of the cornea was bisected and half of it was sectioned in the transverse plane. The other half was prepared in flat sections. The tissues were embedded in ei­ ther paraffin or celloidin for study of the stroma and epithelium. These studies will be reported at a later date.

Fig. 2 (Chi and Kelman). The cornea a few sec­ onds after freezing shows a sharp demarcation be­ tween frozen and nonfrozen areas. (— 80° C, 8mm applicator, 20 sec, BIO, OS.)

tween the cornea and the applicator was loosened. This took about six to eight sec­ onds after freezing was discontinued. The frozen area was completely thawed in an­ other eight to 10 seconds. Immediately after thawing the cornea appeared quite clear. In­ stillation of fluorescein, however, revealed numerous punctate stainings of the affected corneal surface. Four to 24 hours after freezing the affected area showed a large fluorescein stain averaging about 7.5 to 8.0 mm. The

OBSERVATIONS AND RESULTS I. CLINICAL OBSERVATIONS

All animals were examined at regular in­ tervals as described previously. At — 80°C using an 8-mm applicator for 20 seconds, the frozen area of the cornea was about 12 mm in diameter and involved the whole thickness. There was a sharp demarcation line between the frozen and the nonfrozen areas (fig. 2). The frozen area extended Fig. 3 (Chi and Kelman). The frozen area ex­ into the anterior chamber (fig. 3). The ap­ tends into the anterior chamber (arrows). plicator was removed when the adhesion be- (—80°C, 8-mm applicator, 20 sec, B 9 OD.)

EFFECTS OF FREEZING ON OCULAR TISSUES

staining area decreased in size during the next few days and was completely covered by epithelium on the fifth to seventh day. One to two hours after freezing the corneas became hazy due to edema of the stroma in­ volving the posterior layers. At this time the anterior chamber was filled with fibrinous exudate containing white particles. After four hours corneal edema could be seen throughout the full thickness. When the edema reached its maximum about 24 hours after thawing, it was associated with fine striations of Descemet's membrane. The edema gradually subsided over the next sev­ eral days and by the 10th to the 15th days the corneas were usually quite normal in thickness, although varying degrees of opac­ ity remained (figs. 4 and 5). The longest period of persistence of corneal opacity was eight months, when the experiments were terminated (fig. 5). Slitlamp examination revealed that the opacities involved the whole corneal thickness, with most of the lesions showing denser opacity of the deep layers. The fibrinous exudate in the anterior chamber was completely absorbed by the end of the first week. Both pigmented and white precipitates were observed on the pos-

Fig. 4 (Chi and Kelman). Five months after freezing. Note large central corneal nebula and smaller, deep dense opacity. (— 80° C, 8-mm appli­ cator, 20 sec, B 59 OD.)

633

Fig. S (Chi and Kelman). Eight months after freezing there is a large, central, superficial corneal opacity and denser opacity of the deep layers. (—80°C, 8-mm applicator, 20 sec, B IS OS.)

terior surface of the cornea and the anterior lens capsule, but after a few weeks they gradually disappeared. At — 80°C using a 4-mm applicator, and at — 20°C with an 8-mm applicator, the fro­ zen areas were about six and eight mm, re­ spectively. They involved the whole thick­ ness of the cornea. The temporary edema which ensued after thawing lasted for seven to 10 days, after which the cornea regained its normal appearance and transparency on both gross and slitlamp examination. Immediately after thawing the epithelium of the affected zone showed numerous punc­ tate stainings with fluorescein, which be­ came one large staining area averaging 5.0 by 6.0 mm by the next day. This area was covered completely in two to three days. These eyes demonstrated a mild aqueous flare which disappeared in a few days. Using the 8-mm applicator and a temper­ ature of — 10°C, the frozen area was about eight mm in diameter and involved only the superficial layers of the corneas. Punctate staining with fluorescein in the affected area could be observed by slitlamp examination for a few days. Otherwise the corneas ap­ peared normal. Neither vascularization of the cornea nor secondary infection occurred after any of

HELEN H. CHI AND CHARLES D. KELMAN

634

Fig. 6 (Chi and Kelman). In a flat preparation taken five minutes after freezing the endothelial cells show aggregation of the nuclear chromatin into fine particles. The nuclei retain the normal kidneyshape, but are smaller than the normal endothelial nuclei shown on the left in the same magnification. (— 80 °C, 8-mm applicator, 20 sec, B 7 OD, hematoxylin, χ1280.)

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the cold applications in these experiments. Corneal vascularization may take place when the freezing involves the limbal area.2'5 In our experiments the freezing was produced in the central part of the cornea. I I . HlSTOLOGIC THELIUM

FINDINGS

IN

THE

ENDO­

This study was based on 116 eyes re­ moved at intervals of from five minutes to eight months after freezing at selected tem­ peratures as previously outlined.

1. At —80°C, using either the 4-mm or the 8-mm applicator, five minutes after freezing the endothelium in the affected area showed aggregation of the nuclear chromatin into fine particles. The nuclei retained their normal kidney-shape, but appeared smaller (fig. 6 ) . In the eyes of animals killed four hours later some endothelial nu­ clei showed marked shrinkage and stained more intensely basophilic (pyknosis) (fig. 7) ; other nuclei lost their ability to stain differentially with basic dyes (karyolysis)

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Fig. 7 (Chi and Kelman). Four hours after freezing some endothelial nuclei show pyknotic changes (arrows). Compare with the normal endothelial cells on the left. (—80°C, 8-mm applicator, 20 sec, B 1 OS, hematoxylin, χ 1280.)

EFFECTS OF FREEZING ON OCULAR TISSUES

635

so that they left only a stained nuclear conlour (fig. 8 ) , and still others revealed ag­ gregation of nuclear chromatin in coarse particle«! and fragments (karvorrhexis) (fig. 9). E\entually the mdnthelial celk appeared 1o have disintegialed and disappeared.

Fig. 8 (Chi and Kelman). Four hours after freezing the endothelial cells lose their ability to stain with basic dyes—karyolysis. (— 80° C, 8-mm applicator, 20 sec, B 1 OD, hematoxylin, χ1280.)

Fig 9 (.Chi ruid Kelman). AUci four hours after freezing. These endothelial cells reveal aggrega­ tion of nuclear chromatin in coarse particles (ar­ rows) and fragments (double arrows)—karyorrhexis. (—80°C, 8-mm applicator, 20 sec, B 2 OS, hematoxylin, X1024.)

Fig. 10 (Chi and Kelman). Flat preparation of the corneal endothelium 17 hours after freezing shows sharp demarcation between frozen and nonfrozen areas. (— 80° C, 8-mm applicator, 20 sec, B 4 OS, hematoxylin.)

HELEN H. CHI AND CHARLES D. KELMAN

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Fig. 11 (Chi and Kelman). Seventeen hours after freezing. (—80°C, 8-mm applicator, 20 sec, B 3 OS, hematoxylin.) (A) The endothelium is completely lost in the frozen area (b and c-zones) but stromal cells remain in the peripheral third (b-zone). There are spotty areas of migration of endothelial cells from the adjacent normal endothelium (a-zone) ( χ 8 0 ) . (Β) High-power view of the marked area of (A) (χ320). (C) High-power view of b-zone in (A), showing pyknotic changes and aggregation of nuclear chromatin of stromal cells ( χ 1024).

EFFECTS OF FREEZING ON OCULAR TISSUES

637

Fig. 12 (Chi and Kelman). Twenty-four hours after freezing various stages of mitosis (arrows) are frequently seen among the endothelial cells at some distance from the frozen area. (—20°C, 8mm applicator, 20 sec, B 49 OS, hematoxylin, X400.)

By the 17th hour the endothelium was completely lost in the frozen area, except for an occasional area in which some cellu­ lar debris remained. The acellular area was sharply demarcated and measured an aver­ age of 8.5 by 8.2 mm in the 8-mm applica­ tions (fig. 10) and 4.0 by 4.5 mm in the 4-mm applications. At this time, some stromal cells showed pyknotic changes and others revealed aggregation of the nuclear chromatin in the peripheral third of the fro­ zen area where the endothelium was com­ pletely destroyed (fig. 11). The central two thirds of the area was cell-free, with corn-

Fig. 13 (Chi and Kelman). One week after freezing the newly formed endothelial cells are of various sizes and shapes with oval or round nuclei. Note tiny vacuoles between the cells. (—80°C, 8-mm appli­ cator, 20 sec, B 10 OS.) Compare with inset of normal endothelial cells in the same magnification. (Hematoxylin, χ1280.)

638

HELEN H. CHI AND CHARLES D. KELMAN



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EFFECTS OF FREEZING ON OCULAR TISSUES

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Fig. 15 (Chi and Kelman). Four hours after freezing there are some apparently normal endothelial cells (arrows) among the endothelial cells showing aggregation of the nuclear chromatin in very fine and some coarse particles. (—20° C, 8mm applicator, 20 sec, B 13 OS, hematoxylin, X1280.)

plete loss of both endothelial and stromal cells (fig. 11-A). Simultaneously there were spotty areas where migration of endothelial cells from the adjacent normal endothelium had begun (fig. 11-A and B ) . Twenty-four hours after freezing, the en­ dothelial cells showed more evidence of re­ pair through migration and multiplication of cells by mitosis. The migrating endothelial cells were enlarged. They seemed to migrate individually with a considerable space be­ tween the most advanced and the next cell. Various stages of mitosis (fig. 12) could frequently be seen among the cells at some distance from the acellular area. During the next 48 hours the endothelial cells continued to multiply and migrate over the frozen area and these newly formed cells usually com­ pletely covered the lesions by the seventh day after the 8-mm applications and by the third day after the 4-mm applications. Their number and appearance, however, were still not normal. The unevenly distributed en­ dothelial cells were of various sizes and shapes with oval or round nuclei (fig. 13). Four weeks after freezing the endothelium had still not regained its normal pattern and appearance. s

The endothelium of the corneas removed eight months after freezing showed a con­ siderable number of bizarre, large nuclei (macronuclei) of irregular sizes and shapes in the formerly frozen area. (fig. 14). 2. Lesions produced with an 8-mm appli­ cator at —20°C showed generally similar changes but in a much milder degree. Five minutes after freezing there were no per­ ceptible changes of the endothelium under light and phase-contrast microscopic exami­ nation but, at four hours, most of the en­ dothelial cells in the frozen area showed some degree of aggregation of the nuclear chromatin in very fine and some coarse particles (fig. 15). The nuclei with fine particles of chromatin usually retained their normal shape and size. Some cells were pyknotic. Even though most of the cells showed some degree of necrosis, some apparently normal endothelial cells could still be found (fig. IS). In the 17- to 24-hour slides the endothel­ ium had completely disappeared from the lesions which now measured, on an average, 4.9 by 5.2 mm. Migration and multiplication of cells by mitosis from the adjacent normal endothelium had begun and the appearance

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Fig. 14 ('Chi and Kelman). Eight months after freezing a considerable number of endothelial cells have large, bizarre nuclei of irregular shapes and sizes (arrows) in the formerly frozen area. (A) —80°C, 8-mm applicator, 20 sec, B IS OS. (B and C) —80°C, 8-mm applicator, 20 sec, B IS OD. Compare with (D) which shows normal endothelial cells in the same magnification. (Hematoxylin, χ1280).

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HELEN H. CHI AND CHARLES D. KELMAN

the applicator. The temperatures are charted on the recorder to insure that the — 80°C temperature has been maintained. It is interesting to note that four hours after freezing at — 20°C, there were appar­ ently normal endothelial cells among the many cells in varying degrees of necrosis. It may be that these cells were in an especially insensitive state at the time of exposure, possibly because of a difference in the meta­ bolic stage or age, for instance. It is generally known that sensitivity to low temperatures differs in different types of cells. In our experiments, 17 hours after freezing with temperatures of — 20°C and —80°C, the endothelium is completely lost in the region of the freezing, but the stro­ COMMENTS Previous investigators reported that the mal cells at the peripheral third of the area cornea regained its normal transparency are still present, though they show pyknotic after damage of the corneal cells by changes and aggregation of nuclear chromafreezing.1'2·4 These reports, however, were tin in various sizes of particles. The overly­ based on the use of applicators measuring ing stromal cells were nearer to the applica­ 2.0, 5.5 and 6.0 mm with temperatures of tor than the endothelial cells, but the latter - 7 8 ° C and - 7 2 ° C for three to five and 60 showed complete destruction sooner. This seconds. In our investigation a temperature finding indicates that the endothelial cells of — 80°C applied for 20 seconds with an are more sensitive to freezing than the stro­ 8-mm applicator produced corneal opacity mal cells. lasting for the duration of the experiment The results of freezing determined for (8 months). It is generally known that the rabbit corneas in these experiments are not severity of the lesions produced depends necessarily fully applicable to the human upon the temperature, size and duration of cornea, because rabbit corneas are thinner the cold application. In comparison with and the sensitivity to freezing is different in Maumenee and Kornblueth's experiments, different species. Nevertheless the results of our temperatures were lower, applied for these experiments indicate that caution must longer periods and over a larger area. be exercised in the clinical application of At first glance the difference between the cryogenic treatment of such corneal pathol­ — 78°C used by Maumenee and Kornblueth ogy as pterygia, tumors and downgrowth of and the —80°C which we used may seem corneal epithelium in the anterior chamber, slight but actually the difference is more especially when the temperatures to be used than the 2°C-discrepancy would indicate. are as low as — 80°C or lower and the pa­ In their technique the applicators were thology involves as much as 60% of the cor­ cooled to — 78°C by immersion for a short nea. time in a beaker of absolute alcohol contain­ The question arises as to whether the per­ ing solidified carbon dioxide. During the sistent corneal opacity after freezing with three to five seconds' application the temper­ — 80°C is secondary to the destruction of a ature continuously rose. We used the Coo­ relatively large area of corneal endothelium per unit containing a microthermocouple causing prolonged edema of the stroma, or a which monitors the temperature at the tip of primary damage of the cells and other com-

was similar to that in the — 80°C cases de­ scribed previously. The injured area was completely repopulated by the newly formed endothelial cells in 72 hours but, after four weeks, the endothelium had still not re­ gained the normal cell pattern. 3. Using —10°C and the 8-mm applica­ tor, there was no perceptible endothelial change in any of the corneas removed at various intervals after freezing. Vascularization of the cornea did not occur in any specimens. The corneal cells were destroyed with minimal inflammatory reaction. The detailed epithelial and stromal changes will be described in a separate re­ port.

EFFECTS OF FREEZING ON OCULAR TISSUES

ponents of the stroma. Cogan6 reported that permanent corneal opacity sometimes de­ veloped after vigorous mechanical injury of the endothelial cells by implantation of a magnetic "flea" in the anterior chamber. Furthermore, in our experiments, the cor­ neal opacities occurred only in the 8-mm ap­ plications. However, using the same temper­ ature of —80°C for 20 seconds, but the smaller, 4-mm applicator, the temporarily edematous corneas regained normal tran­ sparency. These findings indicate that if the edema persists long enough, the opacity of the cornea will be permanent and irrevers­ ible. It is very important for the sur­ rounding healthy endothelium to regenerate and cover the damaged area in a relatively short time, before the stromal changes can become permanent. In cryogenic therapy of corneal lesions, if the pathology involves more than 60% of the cornea and temperatures of — 80°C or lower are indicated, two or three treatments using smaller applicators at different times may be preferred to one large application. The interval between the two applications should be long enough to allow the damaged area to be completely covered by the newly formed endothelial cells from the sur­ rounding normal endothelium.

641

Applications of — 80°C, using a 4-mm applicator and — 20°C using an 8-mm appli­ cator, caused temporary edema of the cor­ neas which regained normal transparency after seven to 10 days. At — 10°C, an 8-mm applicator applied for 20 seconds causes no visible effect ex­ cept for punctate staining of the corneal surface in the frozen area for a few days. Histologically, in cold applications using temperatures of - 8 0 ° C and - 2 0 ° C for 20 seconds, destruction of the corneal endothe­ lial cells is shown by aggregation of the nu­ clear chromatin, pyknosis, karyolysis and karyorrhexis, and followed by a repair pro­ cess. There is no perceptible endothelial change after the — 10°C applications. 210 East 64th Street (10021). ACKNOWLEDGMENT

The assistance of Mr. William Rice, Mrs. Kety Sapounova and Miss Irene Hughes is gratefully acknowledged.

REFERENCES

1. Mauraenee, A. E. and Kornhlueth, W. : Regen­ eration of the corneal stromal cells : I. Technique for destruction of corneal corpuscles by applica­ tion of solidified (frozen) carbon dioxide. Am. J. Ophthal., 31:699, 1948. 2. Regeneration of the corneal stromal cells : II. Review of literature and histologie SUMMARY AND CONCLUSION By means of a brass applicator eight mm Study. Am. J. Ophth., 32:1051, 1949. 3. Dunnington, J. H., and Smelser, G. K. : In­ in diameter fitted to the Cooper cryosurgery corporation of S35 in healing wounds in normal system, the central area of rabbit corneas and devitalized corneas. Arch. Ophth., 60:116, were frozen with temperatures of — 80°C, 1958. 4. Polack, F. M. : Isotopic labeling of corneal - 2 0 ° C and - 1 0 ° C for 20 seconds, and a stromal cells prior to transplantation. Trans­ 4-mm applicator was used at a temperature plantation, 1:83, 1963. 5. Sudarsky, R. D., Hulquist, R., and Chi, H. of - 8 0 ° C . H. : Cryogenically induced iris atrophy, iridectoAfter freezing with the 8-mm applicator my and cataract in rabbits. Am. J. Ophth., 60:217, at — 80°C for 20 seconds, various degrees 1965. 6. Cogan, D. G. : A new method for studying of corneal opacity occurred which persisted endothelial regeneration: Biomicroscopic observa­ for at least eight months, the period of these tion on normal and vitamin C deficient animals. Ophthalmologica, 118:440, 1949. experiments.