PRESERVATION OF CORNEAL GRAFTS BY FREEZING

237 which is associated with a total, or almost total, absence An excess of of alpha cells in the pancreatic islets. is not in the liver in this depositedhypoglyglycogen caemic syndrome. Conclusion

The intravenous injection of raise the blood-sugar level in storage disease of the liver. I

am

grateful

to Dr. W. R.

glucagon (HGF) did not a child with glycogen-

Kirtley,

of Eli

Lilly

&

Co.

Ltd., for supplying the HGF used in this investigation. REFERENCES

Andersen, D. H. (1952) In Carbohydrate Metabolism. Baltimore. Bridge, E. M., Holt, L. E. jun. (1945) J. Pediat. 27, 299. Cori, C. F. (1952) In Carbohydrate Metabolism. Baltimore. Crawford, T. 1946) Quart. J. Med. 15, 285. de Duve, C. (1953) Lancet, ii, 99. Forbes, G. B. (1953) J. Pediat. 42, 645. Lamer, J., Illingworth, B., Cori, G. T., Cori, C. F. (1952) J. biol. Chem. 199, 641. MeQuarrie, I., Bell, E. T., Zimmermann, B., Wright, W. S. (1950) Fed. Proc. 9, 337. Staub, A., Sinn, L., Behrens, O. K. (1953) Science, 117, 628. Stetten, D. jun., Boxer, G. E. (1944) J. biol. Chem. 155, 231. Thorn, G. W., Emerson, K. jun. (1950) In Harrison, T. R. Principles of Internal Medicine. Philadelphia; p. 716. Ulstrom, R. A., Ziegler, M. R., Doeden, D., McQuarrie, I. (1952) Metabolism, 1, 291. Van Creveld, S. (1939) Medicine, Baltimore, 18, 1. — (1952) Arch. Dis. Childh. 132, 113. von Gierke, E. (1929) Beitr. path Anat. 82, 497.

PRESERVATION OF CORNEAL GRAFTS BY FREEZING H. H. G. EASTCOTT M.S. Lond., F.R.C.S. ASSISTANT

DIRECTOR, SURGICAL

UNIT

A. G. CROSS M.A.,

M.D. Camb., F.R.C.S.

CONSULTING OPHTHALMIC SURGEON

A. G. LEIGH M.D. CONSULTING

Lpool, F.R.C.S. OPHTHALMIC SURGEON

D. P. NORTH Lond., D.O.M.S.

M.B.

SENIOR OPHTHALMIC REGISTRAR ST.

MARY’S HOSPITAL,

LONDON

THE Corneal. Grafting Act (1952) has simplified the of obtaining cornese for homografting in man, but difficulty often arises in obtaining suitable material at the precise time when it is needed. Present methods of storing corneal grafts have the common feature that the tissues are refrigerated above their freezing-point. This limits their storage life, and it is necessary to reject unused grafts after a few days. It is therefore difficult to build up a reserve or selection of grafts ; each must be taken shortly before the operation for which it is intended. A further inconvenience is the necessity for cutting lamellar grafts either from the eye in situ on the cadaver or from the excised whole eye. attempts have been made to store corneal grafts over long periods, and various methods have been tried in animals. Weiss and Taylor (1944), Katzin (1947), Leopold and Adler (1947), and Smelser and Ozanics (1946) all used rapid freezing in liquid nitrogen, with and without isopentane as an intermediate heat-transfer medium. In the method described in the first three of these papers the grafts after freezing were dried in vacuo over PzOs at -40°C. They were reconstituted before use in most cases by the addition of isotonic sodium-

legal aspects

Many

chloride solution. Smelser and Ozanics did not dry their material but stored it at -195°C for periods of 1 hour up to 5 days. The grafts were then rapidly thawed by immersion in isotonic sodium-chloride solution at 39°C. When thus prepared for implantation, both the frozen and the freeze-dried grafts were found to be clear and of normal consistence and they handled well. Loosening of the epithelial layer was noticed after the thawing of the frozen cornea. The great majority of these grafts healed well after implantation, but in most of them translucency was lost within the first 3 weeks. No lasting transparency was demonstrated in a total of 106 animal operations. We therefore considered the possibility of storing corneal grafts by freezing, using the glycerol-saline technique (Polge et al. 1949) which has been shown in other tissues to be compatible with a high percentage of surviving cells. We describe here the method which we have found to give satisfactory results in the preservation of human corneal tissue for grafting. Method

The Donor Cadaveric material has been used throughout this investigation. The material was obtained as soon as possible after death and never more than 12 hours later.

Graft-takinq A sterilised pack was kept ready in the hospital and contained : (1) eye speculum, (2) 2 pairs -of fixation forceps, (3) Graefe -knife, (4) large scalpel, (5) 2 bijou bottles, and (6) 2 sterile towels. The corneal material was taken under aseptic precautions. A lid speculum was inserted and the eye was irrigated with sterile normal saline. When taking corneae for fnll-thickness grafts a Graefe section was made at the upper part of the cornea ; the cut edge was grasped and raised with fixation forceps ; and the knife was reversed to complete the incision below. Experience showed that it was difficult to cut a satisfactory lamellar graft from an excised cornea. A technique was evolved therefore in which the donor material was cut direct from the cadaver eye in situ. A rectus-muscle insertion was grasped by the forceps in the left hand to steady the eye. Using the large scalpel with a new blade in the right hand, and ensuring that both blade and cornea were wet, a lamella was shaved off the cornea. With practice a lamella some 0-5 mm. in thickness and involving some two-thirds of the area of the cornea could be obtained (fig. 1). Grafts of varying thickness are cut so that there is a selection available to meet the requirements of each particular case.

Lamellae and full-thickness grafts were placed in sterile bijou bottles and were covered with an excess of 15% glycerol in Ringer saline (pH 6). After standing thus for 1 hour at room-temperature, most of the glycerol saline was decanted, leaving only just enough to cover the graft. The screw-cap was tightened as firmly as possible, and the bottles were immersed in a container of carbon dioxide and alcohol freezing mixture at -79°C. They were subsequently stored at this temperature after being transferred to a dry container. Both containers were kept surrounded with solid carbon dioxide, held in a lagged Perspex’ box within a standard commercial deep-freeze. This apparatus has been described elsewhere (Eastcott 1953) as a convenient method for ensuring a constant low temperature for the preservation of frozen arterial grafts. The storage-times for the 12 grafts which have been used up to the time of writing are shown in the accompanying table. Older grafts are now available for further trial. Thawing was rapid in all cases. The bottle containing the frozen cornea was taken from the deep-freeze, immersed immediately in a water-bath at 40°C, and

238 as soon as the medium and its graft could be The medium was decanted, its to have thawed. was estimated with a Cambridge pH meter, and

Results

removed seen

pH

fresh sterile Ringer solution was substituted. The grafts taken to the operating-room, and immediately before use the donor material was transferred to a normal saline solution contained 1000 units. of penicillin per ml. were

OperatÜ’e Technique After the donor material had been thawed the technique of grafting was the same as that with fresh material. The points of importance which we wish to emphasise are :

1. The grafts were cut by the punch technique, to sever the cornea! fibres without distortion. 2. The recipient cornea was cut with the same trephine that had cut the donor cornea. 3. The graft was held in place by indirect sutures, for we consider that direct sutures between the graft and the recipient cornea cause unnecessary trauma to the graft. Only in the larger lamellar grafts did we use direct sutures.

Characteristics Both lamellar and full-thickness grafts appeared normal in every way after thawing, except that loosening of the epithelium was noticed in 2 cases. pH of Thatved lyledium This was found to vary between 4-0 and 6-2 (see table). The pH was unrelated to the duration of storage; nor did the pH of the medium appear to influence the clinical result after grafting. 0 perafÚ’c Results Lamellar grafts.-Of the 5 lamellar grafts used, all have remained clear up to 10 months after operation. P’ull-thickness grafts.-7 full-thickness grafts were applied. In each of these cases effective union was obtained between the graft and the recipient cornea. The postoperative appearance of these grafts was similar, for they showed considerable opacification which was slow to disappear.

Physical

.-

’

Of these opaque ;

the

grafts

1

(case 3,

see

table)

became

completely

opacification progressed steadily until the graft was replaced by an opaque mass of fibrous tissue. Subsequently the eye was regrafted with fresh material and full vision was obtained.

Case 4 similarly became opaque and was successfully regrafted with fresh material. In this case the graft with frozen material was the second perforating graft applied to that eye ; the first graft (fresh material) had become opaque

in

the

absence

of

any

recognised postoperative complication. In cases 10 and 11 the grafts used to replace the defect have remained clear, though in this position vision was not influenced. Case 2 had improvement of her vision to 6/24 after grafting for corneal scarring due to interstitial keratitis.

Case 5 has a completely clear graft, and the patient is now awaiting a cataract extraction. Case 12 has

regained 6/6

unaided vision

(fig. 2). Discussion

All the lamellar grafts in this series have remained completely clear. Their postoperative behaviour differed in no respect, The fullfrom that of fresh material. thickness grafts are less satisfactory ; for, despite complete success in 2 cases and partial success in 3 cases, these do not appear to behave in the same manner as grafts of fresh material. Considerable opacification was seen at the first dressing in all cases ; and this, even in the successful cases, took some time to clear. It was impossible to say in which cases complete clearing could be expected. It is likely that the’ freezing process, while not harming the substantia propria, even when this is cut relatively thick (05 mm.), does damage- the more delicate -endothelium so that the normal nutritional exchanges between the aqueous humour and the cornea are disturbed with resultant opacification of the graft. One of us (Leigh 1954) has recently shown that sudden opacification of a perforating graft can occur when the endothelium is obliterated by a growth of fibrous tissue over the posterior surface of the graft. It is also interesting to recall that in some of the frozen grafts a loosening of the Fig. I-Method of taking lamellar corneal grafts from cadaver eye in situ.

epithelium

was seen on

thawing.

’. ,

239 It seems probable that this periods. to the cornea, though direct applies proof has not yet been obtained. Secondly, if it be granted that a corneal graft should be alive at the time of transplantation, does it survive in the recipientQ

Green (1940) showed that the anterior chamber can maintain tissue-grafts from another individual, and Billingham and Boswell (1953) have found that homografts of split skin survive when placed on the cornea if vascularisation does not develop. These findings suggest that in man the glycerol-frozen corneal graft not only survives the processes of storage but also after a successful transplantation may remain alive in the recipient.

Summary Partial-thickness corneal grafts stored at 79°C after immersion in 15% glycerol in Ringer’s saline have been found to give results which equal those obtainable with fresh material. 6/6 unaided. It is also nossible to obtain comnlete transparency in full-thickness corneal grafts stored in the same way. Further study is needed to determine and eliminate the causes of failure in this group. It seems that by this method lamellar grafts can be stored indefinitely. Thus wastage is very greatly reduced. -

Fig. 2-Full-thickness

corneal

graft

which has restored vision to

In this hospital frozen corneal material is in routine for lamellar grafts. Since the technique of obtaining this type of graft is simple and causes no mutilation of the cadaver a considerable quantity of lamellae are at our disposal in the deep-freeze, and we have no difficulty in selecting a suitable lamella of requisite thickness for use

a

particular

case.

We are unable, however, to advocate the routine use of frozen full-thickness grafts. We are engaged in the study of the problem of opacification in these cases. After homografting, cornea, in common with most other tissues, has been considered to be replaced by the recipient’s tissues. It may be necessary to review this opinion in the light of recent observations. Firstly, no corneal graft has been shown to remain translucent after preservation by any method which would have killed its cells, although the tissue-proteins were otherwise fresh and undenatured. Freezing in glycerol solution is the only method known at present by which normal mammalian cells can be stored frozen for indefinite

We are indebted to Prof. W. D. Newcomb and to Prof. C. G. Rob for their help in providing facilities for the collection and banking of these grafts. ,

REFERENCES

Billingham,

R. E.

B., Boswell, T. (1953) Proc.

roy. Soc.

B, 141,

392.

Eastcott, H. H. G. (1953) Ann. R. Coll. Surg. Engl. 13, 177. Green, H. S. N. (1940) J. exp. Med. 71, 305. Katzin, H. M. (1947) Amer. J. Ophthal. 30, 1128. Leigh, A. G. (1954) Brit. J. Ophthal. 38, 10. Leopold, I. H., Adler, F. H. (1947) Arch. Ophthal., N.Y. 37, 268. Polge, C., Smith, A. U., Parkes, A. S. (1949) Nature, Lond. 164, 666.

Smelser, G. K., Ozanics, V. (1946) Proc. Soc. exp. Biol., N.Y. 62, 274.

Weiss, P., Taylor, A. C. (1944) Anat. Rec. 88, suppl. p. 49.

DATA ON CASES AND GRAFTS