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| ARTICLE |
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| Year : 1962 | Volume
: 10
| Issue : 1 | Page : 1-8 |
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Corneal heteroplasty
A Dubois Poulsen
Paris, France
| Date of Web Publication | 18-Mar-2008 |
Correspondence Address:
A Dubois Poulsen
Paris
France
 Source of Support: None, Conflict of Interest: None  | Check |
How to cite this article:
Poulsen A D. Corneal heteroplasty. Indian J Ophthalmol 1962;10:1-8 |
Grafting of corneal tissues taken from a different animal than the recipient one, has for a long time been considered as biologically impossible and it seemed doomed to be forgotten. Recent developments have taken place in France on this question. The name of Pr. Payrau is in our country associated with these works and it is his conclusions that we shall endeavour to elaborate in this lecture.
The keratoplasty story proceeds through three successive stages.
It began in 1818 with animal experimentation by Reisenger soon followed by the works of Drogshagen, Thome, Feldman, Pawle, Munk; Dieffenbach and by the first attempts on man (Kissam 1838, Wutzer, Mulhauer). Each of these authors tried to use animal grafts (pig and sheep). The results were bad.
The second period is that of oblivion. Some episodic works testify that the idea was not dead, but it was considered merely as a vain imagination.
The third period in which we are still living is that of homoplasty. Notwithstanding some rare success in heteroplasty, Fuchs established, the dogmatic necessity of homoplasty and Magitot did, with pretty good reasons, condemn corneal heteroplasty. The considerable development of corneal grafting and the great improvement until now realised are due to this opinion. Nevertheless study of immunologic reactions of grafts permitted, during recent years, new attempts on different fundamental principles. Special preparation of grafts led Payrau and his collaborators to perform heterokeratoplasties on man and for the first time with clinical success.
Many problems have to be solved.
(1) Is corneal heteroplasty clinically possible and if so what are the inhibiting or facilitating factors?
(2) Is it possible on man and in this case what are its advantages and disadvant. ages over homoplasty?
It is sure that heteroplasty is experimentally possible. Authors previously quoted did have good results. Payrau had successful results in 466 keratoplasties on animal cornea.
It may well be stated that heteroplasty is possible, but successes depend much on conditions of implantation. This fact is an explanation of previous failures and of the global condemnation of heteroplasty. Rabbit is a bad recipient animal, and penetrating keratoplasty an unsuccessful method.
| Choice of Materials | |  |
The first notion to be put forward is the fact that results are much different according to the species of animal in experiment.
A first type (unfavourable) may be represented by lamellar grafting of fresh pig's cornea on a rabbit's eye. The results are always bad. Graft is eliminated during the first week in 25% of the cases, or after a good coaptation period, that is about ten or twelve days, inflammation takes place soon followed by dense and definite opacifica tion.
A second type (favourable) is presented by lamellar grafting of fresh calf cornea on a dog's eye. Evolution is always good, graft is well incorporated and transparency is thoroughly good after the third month.
To the unfavourable type belongs transplantation of fresh calf's, dog's or man's cornea on rabbit's eyes. On the other hand, transplantation of fresh rabbit's cornea on dog's eyes belongs to the second favourable type.
This demonstrates the necessity for a choice of donor and recipient to perform good lamellar heteroplasty.
Transfixing or penetrating heteroplasty seems destined to be defeated by secondary opacification. It must be realised, however, that post-operative immobilisation is quite an impossibility with animals.
Many authors (Babel and Bourquin Choyer) tried to facilitate tolerance by intra-lamellar keratoplasty. Their results are also poor.
| Lamellar Heterotransplants | | |
Payrau studied anatomic evolution of lamellar calf corneal transplant on dog's eye. This type of heteroplasty is always remarkably tolerated. It must be noted that there is no Bowman's membrane in dog and calf. Stroma keeps a regular form, incorporation is perfect in the first twenty-four hours, as well as on the bed as on the borders of the graft. There is no epithelial spur. Stroma soon becomes indistinguishable from the neighbouring cornea. Calf corneal epithelium is different from dog's epithelium. It is much thicker, with eight to ten layers of polyedric cells covered with keratine. The dog's epithelium has only five to six layers.
When graft is fresh, it becomes irregular and towards the third month it is generally thicker than the recipient cornea epithelium. But it has characteristics of a calf epithelium. On the contrary, when graft is silico-desiccated (see later), desquamation is less constant but it keeps the character of a highly keratinised epithelium of a calf. Nevertheless, we know that silicodesiccated cells are dead so it is necessary to state that alter implantation the cellular epithelial frame is inhibited by cells coming from the recipient cornea. The junction zone between recipient and donor cornea is a very discrete one.
On the other hand, anatomic studies showed the high quality of lamellar lyophilisated corneal transplants of a) pig on dog, b) pig on rabbit, c) rabbit on dog.
But dog, pig and rabbit's corneae are not very different from each other so that it may well explain the fact that morphology observed of calf and dog keratoplasty is not observed in these cases.
As a conclusion, it may be said that lamellar heteroplasty is histologically marked by):
(a) An intimate union of the two stromas.
(b) A good continuity between the two epithelia with no spur at their junction, as well with fresh as with desiccated grafts.
(c) Persistence of the thickness. If we use as donor, desiccated cornea, of a calf which has a thicker epithelium, it contrasts with that of the recipient cornea as regards thickness.
It is in fact the only disadvantage belonging to corneal heteroplasty.
Intimate union, rare epithelial spurs, are observed also with the same frequency in experimental heteroplasty with fresh grafts as with lyophilised heterografts. The spur is on the contrary constant with fresh heterografts.
Penetrating heteroplasty is, as we have already said, a very bad method. After an inflammatory stage, graft is completely opacified with neovascularisation.
It is now necessary to study the factors which lead to success or failure in heterografts.
| Failure Factors | | |
There are two reasons of failures.
In the first case it is an inherent absence of incorporation. It is almost constant with rabbit and especially with pig material. The mechanism is not well known. May be pig's cornea is too much swollen by too high and easy dehydration.
In the second case, failure seems to be the manifestation of an immunologic conflict. It is a case of an antigenic reaction .
It is nowadays well demonstrated, by study of cutaneous and tumor homoplasty in mice that the homograft reaction leading to reject the graft is due to an acquired recipient sensitivity towards the implant.
A first grafting sensitizes mouse against a second graft taken on a mouse of the same pure lineage as the first.
It is what Medawar calls "the second set phenomenon" which is characterised by earlier elimination of the second graft.
The foreign grafted tissue contains transplantation antigens (T antigens) which confers allergy towards grafting of the tissue.
It is possible to extract from tissues these antigenic substances. They keep their properties outside the living cells.
Intolerance to heteroplasty is an immunologic reaction too. It has a latency period of ten to twenty days before the beginning of the inflammatory process. It is possible to sensitize the eye by a first grafting, sensitization being manifest with shortening of latency period when second grafting is performed and with increasing intolerance.
This "second set phenomenon" is well marked when grafting calf or pig on rabbit's eye or when grafting calf and rabbit on dog's eyes. Desiccation lessens tissue incompatibility in fairly good proportions.
Payrau tried by laboratory studies to recognize the anatomic localisation and the chemical nature of the hetero-graft-antigens.
Intravenous injection of entire crushed cornea in four rabbits did not give any sensitization phenomena.
Therefore the local intracorneal route was next used. Rabbits were prepared by intralamellar inclusion of a fragment of calf cornea in left eye or by one to three intracorneal injections of different ox-corneal chemical extracts, (a) stroma extracts, (acido-soluble collagen, mucopolysaccharide), (b) epithelial extracts (total corneal epithelial, epithelial corneal desoxyribonucleoproteins).
Five days to three weeks later, the animals received a lamellar fresh bilateral keratoplasty.
It was observed that stroma extracts (collagen-mucopolysaccharides) never gave any sensitization. On the grafted (left) eye as well as on the experimental (right), the results were the same as that observed by grafting of calf's cornea on rabbit's eye:- that is to say there was vascular reaction and opacification.
On the contrary, epithelial extracts gave very positive results. Reactions on the prepared (right) eye were extremely severe, much stronger than on the left eye. After eight days, the swollen cornea was invaded by pannus vessels and became as red as a cherry. After two months, graft became opaque, irregular and pigmented.
With intralamellar implantation in rabbit's cornea BASU and ORMSBY demonstrated that if epithelium is removed, reactions are much weaker with homo as well as with heterografts (Streiff and Shapiro, Lieb and Lerman, Sykes and Girard).
Implantation in rabbit's cornea of epithelium stroma and endothelium leads Girard to the conclusion that epithelium is less tolerated than the two other tissues.
Fielding, Basu and Ormsby (1960) injected chemical fractions of ox cornea in rabbit's veins in order to develop serologic antibodies. They found that epithelial cornea extracts are much more antigenic than corneal extracts without epithelium. So, in cornea, transplantation (T antigens) as well as humoral antigens (H antigens) are localised in epithelial layers, that is to say in cells and not in stroma. They may be found especially in cell nuclei. This fact and certain biological properties led Medawar to think that they may well have some relations with desoxyribonucleoproteins. In fact, mucoproteins are in question.
Payrau tried to find relations between T corneal antigens and nucleic acids by grafting of cornea incubated in nuclease solutions (D Nase and R Nase). No difference was observed. After the DNP fractions are extracted from epithelium, corneal cells do not give any sensitization phenomenon and on the contrary chances of success of heterografting seem to improve.
Conclusion: It may be said that if cornea is considered with tendons, bones and vessels, one of the tissues that may be grafted with success, may be it is not because the graft is dead (or dies after implantation) but because the graft tissue is of a conjunctival nature, poor in cells, free from a fundamental substance and without vessels.
Factors which lead to failure in grafting are located in cells but are not linked to cellular life for, deprived of life, cell extracts have as great an antigenic power as living cells. It may well be expected that depriving graft of the major part of its cells by removing epithelium will reduce immunologic tissue incompatibility.
| Favouring Factors | | |
We already demonstrated that affinity between species is the most important point. But authors do not come to an agreement upon the question which are the species able to give the weaker reactions after transplantation. Magitot and Lacoste thought that best results were obtained with species near to each other (rabbit, guinea pig) and the worst with very different ones (rabbit and chicken for instance) Magitot questioned himself about possible success of grafting chimpanzee's cornea on man. But Nesterov was unsuccessful with transplantation of cat's, dog's and pig's corneze on rabbit's eyes when chicken cornea gave a good result with rabbit in nine of twenty-four cases. This is in contradiction with Magitot's assertions. Tsu Tsui and Watanale thought that heteroplasty from mammals on mammals was less successful than grafting from bird or fish to mammals-Payrau dealing only with mammalians stated that certain species are favourable and others unfavourable.
Dog is a good recipient animal. It accepts rabbit's and calf's grafts.
Rabbit is a bad recipient for man, calf, pig and dog.
Pig is a bad donor and it is always badly accepted by other species. The worst results are obtained with pig's cornea on rabbit's cornea.
Calf and rabbit are excellent donors for dog.
Calf and dog are good donors for man, on the contrary pig is badly tolerated. The dog thus becomes man's best friend in more respects than one.
So SPECIES AFFINITY is one of the main factors of success.
RECIPIENT QUALITY is the second factor. Unfortunately we do not know works about recipient preparation to heteroplasty, but why not transpose to corneal grafting, technics which have proved to be useful and tend to improve recipient tolerance.
Immunologic tolerance to cutaneous homoplasty may be obtained in mice of pure lineage by injection to the future recipient during its life, of living cells coming from mice of the donor's lineage.
The enhancement phenomenon consists in the longer survival of tumors transplanted on mice after previous injection of cells coming from the same tumoral tissue killed by heating, freezing, lyophilisation or injection of tissues extracts.
Cutaneous homoplasty may be prolonged by intravenous injection of epidermic cells of the future donor.
Is it possible to adopt same procedures with heteroplasty ?
If transplantation antigens in heteroplasty are specific of the donor species, there is no good reason why it should be otherwise.
It may be added to this consideration, post-operative use of local or general corticoids which have a good anti-inflammatory effect and also lessens the important secondary vascularisation.
Prescription of strong doses of Vitamin B., is always used a long time before and after grafting on vascularised cornea.
QUALITY OF THE GRAFT may modify the reactions to heteroplasty and these factors seem to be the most important ones.
The age of the donor has not the well known classical value which was attributed to it.
Epithelium abrasion lessens recipient immunologic reactions and facilitates good healing of the graft.
A certain amount of TREATMENT OF THE GRAFT BEFORE TRANSPLANTATION, gave recently some gratifying results.
Incubation of the donor's cornea in the recipient serum was tried but in that way, the graft gets too much swollen by hydration.
Conservation in vivo of the future graft in intralamellar corneal space of rabbit's eye gives good results when graft is transplanted for the second time to another rabbit. Graft seems to lose its H antigens and may be its T antigens.
But the best method nowadays is certainly tissular conservation by strong dehydration which brings complete death to the cells. (Payrau, Boenl and Guyard, 1958) (Payrau and Pouliquen, 1959-1960). It causes a considerable diminution of T antigens.
For five years Pr Payrau has been studying the attenuation of graft immunologic reactions by cryodesiccation or silico-desiccation. He was convinced of possibilities of success by two types of experiments.
In the first, he performed successive grafts on animal cornea in search of sensitization reactions.
In the second, he performed on the same cornea several implantations with grafts of different nature and observed their evolution.
In. the first type of experiments he put in a dog's cornea a heterogenous lamellar fresh graft of rabbit or pig. A second graft of the same species three to eight weeks later gave the proof of the sensitization, by an enormous reaction with opacification, vascularisation, iris hyperemia. This is a demonstration of the sensitizing power of the first graft.
The same experiment was then done with prepared grafts, using either lyophilisated or silico-desiccated grafts for the first implantation and testing sensitization by a second fresh graft, or making the first implantation with fresh cornea and doing three to eight weeks later a second heteroplasty with desiccated grafts.
In each case, grafts were well tolerated, not prominent with no conjunctival reaction.
These experimental results have very often been corroborated as well with silico or lyophilisated grafts as with lamellar or intralamellar grafts. These prepared grafts are less antigenic than fresh corneas.
The second type of experiments was based on the observation on evolution of grafts of different nature implanted on the same cornea, in other words in a medium developing towards them the same immunologic properties. The same characteristic evolution was always observed, that is the fresh lamellar graft developed a pannus with a huge neovascularisation, the silicodesiccated graft was well tolerated without any reaction or vascularisation. The neighbouring cornea remained clear and transparent. After two months silico-desiccated graft became invisible, the fresh graft got cloudy.
It was thought that diminution of antigenic power may be due to elimination in washing water of certain corneal proteins rendered soluble by desiccation.
It was then reasonable to try heteroplasty on animals and then in man.
Lamellar heteroplasty on animals with desiccated grafts affords two important notions.
(1) There is a remarkable attenuation of inflammatory reactions whatever be the species used in the experiments. Eye is quiet, graft is well incorporated and evolution is simple.
(2) There is an attenuation, but not a suppression of immunologic conflicts for the characteristic reaction of the species used are maintained exactly the same as formerly described. On rabbit's eye is observed the same bad evolution with pig, calf or man's desiccated grafts. On the dog, one can see the same favourable evolution with calf's cornea.
As with fresh corneas, transfixing heteroplasty performed with desiccated grafts are very disappointing. Cornea are soon opacified and full of vessels. The constancy of bad results led us to abandon this technic.
To sum up, the best technic for reducing the intensity of immunologic reactions is the preparation of grafts with epithelium abrasion and with conservation by desiccation, to which must be added the choice of the donor's species.
| Results | | |
The properties of lyophilisation and silico-desiccation now known, allowed trial of heteroplasty in man. Only lamellar grafts of corneal tissue, dehydrated and killed according to these methods, having a thoroughly good histologic structure, have been used and never fresh corneae.
Payrau performed thirty-two corneal heteroplasty. Personally we have done 20.
As it was demonstrated by animal experimentation, the donor's species is a very important factor.
Pig's desiccated cornea gives little inflammatory reaction but the evolution is always the same. The graft is well incorporated but gets opacified. Pig's grafts must be abandoned.
Quite different are the results with dog's cornea. Payrau performed sixteen graftings with dog's cornea in mild cases for therapeutic or optical reasons (one pterygium, seven herpes) and in six cases for trophic purposes.
One graft was eliminated but with the other ones, the follow up was particularly quiet. Graft was transparent as well as the neighbouring cornea. Therapeutic result was excellent. Pterygium did not relapse and herpes was definitely cured.
It is interesting to note that dog is refractory to herpes and that dog's cornea is certainly the best material to be used in the treatment of this severe disease.
Optical result is more satisfying, grafts are transparent and now have been so for more than three years.
Eight grafts were performed for trophic purposes on leucomatous and highly vascularised lesions.
In six cases no vascularisation enhancement was observed although it was strongly developed. In one case graft had an excellent action against vessels which disappeared in a few weeks.
Sixteen grafts have been performed with calf's desiccated corneae.
Five graftings for therapeutic purpose, against herpetic keratitis gave same results as with dog's cornea. Same tolerance, same evolution without any incident, same good result, without any relapse have been taking place for more than eighteen months. The optical result proved to be good but transparency is a little worse than with dog's cornea.
Nine graftings have been performed for trophic purposes. Each of them have been done on very badly opacified and vascularised cornea. These graftings are more recent than heteroplasty with dog's cornea so it is difficult to give a valuable opinion, but it seems when considering the oldest cases (eighteen to twenty-two months) that their trophic action is less important than that of dog's cornea.
In two recent cases, Payrau tried to treat severe chemical corneal burns with porcelain opacity and high vascularisation. Fresh homografting was contraindicated. The graft has been so far tolerated (one of dog, the other of calf), respectively for ten and five months. Unfortunately, stroma still remains opaque.
One transfixing keratoheteroplasty was performed on man with a calf silico-desiccated graft. (Giani and Ligorio). These authors have been astonished by good coaptation without any reaction. But the optical result was bad.
It may well be concluded that lamellar corneal heteroplasty is more than experimentally possible. It already gives both good optical and histologic results.
| Preparation of Desiccated Grafts | | |
For this purpose dog's and calf's cornea are selected.
Freezing at very low temperature is a good means for conservation, but it is necessary to possess a good permanent installation to produce cold and it is impossible to forward fresh grafting material. Lyophilisation or cryodesiccation is a better technic. It consists in a congelation at low temperature, followed by sublimation of tissular water in vacuum. Material so desiccated and kept in vacuum may be indefinitely conserved and expedition is very easy. Efficiency is absolutely total.
But the method is not simple, apparatus is expensive, twelve to fifteen hours are necessary to perform good sublimation. Grafts must be rehydrated by serum immersion one hour before operation.
Silico desiccation is a more simple technic. It is a mild desiccation and is not expensive.
The cornea to be conserved is wrapped in a cellophan paper and then placed in a bottle containing a silica microporous gel. The bottle is corked. When the gel is dry, it is blue, but becomes red when in contact with humidity. The blue colour of the product is then the guarantee of a good conservation. Naturally, the bottle, the silica gel, and the cellophan sheet are previously sterilized.
Tissues so treated are almost totally dehydrated. They are completely dead. Their histologic structure is remarkably well preserved. They have lost their antigenic power. A good technic is to add to silico-desiccation an alcohol bathing to sterilise the corneal tissue either before or after desiccation. The graft must be rehydrated before operation.
Sclera may be prepared too with this technique. It gave to Payrau the good results we have reported.
| Conclusion | | |
Corneal heteroplasty is possible with thoroughly good results.
Intolerance reaction to graft is an acquired immunologic reaction, the antigens of which must be found in the cell constituents mainly in graft's epithelium.
The intensity of this reaction may be lessened by choice of the species in question, by good preparation of the graft and by a previous treatment of the recipient animal.
Lyophilisation or silico-desiccation lessen the antigenic power of the graft and by this fact make heteroplasty more similar to homoplasty.
Used on man, grafts of dog or calf preserved by these methods, give good optical, therapeutic, trophic and antivascular results.
The best results are obtained with dog's cornea. The optical role is demonstrated but is not constant. Transfixing grafting must be abandoned.
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