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Heterograft of fish Cornea into Rabbit Eyes*
HETEROGRAFT O F F I S H CORNEA INTO RABBIT EYES* J U N TSUTSUI, M.D.,
AND SAEKO WATANABE, B . S C .
Okayama-Shi, Japan
of the grafts were pretreated in rabbit serum for 24 hours in a refrigerator at 4°C. At the time of grafting a transverse in cision five-mm. long and about 0.25-mm. deep was made with a fine knife at the upper Hmbus, slightly on the corneal side. The cornea was split from the incision with the help of a spatula with a thin edge, making an interlamellar pocket sufficiently large to ac commodate the grafted disc, eight mm. in diameter. The graft was inserted into the intracorneal pocket with help of the spatula, care being taken to spread the graft as uni formly as possible. The transverse incision was closed with two sutures just after the insertion of the graft. In order to avoid an immunologie influence, the grafts were placed in both eyes at the same time. In most cases, the corneas were taken from the fish stored in ice for two to five days. In other instances they were obtained from the living fish. In the main experiments 19 eyes in 12 rabbits were grafted with fish corneas and as a con trol, human (two eyes), ox (two eyes) and dog (one eye) grafts were used. The period of observation was two to 10 months on 12 eyes in eight animals. For a histologie exam ination in the early stage seven eyes in four animals were enucleated after 3, 10, 20, and 30 days. Postoperatively all the cases were treated with antibiotics for three weeks. Topical administration of cortisone was con tinued for three months in most cases.
More than a hundred years ago keratoplasty with heterografts was first attempted. During the past century many attempts were made on human eyes but there was no chance of a successful corneal graft unless the donor material came from the same species. Corneal heterografts are still believed to be impossi ble by most ophthalmologists. The pioneer work on heterografts was done by Babel and Bourquin,1 and Choyce2 in 1952, and their works stimulated a reinvestigation of this problem. The clinical use of chicken cornea to human eyes was also attempted by Kuwahara3 with some success. Kamata 4 reported a study of corneal heterografts of chicken cornea into rabbits' eyes. Basu and Ormsby 5 reported interesting evidence that the anti gen-antibody reaction of rabbit's cornea fol lowing a corneal heterograft was less fre quent with an avian cornea than with a mam malian cornea. Their result suggested that the further apart phylogenetically the two tissues, the less likely it is that a donor re cipient reaction will develop. This led to our experiments on corneal heterografts of fish cornea into rabbit eyes. METHOD AND MATERIAL
Adult rabbits weighing from 2.5 to 3.5 kg. were used as recipients. Graft tissue was ob tained from various kinds of fish: perch, mackerel, seriola, graymullet and bonito. Us ing Castroviejo's trephine a lamellar disc of the cornea, eight mm. in diameter and 0.3 to 0.8 mm. thick, was prepared and sterilized in a solution of penicillin, streptomycin, and sulfisomidin for one-half to two hours. Some
RESULTS
* From the Eye Department and Eye-Bank Labo ratory, Okayama Rosai Hospital. We are indebted to Dr. Frederick C. Blodi of the State University of Iowa and Dr. S. Tsuda of Okayama Rosai Hos pital for valuable suggestions and for aid in the preparation of this manuscript. Presented at the XVIII International Congress of Ophthalmology, Brussels, September, 1958.
In the preliminary experiment the opera tive technique was worked out on 10 animals and finally the method already described ap peared to be the most suitable. These first animals were excluded from the following data. In the main experiment interlamellar heterografts were done on 19 eyes in 12 rab bits. Seven eyes in four rabbits were ex cluded from the clinical results because of
363
364
JUN TSUTSUI AND SAEKO WATANABE
Fig. 1 (Tsutsui and Watanabe). Rabbit 23, left eye. Perch interlamellar graft, first postoperative day, slight opacity of graft and iris congestion. Resulted in transparent graft after two months.
Fig. 3 (Tsutsui and Watanabe). Rabbit 5, left eye. Mackerel interlamellar graft, 10th postopera tive day, no active inflammation, transparent graft.
the early enucleation for histologie examina tion within one month. Therefore clinical ob servation was performed on 12 eyes in eight rabbits for two to 10 months.
sided soon and the transparency of the grafts returned during the 10th to 20th postopera tive days (fig. 3). There was a variable degree of vascularization from the upper limbus around the op erated area but in no case was the central area vascularized. After three to four weeks these vascularizations almost disappeared but they could still be detected by slitlamp exam ination. After one or two months the con dition of the corneas became stable. The grafts maintained their transparency for a long time, perhaps permanently (figs. 4 and 5). Temporary opacity and inflamma tion never appeared after three weeks. In some cases a small fault of the surgical tech nique—for example, perforation of the su perficial layer of the recipient cornea, folding of the graft in the interlamellar pocket or too
I. CLINICAL RESULTS
1. Transparent heterograft. The transpar ency of the heterogeneous grafts showed a slight alteration during the postoperative course but finally 10 grafts out of 12 were almost transparent. The postoperative course was similar in each case. During the first several days slight irritative manifestations such as edema of the graft, traumatic pannus around the incision, and ciliary and iris con gestion appeared in most cases (figs. 1 and 2). These immediate reactions, however, sub-
Fig. 2 (Tsutsui and Watanabe). Rabbit S, left eye. Mackerel interlamellar graft, fourth post operative day, slight pannus from upper limbus, transparent graft. Resulted in transparent graft after 10 months.
Fig. 4 (Tsutsui and Watanabe). Rabbit 5, left eye. Mackerel interlamellar graft, 67th postopera tive day, transparent graft.
HETEROGRAFT OF FISH CORNEA
Fig. 5 (Tsutsui and Watanabe). Rabbit S, left eye. Mackerel interlamellar graft, 164th postopera tive day, transparent graft.
thick a graft (over 0.7 mm.)—might pro duce a localized opacity and vascularization. These opacities were not extensive and re mained as a small nebula. 2. Opacified heterografts. In two cases out of 12 ( 16.6 percent) the grafts became opaque more or less immediately after the operation. The opacification was extensive and covered the whole graft with a pannus-type vascu larization (fig. 6). After a week, the grafted cornea became necrotic and the inflammation extended to the iris and ciliary body. Finally the cornea developed into a dense leukoma with extensive vascularization. In one case, the cause of the opacified graft was found to be an infection by Pseudomonas aeruginosa and in the other eye it was too thick a graft with a poor fit into the interlamellar pocket.
Fig. 6 (Tsutsui and Watanabe). Rabbit 8, right eye. Mackerel interlamellar graft, fifth postopera tive day, opaque graft.
365
Fig. 7 (Tsutsui and Watanabe). Rabbit 12, right eye. Dog interlamellar graft, sixth postoperative day, opaque graft.
3. Control experiments. As control exper iments human (two cases), dog (one case) and ox (two cases) corneas were grafted interlamellarly into rabbit cornea with the same technique as already mentioned. The grafts were clear during three to 10 days and then the opacification of the entire graft developed with haziness of the corneal stroma in the recipient eye (fig. 7). In the case of the dog cornea the graft became necrotic. Large permanent opacities remained in all cases. I I . HlSTOLOGIC RESULTS
Enucleation of the grafted eyes was done on 3, 10, 12, 13, 20, 67, 72, and 154 days postoperatively. The graft was transparent in 10 cases and opaque in two cases at the time of enucleation. In the control experi-
Fig. 8 (Tsutsui and Watanabe). Rabbit 17, right eye. Bonito interlamellar graft, third postoperative day, no donor-recipient reaction, transparent graft.
366
JUN TSUTSUI AND SAEKO WATANABE was a violent inflammatory reaction. The epithelial layer became thick and irregular. The corneal stroma was irregular in the ar rangement of the fibers with numerous in flammatory cells and vessels. The presence of a graft could not be clearly determined. There were many inflammatory cells in the anterior chamber (fig. 12).
Fig. 9 (Tsutsui and Watanabe). Rabbit 11, left eye. Graymullet interlamellar graft, 30th postopera tive day, no donor-recipient reaction, transparent graft.
3. Control experiments. The histologie findings of this group were similar to those of the opacified grafts of fish cornea. A vio lent inflammatory reaction, necrosis and de struction of the grafts with a remarkable vascularization were present (fig. 13). DISCUSSION
ments, a dog and a human heterograft were examined histologically. 1. Transparent heterografts. The corneas which were enucleated between one week and one month showed the recipient corneal stroma to be unaffected by the introduction of the foreign body. Only few inflammatory cells or vessels were noted. T h e grafted fish cornea was enclosed between lamellae of nor mal aspect, though the nuclei appeared rare fied and showed hardly any pyknosis or other changes. The grafted stroma showed a ho mogeneous structure with very fine fibers and no destructive or inflammatory changes (figs. 8 and 9 ) . After two months the grafted cornea showed as a characteristic finding the development of a nuclear layer in the superficial area of the graft. These nuclei may come from the recipient corneal stroma as an organization of the grafted tis sue by the recipient tissue which seems to start during the second and third postopera tive months (fig. 10). After five months the structure of the grafted cornea became so similar to the host cornea that the existence of the heterogeneous tissue was no longer detectable. Particularly, the arrangement of the nuclei and of the corneal fibers was very similar to the recipient cornea (fig. 11). 2. Opacified heterografts. T w o cases of opacified heterografts were examined his tologically at the end of one month. There
W e utilized the idea of Basu and Ormsby 5 and extended it in our series of experiments on corneal heterografts using fish cornea. In a total of 12 cases of interlamellar corneal heterografts, 10 cases healed with a trans parent graft. The rate of successful trans plantation was quite high in our experiments. On the one hand Babel and Baurquin 1 had about 50 percent successful heterografts us ing human and cat cornea in rabbits. O n the other hand, Basu and Ormsby 5 obtained a 66.6 percent success in avian grafts and a 51.8 percent in mammalian grafts. In fish grafts we were successful in 83 percent of
Fig. 10 (Tsutsui and Watanabe). Rabbit 23, left eye. Perch interlamellar graft, 67th postoperative day, no donor-recipient reaction, nuclei of stroma cells appear in the superficial layer of the graft, transparent graft.
HETEROGRAFT
Fig. 11 (Tsutsui and Watanabe). Rabbit 6, left eye. Bonito interlamellar graft, 151st postoperative day, well organized graft with replaced corneal stroma cells, transparent graft the eyes. In our small number of control ex periments using mammalian cornea none of the grafts remained transparent. Conse quently, we also believe that there is evidence that the farther apart phylogenetically the donor and the recipent are the less likelihood there is that a donor-recipent reaction de velops. The factors necessary to obtain a clear graft seemed to be: No characteristic correlation was found between a successful graft and the species of fish used. T h e postoperative donor-recipient reaction seemed to be more intensive when a fresh
Fig. 12 (Tsutsui and Watanabe). Rabbit 16, left eye. Seriola interlamellar graft, 30th postoperative day, extensive donor-recipient reaction with irregu lar stroma cells and numerous inflammatory cells, opaque graft.
Fig. 13 (Tsutsui and Watanabe). Rabbit 12, left eye. Dog interlamellar graft, 30th postoperative day, extensive donor-recipient reaction with nu merous inflammatory cells, necrotic graft. graft was used and less intensive in older grafts. T h e cornea taken from fish stored for a few days in ice appeared to be ade quate. If the cornea was stored for more than 10 days the results were unfavorable. This evidence suggests that an adequate denaturation of the proteins may decrease the antigenicity. A preoperative serum treatment of the graft appeared to have no influence on the fish cornea. Kamata 4 reported that in using chicken cornea into rabbits' eyes the preop erative serum treatment was very effective in preventing a donor-recipient reaction. The antigenicity of fish protein may be so weak that no definite donor-recipient reaction de velops. Postoperative use of cortisone appears to be effective in preventing or reducing the development of a donor-recipient reaction but it is not absolutely necessary since some cases healed with a transparent graft without the use of cortisone. One of the most important factors to obtain a clear graft is accurate surgical tech nique. In our method a graft of less than 0.6 mm. is adequate. A graft of over 0.7 mm. may easily cause opacification and vascularization. Folding of the graft in the in terlamellar pocket is the cause of a localized opacity and vascularization. A perforation of the superficial layer of the recipient cornea
368
JUN TSUTSUI AND SAEKO WATANABE
also gives an unfavorable result. Conse quently we might be able to obtain a clear graft in all instances if our surgical tech nique were perfect and if the graft had ade quate freshness and thinness. The question remains open why these transparent heterografts are obtained in such a high percentage using fish cornea. We can only explain it from the phylogenetic view-point. Further studies on the protein chemistry and séro logie factors may give more concrete an swers. The organization of the grafted tissue into the recipient cornea was observed by his tologie examination. In the first month the nuclei of the grafted tissue disappear. During the second and the third months, organization of the graft begins on the surface. Nuclei of stroma cells appear in the peripheral zone of the graft and then in the deeper layers. Thus the organization progresses and finally all cells of the graft are completely replaced by the host cells.
CONCLUSION
For the purpose of clarifying the factors which make corneal heterografts successful fish corneas were transplanted interlamellarly into rabbits' corneas. The grafts, eight mm. in diameter and 0.3 to 0.8 mm. thick, were taken from perch, mackerel, seriola, graymullet and bonito. Some of the grafts were pretreated with a rabbit serum in a refrig erator for 24 hours. After a slight donor-recipient reaction, transparent heterografts were obtained in 10 cases out of 12. In the control experiments using human, dog and ox grafts, all the grafts became opaque during three to 10 days. The histologie reaction of the recipient cornea was slight in the successful hetero grafts. The process of organization of the grafted tissue into the recipient cornea ap peared one month after the operation and was completed five to six months later. Okayama Rosai Hospital.
REFERENCES
1. Babel, J., and Bourqin, J. B.: Experimental research with corneal heterografts. Brit. J. Ophth., 36: 529, 1952. 2. Choyce, D. P.: Successful transplantation of human and cat corneal tissue into rabbit cornea. Brit. J. Ophth, 36:537, 1952. 3. Kuwahara, Y. : Successful transplantation of chicken cornea into human cornea. J. Clin. Ophth, 10:963, 1956. 4. Kamata, W. : An experimental study of heterokeratoplasty. Acta Soc. Ophth, Japan, 61:1098, 1957. 5. Basu, P. K, and Ormsby, H. L.: Corneal heterograft in rabbits. Am. J. Ophth, 44:477, 1957.
PROPHYLAXIS AND T R E A T M E N T O F ENDOPHTHALMITTS* M.
NEVEU,
M.D.
Three Rivers, Quebec AND
A. J. ELLIOT,
M.D.
Toronto, Canada
In the practice of ophthalmic surgery one of the most alarming and distressing condi tions encountered is an acute endophthalmitis following a well-executed surgical proced ure. Unfortunately most of these cases de* From the Department of Ophthalmology, Uni versity of Toronto and Toronto General Hospital.
velop in apparently clean eyes showing no complications, where the co-operation of the patient has been excellent and a normal post operative convalescence could be expected. Occasionally one must undertake surgical treatment of patients in whom chronic infec tions have provided a warning but, all too
AND SAEKO WATANABE, B . S C .
Okayama-Shi, Japan
of the grafts were pretreated in rabbit serum for 24 hours in a refrigerator at 4°C. At the time of grafting a transverse in cision five-mm. long and about 0.25-mm. deep was made with a fine knife at the upper Hmbus, slightly on the corneal side. The cornea was split from the incision with the help of a spatula with a thin edge, making an interlamellar pocket sufficiently large to ac commodate the grafted disc, eight mm. in diameter. The graft was inserted into the intracorneal pocket with help of the spatula, care being taken to spread the graft as uni formly as possible. The transverse incision was closed with two sutures just after the insertion of the graft. In order to avoid an immunologie influence, the grafts were placed in both eyes at the same time. In most cases, the corneas were taken from the fish stored in ice for two to five days. In other instances they were obtained from the living fish. In the main experiments 19 eyes in 12 rabbits were grafted with fish corneas and as a con trol, human (two eyes), ox (two eyes) and dog (one eye) grafts were used. The period of observation was two to 10 months on 12 eyes in eight animals. For a histologie exam ination in the early stage seven eyes in four animals were enucleated after 3, 10, 20, and 30 days. Postoperatively all the cases were treated with antibiotics for three weeks. Topical administration of cortisone was con tinued for three months in most cases.
More than a hundred years ago keratoplasty with heterografts was first attempted. During the past century many attempts were made on human eyes but there was no chance of a successful corneal graft unless the donor material came from the same species. Corneal heterografts are still believed to be impossi ble by most ophthalmologists. The pioneer work on heterografts was done by Babel and Bourquin,1 and Choyce2 in 1952, and their works stimulated a reinvestigation of this problem. The clinical use of chicken cornea to human eyes was also attempted by Kuwahara3 with some success. Kamata 4 reported a study of corneal heterografts of chicken cornea into rabbits' eyes. Basu and Ormsby 5 reported interesting evidence that the anti gen-antibody reaction of rabbit's cornea fol lowing a corneal heterograft was less fre quent with an avian cornea than with a mam malian cornea. Their result suggested that the further apart phylogenetically the two tissues, the less likely it is that a donor re cipient reaction will develop. This led to our experiments on corneal heterografts of fish cornea into rabbit eyes. METHOD AND MATERIAL
Adult rabbits weighing from 2.5 to 3.5 kg. were used as recipients. Graft tissue was ob tained from various kinds of fish: perch, mackerel, seriola, graymullet and bonito. Us ing Castroviejo's trephine a lamellar disc of the cornea, eight mm. in diameter and 0.3 to 0.8 mm. thick, was prepared and sterilized in a solution of penicillin, streptomycin, and sulfisomidin for one-half to two hours. Some
RESULTS
* From the Eye Department and Eye-Bank Labo ratory, Okayama Rosai Hospital. We are indebted to Dr. Frederick C. Blodi of the State University of Iowa and Dr. S. Tsuda of Okayama Rosai Hos pital for valuable suggestions and for aid in the preparation of this manuscript. Presented at the XVIII International Congress of Ophthalmology, Brussels, September, 1958.
In the preliminary experiment the opera tive technique was worked out on 10 animals and finally the method already described ap peared to be the most suitable. These first animals were excluded from the following data. In the main experiment interlamellar heterografts were done on 19 eyes in 12 rab bits. Seven eyes in four rabbits were ex cluded from the clinical results because of
363
364
JUN TSUTSUI AND SAEKO WATANABE
Fig. 1 (Tsutsui and Watanabe). Rabbit 23, left eye. Perch interlamellar graft, first postoperative day, slight opacity of graft and iris congestion. Resulted in transparent graft after two months.
Fig. 3 (Tsutsui and Watanabe). Rabbit 5, left eye. Mackerel interlamellar graft, 10th postopera tive day, no active inflammation, transparent graft.
the early enucleation for histologie examina tion within one month. Therefore clinical ob servation was performed on 12 eyes in eight rabbits for two to 10 months.
sided soon and the transparency of the grafts returned during the 10th to 20th postopera tive days (fig. 3). There was a variable degree of vascularization from the upper limbus around the op erated area but in no case was the central area vascularized. After three to four weeks these vascularizations almost disappeared but they could still be detected by slitlamp exam ination. After one or two months the con dition of the corneas became stable. The grafts maintained their transparency for a long time, perhaps permanently (figs. 4 and 5). Temporary opacity and inflamma tion never appeared after three weeks. In some cases a small fault of the surgical tech nique—for example, perforation of the su perficial layer of the recipient cornea, folding of the graft in the interlamellar pocket or too
I. CLINICAL RESULTS
1. Transparent heterograft. The transpar ency of the heterogeneous grafts showed a slight alteration during the postoperative course but finally 10 grafts out of 12 were almost transparent. The postoperative course was similar in each case. During the first several days slight irritative manifestations such as edema of the graft, traumatic pannus around the incision, and ciliary and iris con gestion appeared in most cases (figs. 1 and 2). These immediate reactions, however, sub-
Fig. 2 (Tsutsui and Watanabe). Rabbit S, left eye. Mackerel interlamellar graft, fourth post operative day, slight pannus from upper limbus, transparent graft. Resulted in transparent graft after 10 months.
Fig. 4 (Tsutsui and Watanabe). Rabbit 5, left eye. Mackerel interlamellar graft, 67th postopera tive day, transparent graft.
HETEROGRAFT OF FISH CORNEA
Fig. 5 (Tsutsui and Watanabe). Rabbit S, left eye. Mackerel interlamellar graft, 164th postopera tive day, transparent graft.
thick a graft (over 0.7 mm.)—might pro duce a localized opacity and vascularization. These opacities were not extensive and re mained as a small nebula. 2. Opacified heterografts. In two cases out of 12 ( 16.6 percent) the grafts became opaque more or less immediately after the operation. The opacification was extensive and covered the whole graft with a pannus-type vascu larization (fig. 6). After a week, the grafted cornea became necrotic and the inflammation extended to the iris and ciliary body. Finally the cornea developed into a dense leukoma with extensive vascularization. In one case, the cause of the opacified graft was found to be an infection by Pseudomonas aeruginosa and in the other eye it was too thick a graft with a poor fit into the interlamellar pocket.
Fig. 6 (Tsutsui and Watanabe). Rabbit 8, right eye. Mackerel interlamellar graft, fifth postopera tive day, opaque graft.
365
Fig. 7 (Tsutsui and Watanabe). Rabbit 12, right eye. Dog interlamellar graft, sixth postoperative day, opaque graft.
3. Control experiments. As control exper iments human (two cases), dog (one case) and ox (two cases) corneas were grafted interlamellarly into rabbit cornea with the same technique as already mentioned. The grafts were clear during three to 10 days and then the opacification of the entire graft developed with haziness of the corneal stroma in the recipient eye (fig. 7). In the case of the dog cornea the graft became necrotic. Large permanent opacities remained in all cases. I I . HlSTOLOGIC RESULTS
Enucleation of the grafted eyes was done on 3, 10, 12, 13, 20, 67, 72, and 154 days postoperatively. The graft was transparent in 10 cases and opaque in two cases at the time of enucleation. In the control experi-
Fig. 8 (Tsutsui and Watanabe). Rabbit 17, right eye. Bonito interlamellar graft, third postoperative day, no donor-recipient reaction, transparent graft.
366
JUN TSUTSUI AND SAEKO WATANABE was a violent inflammatory reaction. The epithelial layer became thick and irregular. The corneal stroma was irregular in the ar rangement of the fibers with numerous in flammatory cells and vessels. The presence of a graft could not be clearly determined. There were many inflammatory cells in the anterior chamber (fig. 12).
Fig. 9 (Tsutsui and Watanabe). Rabbit 11, left eye. Graymullet interlamellar graft, 30th postopera tive day, no donor-recipient reaction, transparent graft.
3. Control experiments. The histologie findings of this group were similar to those of the opacified grafts of fish cornea. A vio lent inflammatory reaction, necrosis and de struction of the grafts with a remarkable vascularization were present (fig. 13). DISCUSSION
ments, a dog and a human heterograft were examined histologically. 1. Transparent heterografts. The corneas which were enucleated between one week and one month showed the recipient corneal stroma to be unaffected by the introduction of the foreign body. Only few inflammatory cells or vessels were noted. T h e grafted fish cornea was enclosed between lamellae of nor mal aspect, though the nuclei appeared rare fied and showed hardly any pyknosis or other changes. The grafted stroma showed a ho mogeneous structure with very fine fibers and no destructive or inflammatory changes (figs. 8 and 9 ) . After two months the grafted cornea showed as a characteristic finding the development of a nuclear layer in the superficial area of the graft. These nuclei may come from the recipient corneal stroma as an organization of the grafted tis sue by the recipient tissue which seems to start during the second and third postopera tive months (fig. 10). After five months the structure of the grafted cornea became so similar to the host cornea that the existence of the heterogeneous tissue was no longer detectable. Particularly, the arrangement of the nuclei and of the corneal fibers was very similar to the recipient cornea (fig. 11). 2. Opacified heterografts. T w o cases of opacified heterografts were examined his tologically at the end of one month. There
W e utilized the idea of Basu and Ormsby 5 and extended it in our series of experiments on corneal heterografts using fish cornea. In a total of 12 cases of interlamellar corneal heterografts, 10 cases healed with a trans parent graft. The rate of successful trans plantation was quite high in our experiments. On the one hand Babel and Baurquin 1 had about 50 percent successful heterografts us ing human and cat cornea in rabbits. O n the other hand, Basu and Ormsby 5 obtained a 66.6 percent success in avian grafts and a 51.8 percent in mammalian grafts. In fish grafts we were successful in 83 percent of
Fig. 10 (Tsutsui and Watanabe). Rabbit 23, left eye. Perch interlamellar graft, 67th postoperative day, no donor-recipient reaction, nuclei of stroma cells appear in the superficial layer of the graft, transparent graft.
HETEROGRAFT
Fig. 11 (Tsutsui and Watanabe). Rabbit 6, left eye. Bonito interlamellar graft, 151st postoperative day, well organized graft with replaced corneal stroma cells, transparent graft the eyes. In our small number of control ex periments using mammalian cornea none of the grafts remained transparent. Conse quently, we also believe that there is evidence that the farther apart phylogenetically the donor and the recipent are the less likelihood there is that a donor-recipent reaction de velops. The factors necessary to obtain a clear graft seemed to be: No characteristic correlation was found between a successful graft and the species of fish used. T h e postoperative donor-recipient reaction seemed to be more intensive when a fresh
Fig. 12 (Tsutsui and Watanabe). Rabbit 16, left eye. Seriola interlamellar graft, 30th postoperative day, extensive donor-recipient reaction with irregu lar stroma cells and numerous inflammatory cells, opaque graft.
Fig. 13 (Tsutsui and Watanabe). Rabbit 12, left eye. Dog interlamellar graft, 30th postoperative day, extensive donor-recipient reaction with nu merous inflammatory cells, necrotic graft. graft was used and less intensive in older grafts. T h e cornea taken from fish stored for a few days in ice appeared to be ade quate. If the cornea was stored for more than 10 days the results were unfavorable. This evidence suggests that an adequate denaturation of the proteins may decrease the antigenicity. A preoperative serum treatment of the graft appeared to have no influence on the fish cornea. Kamata 4 reported that in using chicken cornea into rabbits' eyes the preop erative serum treatment was very effective in preventing a donor-recipient reaction. The antigenicity of fish protein may be so weak that no definite donor-recipient reaction de velops. Postoperative use of cortisone appears to be effective in preventing or reducing the development of a donor-recipient reaction but it is not absolutely necessary since some cases healed with a transparent graft without the use of cortisone. One of the most important factors to obtain a clear graft is accurate surgical tech nique. In our method a graft of less than 0.6 mm. is adequate. A graft of over 0.7 mm. may easily cause opacification and vascularization. Folding of the graft in the in terlamellar pocket is the cause of a localized opacity and vascularization. A perforation of the superficial layer of the recipient cornea
368
JUN TSUTSUI AND SAEKO WATANABE
also gives an unfavorable result. Conse quently we might be able to obtain a clear graft in all instances if our surgical tech nique were perfect and if the graft had ade quate freshness and thinness. The question remains open why these transparent heterografts are obtained in such a high percentage using fish cornea. We can only explain it from the phylogenetic view-point. Further studies on the protein chemistry and séro logie factors may give more concrete an swers. The organization of the grafted tissue into the recipient cornea was observed by his tologie examination. In the first month the nuclei of the grafted tissue disappear. During the second and the third months, organization of the graft begins on the surface. Nuclei of stroma cells appear in the peripheral zone of the graft and then in the deeper layers. Thus the organization progresses and finally all cells of the graft are completely replaced by the host cells.
CONCLUSION
For the purpose of clarifying the factors which make corneal heterografts successful fish corneas were transplanted interlamellarly into rabbits' corneas. The grafts, eight mm. in diameter and 0.3 to 0.8 mm. thick, were taken from perch, mackerel, seriola, graymullet and bonito. Some of the grafts were pretreated with a rabbit serum in a refrig erator for 24 hours. After a slight donor-recipient reaction, transparent heterografts were obtained in 10 cases out of 12. In the control experiments using human, dog and ox grafts, all the grafts became opaque during three to 10 days. The histologie reaction of the recipient cornea was slight in the successful hetero grafts. The process of organization of the grafted tissue into the recipient cornea ap peared one month after the operation and was completed five to six months later. Okayama Rosai Hospital.
REFERENCES
1. Babel, J., and Bourqin, J. B.: Experimental research with corneal heterografts. Brit. J. Ophth., 36: 529, 1952. 2. Choyce, D. P.: Successful transplantation of human and cat corneal tissue into rabbit cornea. Brit. J. Ophth, 36:537, 1952. 3. Kuwahara, Y. : Successful transplantation of chicken cornea into human cornea. J. Clin. Ophth, 10:963, 1956. 4. Kamata, W. : An experimental study of heterokeratoplasty. Acta Soc. Ophth, Japan, 61:1098, 1957. 5. Basu, P. K, and Ormsby, H. L.: Corneal heterograft in rabbits. Am. J. Ophth, 44:477, 1957.
PROPHYLAXIS AND T R E A T M E N T O F ENDOPHTHALMITTS* M.
NEVEU,
M.D.
Three Rivers, Quebec AND
A. J. ELLIOT,
M.D.
Toronto, Canada
In the practice of ophthalmic surgery one of the most alarming and distressing condi tions encountered is an acute endophthalmitis following a well-executed surgical proced ure. Unfortunately most of these cases de* From the Department of Ophthalmology, Uni versity of Toronto and Toronto General Hospital.
velop in apparently clean eyes showing no complications, where the co-operation of the patient has been excellent and a normal post operative convalescence could be expected. Occasionally one must undertake surgical treatment of patients in whom chronic infec tions have provided a warning but, all too