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Studies on Heterotransplantation of Corneas*
STUDIES ON H E T E R O T R A N S P L A N T A T I O N O F CORNEAS* Y. KUWABARA, M . D . Tokyo, Japan
biochemistry and serology. The results ob tained so far will be summarized in this paper.
INTRODUCTION
Since the time of Reisinger (1818), cor neal transplants have been widely per formed, and a considerable number of re ports have been published on this subject. With the establishment of eye-banks, trans plantation is expected to become even more widespread. However, even with the aid of eye-banks, the use of human corneas in our country still presents special problems. For instance, for both religious and senti mental reasons it is quite difficult to obtain corneas either from living persons or ca davers. Also, it is hard to predict exactly when a human eye will be available for transplantation and, once removed, the hu man eyeball can be stored for only a (rela tively) short time. This can cause great in convenience to a patient. If animal corneas could be utilized in stead of human corneas, the clinical signifi cance would be enormous. Operations could be performed at any time, at any place. For many years the problem of animal corneal transplantation was seriously in vestigated and the first attempts were made as early as the middle of the 19th century. Following the classical investigations by Salzer, v. Hippel and others, numerous at tempts, using horse and dog corneas, were made which, despite their efforts, were all unsuccessful. When these reports were examined, it soon became apparent that these experi mental heterotransplantations had been per formed merely to ascertain whether or not they would be successful, and that no funda mental studies had been made. Therefore we made a systematic study of heterotransplantation from the viewpoints of anatomy,
PART I. PRELIMINARY EXPERIMENTS HISTOLOGIC EXAMINATION OF ANIMAL COR NEAS
First, the results of a histologic examina tion of the corneas of several animals were compared. The corneas, which were obtained from humans, monkeys, cattle, horse, swine, cats, rabbits and chickens, were fixed in 10percent formalin and then were either pre pared in paraffin or frozen. After staining them by hematoxylin-eosin, PAS or Mallory stain, a comparative study was performed. The thickness of human corneas was 0.75 mm., as is generally accepted. Corneas of monkeys and cats were as thick as human corneas. Those of cattle, horse and dogs were thicker, while those of rabbits and chickens, measuring 0.5 mm., were thinner than human corneas. In the interest of brevity, we shall limit our comparison of histologic structures to those findings which are especially remark able. Among them was the discovery that the spaces in the parenchyma that contained the corneal tissue fluid varied in width from animal to animal: these spaces were narrow and scarce in humans, cattle swine, horse dogs and cats, while they were wide and numerous in monkeys and chickens. The cornea receives no vascular supplies and is believed to obtain nutrients from the tissue fluid. Therefore, it is inferred that in the animals that have corneas with wide and numerous spaces, conditions are favorable for good circulation and active metabolism. Consequently, it is conjectured that, from the histologic standpoint, chicken corneas, with their wide and numerous corneal
* From the Medical School of Keio University. This paper was translated from the Japanese by Robert W. and Seiko S. Chamberlin, New York. 911
Y. KUWABARA
912
spaces, should be suitable for transplanta tion. METABOLISM IN ANIMAL CORNEAS
For purposes of heterotransplantation, it is undoubtedly necessary to utilize animal corneas in which metabolism is active. In the preceding histologie study it was pointed out that the animals studied could be classified into two distinct groups, those with wide corneal spaces and those with narrow ones. It was also assumed that me tabolism would be most active in corneas with wide spaces. Chickens were chosen as representative of animals with wide corneal spaces while rabbits, dogs and cats were chosen as representative of animals with narrow corneal spaces. Metabolism was measured in these ani mals by use of the oxidation manometer. The results were as follows: Chicken Rabbit Cat Dog Human adult
-2.95 -1.22 -0.95 -1.20 —1.20
2.29 0.74 0.63 0.73 1.15
In rabbits, cats and dogs, which have nar row spaces in their respective corneal paren chymas, both oxidation and glycolysis (not measured in the dogs) were quite similar. However, in the chickens, having wide spaces, both oxidation and glycolysis were about twice as active as they were in ani mals of the other group. These results con firmed our hypothesis that metabolism was more active in animals with wide corneal spaces. BIOCHEMICAL STUDY
The problem of heterogeneous protein re action has long been discussed in relation to heterotransplantation. However active me tabolism in the cornea itself may be, if there is a great difference in protein composition between the recipient's and the donor's cor neas, transplantation would be unsuccessful. For this reason, a close similarity in the composition of corneal proteins between
donor and recipient is highly desirable. A biochemical study was made on the corneas of rabbits, chickens, dogs, cats, hu mans and monkeys. The free amino acids in the corneas and those in the protein hydrolysates were analyzed by the paper Chro matographie method and a remarkable dis covery was made. In total amino acid con tent, man (316.0) and chicken (319.08) were quite similar. Human and chicken corneas as compared with those of monkeys, dogs, cats and rab bits, contained slightly more sulfo-amino acids and less tyrosine. Of all the animals tested, the amino acid content of the corneas of chickens was most similar to that of man. TISSUE CULTURE STUDY
This experiment was performed for the purpose of examining dynamically the his tologie features of tissue reactions during heterotransplantation. Using the hangingdrop method, a one-mm. square of chicken cornea and a one-mm. square of rabbit cor nea were simultaneously cultured at a twomm. interval. Previously, it had been anticipated that such a simultaneous culture of heterogene ous corneal tissues would result in the usual heterogeneous protein reactions, such as de generation, necrosis and phagocytosis, as the proliferating tissues from the two grafts came into contact. However, contrary to this assumption, no heterogeneous protein reactions occurred at the contacting sur faces and both proliferating tissues fused together, leaving no border between them. Now, corneas, having no blood vessels, must be nourished by the tissue fluid. Though the corneas of different animals draw their nutrients from different tissue fluids, we observe from this experiment that they can proliferate in the same culture medium. On the other hand, corneal tissue, like tendon, is said to have little antigenicity. It is therefore concluded that, when a hetero geneous protein reaction occurs during het-
HETEROTRANSPLANTATION OF CORNEAS erotransplantation, it is caused not by the proteins in the cornea but by those in the tissue fluid. A SEEOLOGIC STUDY
O u r observations on tissue culture led us to the assumption that antigenicity was due not to the cell protein in the cornea itself but to the protein in the fluid filling the tis sue spaces in the cornea. T h e following se ries of experiments was carried out to con firm this assumption. First, a superficial transplantation of chicken cornea to rabbit cornea was at tempted. As tested by the precipitation su perposition method, our test for antigenantibody reactions, antibodies to chicken corneas were found in rabbit blood. Next, we repeated this experiment, this time using chicken corneas whose fluids had been replaced with fluids similar to the fluid in rabbit corneas. A rigorous replace ment of the former with the latter was found to be difficult. However, in our at tempts to resolve this difficulty, we dis covered through paper chromatography that, in any given animal, the tissue in the cor nea is quite similar to that in the serum. O n the basis of this discovery, the experi mental transplantation was made after the chicken cornea had been immersed in rab bit serum for two days. This time, as is shown in Table 1, no antibodies were found in the serum. F r o m these results it is clear that heterogenous protein reactions, which are the main cause of unsuccessful heterotransplantation of corneas, should be due not to pro teins in the corneal cells but to those in the tissue fluid. This fact has an important sig nificance in relation to heterotransplantation of corneas. PART II.
E X P E R I M E N T S ON A N I M A L S
FIRST EXPERIMENT
It was determined in the preliminary ex periments that failure of heterotransplantation was largely due to antibody reactions
913
among the proteins in the corneal tissue fluid. It was therefore concluded that re placement of tissue fluid in the donor's cor nea with that of the recipient's would be highly desirable. This, of course, is impos sible. A s was determined in our sérologie study, the amino-acid fraction in human serum was similar to that of the fluid in the anterior chamber of the human eye. H o w ever, we wanted to see if a more suitable replacement fluid could be found. This was the object of the present experiment. T h e experiments were performed using chickens and rabbits for subjects. T h e chicken corneas, which had previously been immersed for three days in various fluids, were superficially transplanted to the cor neas of rabbits and the results were ob served. Immersion for three days was adopted because proliferation of tissue be gan on the third day of tissue culture. T h e various fluids used and their respective re sults were: ( 1 ) without treatment, ( 2 ) rabbit serum, ( 3 ) physiologic saline solution, ( 4 ) Ringer's solution, ( 5 ) formalin (formal dehyde), ( 6 ) fowl serum, ( 7 ) deproteinized rabbit serum, ( 8 ) indispensable amino-acid solution and ( 9 ) homolyzed rabbit serum. RESULTS
Conjunctiva! infection, discharge from the eye due to surgical intervention and compression by suturing threads were ob served in all cases. Below, under each item, the description will be limited to whatever changes were peculiar to that particular ex periment. 1. After the operation, after complete vascularization had occurred in the cornea, intense ciliary injection, iridal injection and opacity of the transplanted corneal graft were observed. One month later, the trans planted graft atrophied and became whitish and opaque. 2. After the operation a slight amount of discharge and corneal infection were ob served at first but gradually disappeared
Y. KUWABARA
914
and no sign of iridal injection, corneal opacity or atrophy was observed. One month later all signs of irritation disap peared and no vascularization occurred. The transplanted cornea was quite transparent but a slight linear and ring-shaped opacity was found on the border. This was not per ceived macroscopically but was first de tected by lateral illumination. The fundus was clearly observable through the graft. One year later a follow-up examination showed no change and the cornea re mained transparent. During the clinical period, histologie examinations were regu larly performed. No inflammatory cell in filtration was observed, by the hematoxylineosin stain method, and the transplanted graft of chicken cornea remained on the whole more transparent than the cornea of the recipient rabbit. Though slight granula tion was found at the border between the graft and the recipient's cornea, no vascu larization was detectable.
After about 60 days, the same examina tion was reported and revealed that the homogeneous, transparent graft had fused with the surrounding recipient cornea and it was possible to distinguish between the two only by noticing a slight scar and a trace of granulation at the border. Com plete fusion was presumed to have been es tablished between the transplanted graft and the recipient's cornea. 3. In physiologic saline the chicken cor nea became considerably opaque and almost lost its transparency after a five-day immer sion. After transplantation, ciliary and iridic injection was intense, vascularization and marked opacity were observed. 4. Immersion in Ringer's solution fol lowed almost the same course as did physio logic saline. The results of such immersion, however, were slighter in degree. 5. Salzer had previously reported suc cessful transplantation of horse cornea to rabbit recipients after fixation in formalin.
TABLE 1 DURATION OF ANTIBODY REACTIONS IN THE SERUM OF RABBITS AFTER TRANSPLANTATION OF CHICKEN CORNEAL GRAFTS WHICH HAD BEEN PRETREATED IN RABBIT SERUM
Specimen No.
Size of Corneal Graft (mm.)
Number of Days Following Operation 14
21
28
35
42
49
56
70
84
112
Transparency of Cornea
1
4
Clear, transparent
2
4
Clear, transparent
3
4
Clear, transparent
4
4
Clear, transparent
5
4
Clear, transparent
6
4
Clear, transparent
7
4
Clear, transparent
8
4
Clear
9
4
10
7
11
7
12
7
////////////////////////////////
Cloudy, turbid Clear
//////////////////////////
No antibodies were observed except in specimens 9 and 11. / / / / / / / / / / Duration of appearance of antibodies.
Cloudy Clear
HETEROTRANSPLANTATION OF CORNEAS
915
TABLE 2 DURATION OF ANTIBODY REACTIONS IN THE SERUM OF RABBITS AFTER CHICKEN CORNEAS, TREATED WITH PHYSIOLOGIC SALINE SOLUTION, WERE TRANSPLANTED TO THE RABBIT EYES
Size of Specimen Corneal Graft No. (mm.)
Number of Days Following Operation 14
21
|
28
|
35
42
49
56
70
84
112
Transparency of Cornea
1
4
ΊΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΊ,
Cloudy
2
4
Cloudy
3
4
un minimum immun
4
4
5
4
Slough
6
4
Cloudy
7
4
Slough
8
7
9
7
Cloudy
10
7
Cloudy
11
7
12
7
13
7
14
7
//////////////////////// ////////////////////////////////////
ΊΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙ
Cloudy
Cloudy
////////////////////////////////////
Cloudy Cloudy
minium //////////////////
Cloudy
Cloudy
Cloudy
de ath
111111111 Duration of appearance of antibodies.
However, in the present study, chicken cor nea became moderately opaque after im mersion in formalin. After transplantation, the signs of irritation became quite intense and the attempt was considered unsuccess ful. 6. Considering the nature of tissue fluid in the cornea, it had been supposed that the transplantation of chicken corneas treated in chicken serum to rabbit recipients would produce much the same effect as did the transplantation of untreated chicken cor neas. However, as compared with untreated corneas, both the signs of irritation and opacity were much slighter in degree. This result suggests that chicken serum might contain some factors that are common to both chicken and rabbit serum, therefore in creasing tissue affinity between chicken and rabbit corneas. This point requires further study.
7 and 8. In both cases strong signs of ir ritation were observed, along with ciliary injection, increased discharge from the eye, opacity and degeneration of the transplanted graft. 9. When the chicken cornea treated in homolyzed serum was transplanted into the cornea of a rabbit, the graft became opaque. The presence of erythrocytes thus appeared to be harmful. In conclusion, the serum of the recipient was found to be the most suitable medium for replacement of the donor cornea's tissue fluid. SECOND EXPERIMENT
Having determined that the serum of the recipient was the most suitable replacement medium for chicken corneal grafts which had wide tissue spaces, it became the pur pose of the second experiment to see
916
Y. KUWABARA PART III.
CLINICAL EXPERIENCE
As the systematic study of heterotrans plantation is still under way, clinical appli cation should be avoided until a valid con clusion can be obtained. However, for ref erence, one experience will be described. In the future, after more experience on human corneas is accumulated, more precise reports on-clinical application will be made.
Fig. 1 (Kuwabara). Before operation.
whether this method was also applicable to animal donors whose corneas contained nar row tissue spaces. First, corneas from cats, dogs and hu mans were immersed in rabbit serum for three days and then transplanted to the su perficial corneas of rabbits. With cat corneas strong signs of irrita tion were observed, the corneas became cloudy and opaque. With dog corneas, although the signs of irritation were slighter than with cat cor neas, ciliary infection was intense and the grafts were cloudy. With human corneas the signs of irrita tion were the slightest of the three and opacity in the resultant graft was less marked. As compared with fowl corneas, however, the results were markedly poor and the at tempt was considered unsuccessful. In short, though chicken corneas, having wider corneal tissue spaces, can be successfully trans planted by immersion in the recipient's serum, heterotransplantation is unsuccessful when corneas that have narrow tissue spaces are used. Much as yet remains obscure concerning specific properties of chicken corneas. At present it is assumed that fowl cornea is appropriate for transplantation because, with its wide and numerous parenchymal spaces, it is permeable to fluid, and the re placement of fluid can be achieved readily and completely.
We received at our clinic a 14-year-old boy who complained of blurred vision occurring over a pe riod of several years, with visual acuity gradually diminishing. At the first visit, during the summer of 1955, vision was: O.D., 0.01; O.S., 0.2. Gray punctuated cloudings were aggregated in the super ficial layer of the cornea, indicating typical Groenouw's corneal dystrophy. After superficial transplantation of a human cornea to the right eye, visual acuity was improved to 0.4. In February, 1956, the patient returned com plaining of defective vision in the left eye. This time his vision, O.S., was 0.01. A photograph of this cornea as it was before the operation is pre sented in Figure 1. A chicken cornea, after having been immersed for two days in serum obtained from the human recipient, was superficially transplanted in the same way as was the previously mentioned human cor nea. After the operation, signs of irritation, such as ciliary infection, were slight. One month after the operation (fig. 2) vision, O.S., was 0.3. A oneyear follow-up observation showed no change. CONCLUSION
The results of these fundamental studies on heterotransplantation can be summarized in the following manner: Superficial hetero transplantation of corneas has been success-
Fig. 2 (Kuwabara). After operation.
HETEROTRANSPLANTATION OF CORNEAS fully performed. For purposes of heterotransplantation it is necessary to immerse the corneal graft in the recipient's serum. Finally, chicken corneas with their wide corneal spaces are the most suitable for heterotransplantation.
917
The study of transplantation of the entire cornea is also under way. As experiments are still in progress, further results will be reported in succeeding papers Tokyo Medical College.
T H E USE O F CORNEAS FROM FROZEN W H O L E EYES* FOR EMERGENCY KERATOPLASTY SAMUEL D. MCPHERSON, JR., M.D.,
AND H.
MAXWELL MORRISON, JR.,
M.D.
Durham, North Carolina
The lack of readily available donor ma terial sometimes limits the surgical manage ment of perforating corneal disorders. Al though eyes with simple corneal lacerations or small corneal ulcers without appreciable loss of corneal stroma may be treated ade quately with direct suturing or conjunctival flaps, eyes with large perforations with loss of much corneal substance from infection or trauma sometimes can only be saved with keratoplasty. Difficulty in obtaining fresh donor material on short notice for use in such procedures is not uncommon, and at times this has led to enucleation of eyes which might otherwise have been salvaged. We have recently been interested in the possibility of whole eye preservation through glycerol soaking and fast freezing and, as a result, have had the opportunity to utilize corneas from such eyes in keratoplasties on three eyes which would probably have been lost otherwise. It is the purpose of this paper to report these operations and the method of preser vation used as a means of making preserved donor cornea and sciera readily available where fresh material is not. * From the McPherson Hospital, Durham, and the Division of Ophthalmology, University of North Carolina School of Medicine, Chapel Hill. This study was supported by a grant from the North Carolina State Association for the Blind. Presented at the 97th annual meeting of the Ameri can Ophthalmological Society, Hot Springs, Vir ginia, May, 1961.
METHOD OF PRESERVATION
Whole eyes removed with sterile precau tions at autopsy as soon as possible after death were used. The eyes were washed with a solution containing 200,000 units of peni cillin and 0.04 gm. of streptomycin per cc. Under aspetic conditions the eyes were soaked for one hour in 15 cc. of 15 percent glycerol in Ringer's solution in a large py rex test tube cut to half size (fig. 1 ). The ex cess glycerol was then decanted, and the tube was partly immersed in a dry ice-alcohol slush at — 79°C. for five minutes (fig. 2). The test tubes were then placed in screwtop glass bottles and stored in an ordinary deep freeze at — 28° C. PREOPERATIVE PREPARATION OF DONOR MATERIAL
When an eye was required for trans plantation, it was transported to the operat ing room in a dry-ice chest. The glass bottle was removed, uncapped, and the sterile tube containing the frozen eye was passed to the assistant. The test tube containing the eye was immersed in a solution of isotonic sa line at 38°C. The cornea, which was opaque, gradually cleared and, in five minutes, thaw ing of the cornea was complete. In most in stances the cornea was quite clear on thaw ing (fig. 3). SURGICAL TECHNIQUE
All operations were performed
under
biochemistry and serology. The results ob tained so far will be summarized in this paper.
INTRODUCTION
Since the time of Reisinger (1818), cor neal transplants have been widely per formed, and a considerable number of re ports have been published on this subject. With the establishment of eye-banks, trans plantation is expected to become even more widespread. However, even with the aid of eye-banks, the use of human corneas in our country still presents special problems. For instance, for both religious and senti mental reasons it is quite difficult to obtain corneas either from living persons or ca davers. Also, it is hard to predict exactly when a human eye will be available for transplantation and, once removed, the hu man eyeball can be stored for only a (rela tively) short time. This can cause great in convenience to a patient. If animal corneas could be utilized in stead of human corneas, the clinical signifi cance would be enormous. Operations could be performed at any time, at any place. For many years the problem of animal corneal transplantation was seriously in vestigated and the first attempts were made as early as the middle of the 19th century. Following the classical investigations by Salzer, v. Hippel and others, numerous at tempts, using horse and dog corneas, were made which, despite their efforts, were all unsuccessful. When these reports were examined, it soon became apparent that these experi mental heterotransplantations had been per formed merely to ascertain whether or not they would be successful, and that no funda mental studies had been made. Therefore we made a systematic study of heterotransplantation from the viewpoints of anatomy,
PART I. PRELIMINARY EXPERIMENTS HISTOLOGIC EXAMINATION OF ANIMAL COR NEAS
First, the results of a histologic examina tion of the corneas of several animals were compared. The corneas, which were obtained from humans, monkeys, cattle, horse, swine, cats, rabbits and chickens, were fixed in 10percent formalin and then were either pre pared in paraffin or frozen. After staining them by hematoxylin-eosin, PAS or Mallory stain, a comparative study was performed. The thickness of human corneas was 0.75 mm., as is generally accepted. Corneas of monkeys and cats were as thick as human corneas. Those of cattle, horse and dogs were thicker, while those of rabbits and chickens, measuring 0.5 mm., were thinner than human corneas. In the interest of brevity, we shall limit our comparison of histologic structures to those findings which are especially remark able. Among them was the discovery that the spaces in the parenchyma that contained the corneal tissue fluid varied in width from animal to animal: these spaces were narrow and scarce in humans, cattle swine, horse dogs and cats, while they were wide and numerous in monkeys and chickens. The cornea receives no vascular supplies and is believed to obtain nutrients from the tissue fluid. Therefore, it is inferred that in the animals that have corneas with wide and numerous spaces, conditions are favorable for good circulation and active metabolism. Consequently, it is conjectured that, from the histologic standpoint, chicken corneas, with their wide and numerous corneal
* From the Medical School of Keio University. This paper was translated from the Japanese by Robert W. and Seiko S. Chamberlin, New York. 911
Y. KUWABARA
912
spaces, should be suitable for transplanta tion. METABOLISM IN ANIMAL CORNEAS
For purposes of heterotransplantation, it is undoubtedly necessary to utilize animal corneas in which metabolism is active. In the preceding histologie study it was pointed out that the animals studied could be classified into two distinct groups, those with wide corneal spaces and those with narrow ones. It was also assumed that me tabolism would be most active in corneas with wide spaces. Chickens were chosen as representative of animals with wide corneal spaces while rabbits, dogs and cats were chosen as representative of animals with narrow corneal spaces. Metabolism was measured in these ani mals by use of the oxidation manometer. The results were as follows: Chicken Rabbit Cat Dog Human adult
-2.95 -1.22 -0.95 -1.20 —1.20
2.29 0.74 0.63 0.73 1.15
In rabbits, cats and dogs, which have nar row spaces in their respective corneal paren chymas, both oxidation and glycolysis (not measured in the dogs) were quite similar. However, in the chickens, having wide spaces, both oxidation and glycolysis were about twice as active as they were in ani mals of the other group. These results con firmed our hypothesis that metabolism was more active in animals with wide corneal spaces. BIOCHEMICAL STUDY
The problem of heterogeneous protein re action has long been discussed in relation to heterotransplantation. However active me tabolism in the cornea itself may be, if there is a great difference in protein composition between the recipient's and the donor's cor neas, transplantation would be unsuccessful. For this reason, a close similarity in the composition of corneal proteins between
donor and recipient is highly desirable. A biochemical study was made on the corneas of rabbits, chickens, dogs, cats, hu mans and monkeys. The free amino acids in the corneas and those in the protein hydrolysates were analyzed by the paper Chro matographie method and a remarkable dis covery was made. In total amino acid con tent, man (316.0) and chicken (319.08) were quite similar. Human and chicken corneas as compared with those of monkeys, dogs, cats and rab bits, contained slightly more sulfo-amino acids and less tyrosine. Of all the animals tested, the amino acid content of the corneas of chickens was most similar to that of man. TISSUE CULTURE STUDY
This experiment was performed for the purpose of examining dynamically the his tologie features of tissue reactions during heterotransplantation. Using the hangingdrop method, a one-mm. square of chicken cornea and a one-mm. square of rabbit cor nea were simultaneously cultured at a twomm. interval. Previously, it had been anticipated that such a simultaneous culture of heterogene ous corneal tissues would result in the usual heterogeneous protein reactions, such as de generation, necrosis and phagocytosis, as the proliferating tissues from the two grafts came into contact. However, contrary to this assumption, no heterogeneous protein reactions occurred at the contacting sur faces and both proliferating tissues fused together, leaving no border between them. Now, corneas, having no blood vessels, must be nourished by the tissue fluid. Though the corneas of different animals draw their nutrients from different tissue fluids, we observe from this experiment that they can proliferate in the same culture medium. On the other hand, corneal tissue, like tendon, is said to have little antigenicity. It is therefore concluded that, when a hetero geneous protein reaction occurs during het-
HETEROTRANSPLANTATION OF CORNEAS erotransplantation, it is caused not by the proteins in the cornea but by those in the tissue fluid. A SEEOLOGIC STUDY
O u r observations on tissue culture led us to the assumption that antigenicity was due not to the cell protein in the cornea itself but to the protein in the fluid filling the tis sue spaces in the cornea. T h e following se ries of experiments was carried out to con firm this assumption. First, a superficial transplantation of chicken cornea to rabbit cornea was at tempted. As tested by the precipitation su perposition method, our test for antigenantibody reactions, antibodies to chicken corneas were found in rabbit blood. Next, we repeated this experiment, this time using chicken corneas whose fluids had been replaced with fluids similar to the fluid in rabbit corneas. A rigorous replace ment of the former with the latter was found to be difficult. However, in our at tempts to resolve this difficulty, we dis covered through paper chromatography that, in any given animal, the tissue in the cor nea is quite similar to that in the serum. O n the basis of this discovery, the experi mental transplantation was made after the chicken cornea had been immersed in rab bit serum for two days. This time, as is shown in Table 1, no antibodies were found in the serum. F r o m these results it is clear that heterogenous protein reactions, which are the main cause of unsuccessful heterotransplantation of corneas, should be due not to pro teins in the corneal cells but to those in the tissue fluid. This fact has an important sig nificance in relation to heterotransplantation of corneas. PART II.
E X P E R I M E N T S ON A N I M A L S
FIRST EXPERIMENT
It was determined in the preliminary ex periments that failure of heterotransplantation was largely due to antibody reactions
913
among the proteins in the corneal tissue fluid. It was therefore concluded that re placement of tissue fluid in the donor's cor nea with that of the recipient's would be highly desirable. This, of course, is impos sible. A s was determined in our sérologie study, the amino-acid fraction in human serum was similar to that of the fluid in the anterior chamber of the human eye. H o w ever, we wanted to see if a more suitable replacement fluid could be found. This was the object of the present experiment. T h e experiments were performed using chickens and rabbits for subjects. T h e chicken corneas, which had previously been immersed for three days in various fluids, were superficially transplanted to the cor neas of rabbits and the results were ob served. Immersion for three days was adopted because proliferation of tissue be gan on the third day of tissue culture. T h e various fluids used and their respective re sults were: ( 1 ) without treatment, ( 2 ) rabbit serum, ( 3 ) physiologic saline solution, ( 4 ) Ringer's solution, ( 5 ) formalin (formal dehyde), ( 6 ) fowl serum, ( 7 ) deproteinized rabbit serum, ( 8 ) indispensable amino-acid solution and ( 9 ) homolyzed rabbit serum. RESULTS
Conjunctiva! infection, discharge from the eye due to surgical intervention and compression by suturing threads were ob served in all cases. Below, under each item, the description will be limited to whatever changes were peculiar to that particular ex periment. 1. After the operation, after complete vascularization had occurred in the cornea, intense ciliary injection, iridal injection and opacity of the transplanted corneal graft were observed. One month later, the trans planted graft atrophied and became whitish and opaque. 2. After the operation a slight amount of discharge and corneal infection were ob served at first but gradually disappeared
Y. KUWABARA
914
and no sign of iridal injection, corneal opacity or atrophy was observed. One month later all signs of irritation disap peared and no vascularization occurred. The transplanted cornea was quite transparent but a slight linear and ring-shaped opacity was found on the border. This was not per ceived macroscopically but was first de tected by lateral illumination. The fundus was clearly observable through the graft. One year later a follow-up examination showed no change and the cornea re mained transparent. During the clinical period, histologie examinations were regu larly performed. No inflammatory cell in filtration was observed, by the hematoxylineosin stain method, and the transplanted graft of chicken cornea remained on the whole more transparent than the cornea of the recipient rabbit. Though slight granula tion was found at the border between the graft and the recipient's cornea, no vascu larization was detectable.
After about 60 days, the same examina tion was reported and revealed that the homogeneous, transparent graft had fused with the surrounding recipient cornea and it was possible to distinguish between the two only by noticing a slight scar and a trace of granulation at the border. Com plete fusion was presumed to have been es tablished between the transplanted graft and the recipient's cornea. 3. In physiologic saline the chicken cor nea became considerably opaque and almost lost its transparency after a five-day immer sion. After transplantation, ciliary and iridic injection was intense, vascularization and marked opacity were observed. 4. Immersion in Ringer's solution fol lowed almost the same course as did physio logic saline. The results of such immersion, however, were slighter in degree. 5. Salzer had previously reported suc cessful transplantation of horse cornea to rabbit recipients after fixation in formalin.
TABLE 1 DURATION OF ANTIBODY REACTIONS IN THE SERUM OF RABBITS AFTER TRANSPLANTATION OF CHICKEN CORNEAL GRAFTS WHICH HAD BEEN PRETREATED IN RABBIT SERUM
Specimen No.
Size of Corneal Graft (mm.)
Number of Days Following Operation 14
21
28
35
42
49
56
70
84
112
Transparency of Cornea
1
4
Clear, transparent
2
4
Clear, transparent
3
4
Clear, transparent
4
4
Clear, transparent
5
4
Clear, transparent
6
4
Clear, transparent
7
4
Clear, transparent
8
4
Clear
9
4
10
7
11
7
12
7
////////////////////////////////
Cloudy, turbid Clear
//////////////////////////
No antibodies were observed except in specimens 9 and 11. / / / / / / / / / / Duration of appearance of antibodies.
Cloudy Clear
HETEROTRANSPLANTATION OF CORNEAS
915
TABLE 2 DURATION OF ANTIBODY REACTIONS IN THE SERUM OF RABBITS AFTER CHICKEN CORNEAS, TREATED WITH PHYSIOLOGIC SALINE SOLUTION, WERE TRANSPLANTED TO THE RABBIT EYES
Size of Specimen Corneal Graft No. (mm.)
Number of Days Following Operation 14
21
|
28
|
35
42
49
56
70
84
112
Transparency of Cornea
1
4
ΊΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΊ,
Cloudy
2
4
Cloudy
3
4
un minimum immun
4
4
5
4
Slough
6
4
Cloudy
7
4
Slough
8
7
9
7
Cloudy
10
7
Cloudy
11
7
12
7
13
7
14
7
//////////////////////// ////////////////////////////////////
ΊΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙ
Cloudy
Cloudy
////////////////////////////////////
Cloudy Cloudy
minium //////////////////
Cloudy
Cloudy
Cloudy
de ath
111111111 Duration of appearance of antibodies.
However, in the present study, chicken cor nea became moderately opaque after im mersion in formalin. After transplantation, the signs of irritation became quite intense and the attempt was considered unsuccess ful. 6. Considering the nature of tissue fluid in the cornea, it had been supposed that the transplantation of chicken corneas treated in chicken serum to rabbit recipients would produce much the same effect as did the transplantation of untreated chicken cor neas. However, as compared with untreated corneas, both the signs of irritation and opacity were much slighter in degree. This result suggests that chicken serum might contain some factors that are common to both chicken and rabbit serum, therefore in creasing tissue affinity between chicken and rabbit corneas. This point requires further study.
7 and 8. In both cases strong signs of ir ritation were observed, along with ciliary injection, increased discharge from the eye, opacity and degeneration of the transplanted graft. 9. When the chicken cornea treated in homolyzed serum was transplanted into the cornea of a rabbit, the graft became opaque. The presence of erythrocytes thus appeared to be harmful. In conclusion, the serum of the recipient was found to be the most suitable medium for replacement of the donor cornea's tissue fluid. SECOND EXPERIMENT
Having determined that the serum of the recipient was the most suitable replacement medium for chicken corneal grafts which had wide tissue spaces, it became the pur pose of the second experiment to see
916
Y. KUWABARA PART III.
CLINICAL EXPERIENCE
As the systematic study of heterotrans plantation is still under way, clinical appli cation should be avoided until a valid con clusion can be obtained. However, for ref erence, one experience will be described. In the future, after more experience on human corneas is accumulated, more precise reports on-clinical application will be made.
Fig. 1 (Kuwabara). Before operation.
whether this method was also applicable to animal donors whose corneas contained nar row tissue spaces. First, corneas from cats, dogs and hu mans were immersed in rabbit serum for three days and then transplanted to the su perficial corneas of rabbits. With cat corneas strong signs of irrita tion were observed, the corneas became cloudy and opaque. With dog corneas, although the signs of irritation were slighter than with cat cor neas, ciliary infection was intense and the grafts were cloudy. With human corneas the signs of irrita tion were the slightest of the three and opacity in the resultant graft was less marked. As compared with fowl corneas, however, the results were markedly poor and the at tempt was considered unsuccessful. In short, though chicken corneas, having wider corneal tissue spaces, can be successfully trans planted by immersion in the recipient's serum, heterotransplantation is unsuccessful when corneas that have narrow tissue spaces are used. Much as yet remains obscure concerning specific properties of chicken corneas. At present it is assumed that fowl cornea is appropriate for transplantation because, with its wide and numerous parenchymal spaces, it is permeable to fluid, and the re placement of fluid can be achieved readily and completely.
We received at our clinic a 14-year-old boy who complained of blurred vision occurring over a pe riod of several years, with visual acuity gradually diminishing. At the first visit, during the summer of 1955, vision was: O.D., 0.01; O.S., 0.2. Gray punctuated cloudings were aggregated in the super ficial layer of the cornea, indicating typical Groenouw's corneal dystrophy. After superficial transplantation of a human cornea to the right eye, visual acuity was improved to 0.4. In February, 1956, the patient returned com plaining of defective vision in the left eye. This time his vision, O.S., was 0.01. A photograph of this cornea as it was before the operation is pre sented in Figure 1. A chicken cornea, after having been immersed for two days in serum obtained from the human recipient, was superficially transplanted in the same way as was the previously mentioned human cor nea. After the operation, signs of irritation, such as ciliary infection, were slight. One month after the operation (fig. 2) vision, O.S., was 0.3. A oneyear follow-up observation showed no change. CONCLUSION
The results of these fundamental studies on heterotransplantation can be summarized in the following manner: Superficial hetero transplantation of corneas has been success-
Fig. 2 (Kuwabara). After operation.
HETEROTRANSPLANTATION OF CORNEAS fully performed. For purposes of heterotransplantation it is necessary to immerse the corneal graft in the recipient's serum. Finally, chicken corneas with their wide corneal spaces are the most suitable for heterotransplantation.
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The study of transplantation of the entire cornea is also under way. As experiments are still in progress, further results will be reported in succeeding papers Tokyo Medical College.
T H E USE O F CORNEAS FROM FROZEN W H O L E EYES* FOR EMERGENCY KERATOPLASTY SAMUEL D. MCPHERSON, JR., M.D.,
AND H.
MAXWELL MORRISON, JR.,
M.D.
Durham, North Carolina
The lack of readily available donor ma terial sometimes limits the surgical manage ment of perforating corneal disorders. Al though eyes with simple corneal lacerations or small corneal ulcers without appreciable loss of corneal stroma may be treated ade quately with direct suturing or conjunctival flaps, eyes with large perforations with loss of much corneal substance from infection or trauma sometimes can only be saved with keratoplasty. Difficulty in obtaining fresh donor material on short notice for use in such procedures is not uncommon, and at times this has led to enucleation of eyes which might otherwise have been salvaged. We have recently been interested in the possibility of whole eye preservation through glycerol soaking and fast freezing and, as a result, have had the opportunity to utilize corneas from such eyes in keratoplasties on three eyes which would probably have been lost otherwise. It is the purpose of this paper to report these operations and the method of preser vation used as a means of making preserved donor cornea and sciera readily available where fresh material is not. * From the McPherson Hospital, Durham, and the Division of Ophthalmology, University of North Carolina School of Medicine, Chapel Hill. This study was supported by a grant from the North Carolina State Association for the Blind. Presented at the 97th annual meeting of the Ameri can Ophthalmological Society, Hot Springs, Vir ginia, May, 1961.
METHOD OF PRESERVATION
Whole eyes removed with sterile precau tions at autopsy as soon as possible after death were used. The eyes were washed with a solution containing 200,000 units of peni cillin and 0.04 gm. of streptomycin per cc. Under aspetic conditions the eyes were soaked for one hour in 15 cc. of 15 percent glycerol in Ringer's solution in a large py rex test tube cut to half size (fig. 1 ). The ex cess glycerol was then decanted, and the tube was partly immersed in a dry ice-alcohol slush at — 79°C. for five minutes (fig. 2). The test tubes were then placed in screwtop glass bottles and stored in an ordinary deep freeze at — 28° C. PREOPERATIVE PREPARATION OF DONOR MATERIAL
When an eye was required for trans plantation, it was transported to the operat ing room in a dry-ice chest. The glass bottle was removed, uncapped, and the sterile tube containing the frozen eye was passed to the assistant. The test tube containing the eye was immersed in a solution of isotonic sa line at 38°C. The cornea, which was opaque, gradually cleared and, in five minutes, thaw ing of the cornea was complete. In most in stances the cornea was quite clear on thaw ing (fig. 3). SURGICAL TECHNIQUE
All operations were performed
under