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Corneal Heterografts in Rabbits*
CORNEAL P.
Κ.
HETEROGRAFTS
BASU.t D.O.M.S.,
AND HUGH
IN L.
RABBITS* ORMSBY,
M.D.
Toronto, Ontario I n recent years, there has been an in creasing demand for donor eyes for keratoplasty. This has resulted in eflforts to improve methods of storage, but there is still no solution to the problem of obtaining a sufficient supply of suitable eyes from human sources. Since there is little hope that the demand for h u m a n donor corneas will be met for many years, it seemed timely to re investigate the possibiUty of using animal or avian tissues as donor material.
I n a previous report,* we have shown the interlamellar method of grafting to be suita ble for the study of graft opacification. I n the present study we have used both inter lamellar and lamellar methods in parallel series utilizing avian and mammalian donor tissues. METHODS A N D MATERIALS
During the past century there were many reports of the use of heterografts in humans. Since that time, however, it has been gen erally accepted that such grafts are unsuc cessful. T h e new theories of immunity offer an explanation for the opacification of heterografts. If this theory is correct, it seems possible that new therapeutic agents might be used to improve the chances of suc cess in heterografts.
Full grown rabbits weighing from 2.5 to 4.0 kg. from mixed stocks were used as recipients. All operative procedures were performed under intravenous Nembutal anesthesia and full surgical asepsis. Donor corneal tissue was obtained from chicken, duck, turkey, monkey, beef, and lamb. Each recipient received tissues from the same donor, using a n interlamellar graft in one eye and a lamellar graft in the other. All the grafts were fresh and measured 5.0 mm. in diameter by 0.25 mm. in thickness.
T h e present experiments were designed to discover if the opacification occurring in heterografts could be explained by the oc currence of an antigen-antibody reaction, and to find the donor tissue which would be least antigenic when grafted onto rabbits' eyes. Reports in the literature on the experi mental use of heterografts are not numer ous, in recent times. Babel and Bourquin,^ and Choyce^ obtained some clear grafts in rabbits, using the interlamellar technique. Maiunenee' also reported clear grafts fol lowing penetrating keratoplasty, using corti sone postoperatively to suppress the antigenantibody reaction and the vascularization of the graft.
I n the interlamellar method of grafting, a transverse incision 5.0-mm. long and about 0.25-mm. deep was made with a fine scalpel on the limbus, slightly on the scleral side, 2.0 mm. in front or behind the insertion of the superior rectus muscle in the left or right eye respectively. T h e cornea was split from the limbus with the help of a Tooke knife to make an interlamellar pocket suffi ciently large to accommodate a corneal graft 5.0 mm. in diameter without crowding of the tissue. T h e graft was inserted into the intra corneal pocket with the help of nontoothed forceps, care being taken to spread the graft as uniformly as possible and to make sure that no part of the graft remained outside the interlamellar pouch.
•From the Departments of Ophthalmology and Bacteriology, Faculty of Medicine, University of Toronto. t Department of Ophthalmology, Ramakrishna Mission Sevashrama, Vrindaban, U. P., India,
I n the lamellar method of grafting, the bed of the graft measured 5.0 mm. in diameter and 0.25-mm. in depth and the graft was fixed with overlaying stitches. N o lid sutures were used. T h e corneal stitches
477
P. Κ. BASU AND HUGH L. ORMSBY
478
TABLE 1 THE FATE OF CORNEAL HETEROGRAFTS IN RABBITS' EYES (PERCENTAGE)
Interlamellar
Gross Appearance of Grafts
Avian 1. Clear from beginning 2. Clear for 1 - 3 wk., then opaque (a) Opacity temporary (b) Opacity permanent 3. Opaque from beginning
10 80 50 30 10
were removed on the sixth or seventh post operative day. Postoperatively all the cases were treated with antibiotics. T h e minimum period of observation was 10 weeks, and animals were killed at inter vals for histologic examination of the grafts. RESULTS
Postoperatively, the grafted eyes could be classified into three groups. In the first group, the corneal grafts were clear from the beginning, the clarity depending upon .surgical success and tolerance of the donor tissue by the host. In the second group, in which donorrecipient reactions occurred, the grafts were clear for a period of one to three weeks and then developed opacification, varying from a transient cloudiness to a complete and permanent opacity. These eyes were in variably vascularized, and evidenced two types of vessels, ( 1 ) a primary deep type which invaded the graft bed postoperatively.
Fig. 1 (Basu and Ormsby). Clear lamellar graft (chicken).
Lamellar
Mammalian 10 80 36.7 43.3 10
Avian
Mammahan
20 60 20 40 20
10 70 10 60 20
and ( 2 ) a secondary superficial type which invaded the graft rapidly at the onset of cloudiness. In the third group, the grafts were opaque from the beginning and represented surgical failures due to folding of the grafts in the interlamellar pockets, and to faulty fixation in lamellar grafts. T h e percentage of surgi cal failures was twice as high in lamellar (20 percent) as in interlamellar (10 per cent) grafts. Postoperative failures due to infection did not occur because of the use of antibiotics. I n Table 1, it will be seen that the highest percentage of grafts remaining clear from the beginning occurred in avian tissue grafted by the lamellar method (figs. 1 and 2 ) . Temporary opacity occurred more fre quently in interlamellar grafts (fig. 3) than in lamellar grafts, showing that the per centage of recovery following donorrecipient reaction was greater in inter lamellar grafts. T h u s there was a lower per centage of permanent opacities in inter-
Fig. 2 (Basu and Ormsby). Clear lamellar graft (duck).
479
CORNEAL HETEROGRAFTS IN RABBITS
Fig. 3 (Basu and Ormsby). Reaction in interlamellar graft (turkey). lamellar than in the lamellar grafts. It will also be seen that permanent opacities oc curred less frequently in both types of grafts with avian tissue than with the mammalian tissue (fig. 4 ) . I n Table 2 it will be seen that if the surgi cally unsuccessful grafts are disregarded, with avian tissues there were ultimately 66.6 percent clear grafts by the interlamellar and 50 percent by the lamellar method. W i t h mammalian tissues, the percentage of clear lamellar grafts was half that of interlamellar grafts. Mammalian tissues resulted in a lower percentage of clear grafts than did avian tissues. I n Table 3, it will be seen that in the avian group the ultimate transparency of the grafts was better with chicken and duck tissues (figs. 1 and 2) than with turkey. I n the mammalian tissues, monkey tissue was better than beef or lamb. DiscussioK In this series of experiments, donorrecipient reactions occurred in both inter-
Fig. 4 (Basu and Ormsby). Opaque lamellar graft (beef). lamellar and lamellar grafts. Surgical failure was higher in the lamellar than in the inter lamellar series and this difference could be accounted for by the failure of fixation in lamellar grafts. Similarly, the percentage of grafts developing temporary opacities was higher in the interlamellar group, probably due to increased postoperative deep vascularization of graft bed. T h e rate of recovery of clarity, however, was much higher in interlamellar grafts, possibly due to the fact that there was less superficial vascularization in interlamellar than in lamellar grafts. T h e better over-all tolerance of rabbits' eyes for avian donor tissues, as compared to mammalian tissues, suggests that the farther apart phylogenetically the t w o tissues, the less likelihood there is of a donor-recipient reaction developing. This suggests that avian tissues might be better tolerated than mammalian tissues, if they were used for human heterografts. TABLE 3 PERCENTAGE OF CLEAR GRAFTS IN AVIAN AND JIAMMALIAN TISSUES
TABLE 2 Donor
PERCENTAGE OF CLEAR CORNEAL HETEROGRAFTS IN RABBrrS' EYES (SURGICALLY SUCCESSFUL GROUP)
Avian grafts Mammalian grafts
Interlamellar
Lamellar
66.6 51.8
50 25
1. 2. 3. 4. 5. 6.
Chicken Duck Turkey Monkey Lamb Beef
Inter lamellar Grafts
Lamellar Grafts
70 60 SO 60 50 30
50 40 30 30 20 10
P. Κ. BASU AND HUGH L. ORMSBY
480
SUMMARY A N D CONCLUSIONS
1. Interlamellar and lamellar techniques were used in the study of antigen-antibody reactions occurring in heterografts in rabbits' eyes. 2. Antigen-antibody reactions occurred with all donor tissues and were less fre
quent with avian corneas than with mammal ian corneas. Chicken cornea resulted in fewer reactions than either duck or turkey. Reactions with monkey cornea occurred less frequently than with lamb or beef. Banting Institute
(5).
REFERENCES
1. Babel, J., and Bourquin, J. B.: Experimental research with corneal heterografts. Brit. J. Ophth., 36 :S29, 1952. 2. Choyce, D. P.: Successful transplantation of human and cat corneal tissue into rabbit cerneas. Brit. ] . Ophth., 36:1952. 3. Maumenee, A. E.: The immune concept: Its relation to corneal homotransplantation. Atm. New York Acad. Sc., 59 :453, 1955. 4. Basu, P. K., and Ormsby, H. L.: Interlamellar frozen stored corneal homografts in rabbits. Am. J. Ophth., 42:71 (Part II) 1956.
T H E
BICARBONATE-ION
CONCENTRATION
BEFORE A N D AFTER DEATH HARRY GREEN,
Green, Sawyer, and Leopold* recently found that the bicarbonate-ion concentration of the rabbit vitreous humor from an eye removed immediately following death of the animal, and immediately frozen, was higher than that of the arterial blood plasma (25.3 m M / 1 compared with 19.4 m M / 1 ) . Analy sis of frozen sections of vitreous humor showed that the area lying between the cili ary body and the posterior half of the lens was higher (31.3 m M / 1 ) than the periph eral area (23.9 m M / 1 ) and the central core (24.8 m M / 1 ) . I n all three sections, therefore, the bicarbonate-ion concentration was definitely higher than the concentration in the arterial blood plasma. I n the above publication, reference was made to the work of Auricchio and A m b r o •From the Research Department, Wills Eye Hospital, and the Department of Physiological Chemistry, Graduate School of Medicine, Univer sity of Pennsylvania. This investigation was sup ported in part by a grant-in-aid (No. 189C) from the National Council to Combat Blindness.
RABBIT
VITREOUS*
OF T H E A N I M A L
P H . D . , A N D M A Y E R J.
Philadelphia,
OF
MANN,
A.B.
Pennsylvania
sio^ who reported the bicarbonate-ion con centration of the rabbit vitreous humor, re moved following death of the animal, to be lower than that of the arterial blood plasma (19.5 m M / 1 compared with 25.6 m M / 1 ) . T h e conditions under which the vitreous humor was removed from the eye were not described. I n two later publications'" * which came to our attention while our own paper was in press Auricchio reported that the bicarbon ate-ion concentration of the vitreous humor removed under in vivo conditions by cannulation through the sclera of the rabbit eye was higher than that of the blood plasma. H i s data a r e : vitreous humor 28.3 m M / 1 and venous plasma 19.6 m M / 1 , averages of seven samples. T h e previously recorded lower value of the vitreous humor compared to that of a r terial blood plasma was attributed by Auric chio to the fact that the vitreous humor was removed following death of the animal. U n d e r such conditions it was presumed that
Κ.
HETEROGRAFTS
BASU.t D.O.M.S.,
AND HUGH
IN L.
RABBITS* ORMSBY,
M.D.
Toronto, Ontario I n recent years, there has been an in creasing demand for donor eyes for keratoplasty. This has resulted in eflforts to improve methods of storage, but there is still no solution to the problem of obtaining a sufficient supply of suitable eyes from human sources. Since there is little hope that the demand for h u m a n donor corneas will be met for many years, it seemed timely to re investigate the possibiUty of using animal or avian tissues as donor material.
I n a previous report,* we have shown the interlamellar method of grafting to be suita ble for the study of graft opacification. I n the present study we have used both inter lamellar and lamellar methods in parallel series utilizing avian and mammalian donor tissues. METHODS A N D MATERIALS
During the past century there were many reports of the use of heterografts in humans. Since that time, however, it has been gen erally accepted that such grafts are unsuc cessful. T h e new theories of immunity offer an explanation for the opacification of heterografts. If this theory is correct, it seems possible that new therapeutic agents might be used to improve the chances of suc cess in heterografts.
Full grown rabbits weighing from 2.5 to 4.0 kg. from mixed stocks were used as recipients. All operative procedures were performed under intravenous Nembutal anesthesia and full surgical asepsis. Donor corneal tissue was obtained from chicken, duck, turkey, monkey, beef, and lamb. Each recipient received tissues from the same donor, using a n interlamellar graft in one eye and a lamellar graft in the other. All the grafts were fresh and measured 5.0 mm. in diameter by 0.25 mm. in thickness.
T h e present experiments were designed to discover if the opacification occurring in heterografts could be explained by the oc currence of an antigen-antibody reaction, and to find the donor tissue which would be least antigenic when grafted onto rabbits' eyes. Reports in the literature on the experi mental use of heterografts are not numer ous, in recent times. Babel and Bourquin,^ and Choyce^ obtained some clear grafts in rabbits, using the interlamellar technique. Maiunenee' also reported clear grafts fol lowing penetrating keratoplasty, using corti sone postoperatively to suppress the antigenantibody reaction and the vascularization of the graft.
I n the interlamellar method of grafting, a transverse incision 5.0-mm. long and about 0.25-mm. deep was made with a fine scalpel on the limbus, slightly on the scleral side, 2.0 mm. in front or behind the insertion of the superior rectus muscle in the left or right eye respectively. T h e cornea was split from the limbus with the help of a Tooke knife to make an interlamellar pocket suffi ciently large to accommodate a corneal graft 5.0 mm. in diameter without crowding of the tissue. T h e graft was inserted into the intra corneal pocket with the help of nontoothed forceps, care being taken to spread the graft as uniformly as possible and to make sure that no part of the graft remained outside the interlamellar pouch.
•From the Departments of Ophthalmology and Bacteriology, Faculty of Medicine, University of Toronto. t Department of Ophthalmology, Ramakrishna Mission Sevashrama, Vrindaban, U. P., India,
I n the lamellar method of grafting, the bed of the graft measured 5.0 mm. in diameter and 0.25-mm. in depth and the graft was fixed with overlaying stitches. N o lid sutures were used. T h e corneal stitches
477
P. Κ. BASU AND HUGH L. ORMSBY
478
TABLE 1 THE FATE OF CORNEAL HETEROGRAFTS IN RABBITS' EYES (PERCENTAGE)
Interlamellar
Gross Appearance of Grafts
Avian 1. Clear from beginning 2. Clear for 1 - 3 wk., then opaque (a) Opacity temporary (b) Opacity permanent 3. Opaque from beginning
10 80 50 30 10
were removed on the sixth or seventh post operative day. Postoperatively all the cases were treated with antibiotics. T h e minimum period of observation was 10 weeks, and animals were killed at inter vals for histologic examination of the grafts. RESULTS
Postoperatively, the grafted eyes could be classified into three groups. In the first group, the corneal grafts were clear from the beginning, the clarity depending upon .surgical success and tolerance of the donor tissue by the host. In the second group, in which donorrecipient reactions occurred, the grafts were clear for a period of one to three weeks and then developed opacification, varying from a transient cloudiness to a complete and permanent opacity. These eyes were in variably vascularized, and evidenced two types of vessels, ( 1 ) a primary deep type which invaded the graft bed postoperatively.
Fig. 1 (Basu and Ormsby). Clear lamellar graft (chicken).
Lamellar
Mammalian 10 80 36.7 43.3 10
Avian
Mammahan
20 60 20 40 20
10 70 10 60 20
and ( 2 ) a secondary superficial type which invaded the graft rapidly at the onset of cloudiness. In the third group, the grafts were opaque from the beginning and represented surgical failures due to folding of the grafts in the interlamellar pockets, and to faulty fixation in lamellar grafts. T h e percentage of surgi cal failures was twice as high in lamellar (20 percent) as in interlamellar (10 per cent) grafts. Postoperative failures due to infection did not occur because of the use of antibiotics. I n Table 1, it will be seen that the highest percentage of grafts remaining clear from the beginning occurred in avian tissue grafted by the lamellar method (figs. 1 and 2 ) . Temporary opacity occurred more fre quently in interlamellar grafts (fig. 3) than in lamellar grafts, showing that the per centage of recovery following donorrecipient reaction was greater in inter lamellar grafts. T h u s there was a lower per centage of permanent opacities in inter-
Fig. 2 (Basu and Ormsby). Clear lamellar graft (duck).
479
CORNEAL HETEROGRAFTS IN RABBITS
Fig. 3 (Basu and Ormsby). Reaction in interlamellar graft (turkey). lamellar than in the lamellar grafts. It will also be seen that permanent opacities oc curred less frequently in both types of grafts with avian tissue than with the mammalian tissue (fig. 4 ) . I n Table 2 it will be seen that if the surgi cally unsuccessful grafts are disregarded, with avian tissues there were ultimately 66.6 percent clear grafts by the interlamellar and 50 percent by the lamellar method. W i t h mammalian tissues, the percentage of clear lamellar grafts was half that of interlamellar grafts. Mammalian tissues resulted in a lower percentage of clear grafts than did avian tissues. I n Table 3, it will be seen that in the avian group the ultimate transparency of the grafts was better with chicken and duck tissues (figs. 1 and 2) than with turkey. I n the mammalian tissues, monkey tissue was better than beef or lamb. DiscussioK In this series of experiments, donorrecipient reactions occurred in both inter-
Fig. 4 (Basu and Ormsby). Opaque lamellar graft (beef). lamellar and lamellar grafts. Surgical failure was higher in the lamellar than in the inter lamellar series and this difference could be accounted for by the failure of fixation in lamellar grafts. Similarly, the percentage of grafts developing temporary opacities was higher in the interlamellar group, probably due to increased postoperative deep vascularization of graft bed. T h e rate of recovery of clarity, however, was much higher in interlamellar grafts, possibly due to the fact that there was less superficial vascularization in interlamellar than in lamellar grafts. T h e better over-all tolerance of rabbits' eyes for avian donor tissues, as compared to mammalian tissues, suggests that the farther apart phylogenetically the t w o tissues, the less likelihood there is of a donor-recipient reaction developing. This suggests that avian tissues might be better tolerated than mammalian tissues, if they were used for human heterografts. TABLE 3 PERCENTAGE OF CLEAR GRAFTS IN AVIAN AND JIAMMALIAN TISSUES
TABLE 2 Donor
PERCENTAGE OF CLEAR CORNEAL HETEROGRAFTS IN RABBrrS' EYES (SURGICALLY SUCCESSFUL GROUP)
Avian grafts Mammalian grafts
Interlamellar
Lamellar
66.6 51.8
50 25
1. 2. 3. 4. 5. 6.
Chicken Duck Turkey Monkey Lamb Beef
Inter lamellar Grafts
Lamellar Grafts
70 60 SO 60 50 30
50 40 30 30 20 10
P. Κ. BASU AND HUGH L. ORMSBY
480
SUMMARY A N D CONCLUSIONS
1. Interlamellar and lamellar techniques were used in the study of antigen-antibody reactions occurring in heterografts in rabbits' eyes. 2. Antigen-antibody reactions occurred with all donor tissues and were less fre
quent with avian corneas than with mammal ian corneas. Chicken cornea resulted in fewer reactions than either duck or turkey. Reactions with monkey cornea occurred less frequently than with lamb or beef. Banting Institute
(5).
REFERENCES
1. Babel, J., and Bourquin, J. B.: Experimental research with corneal heterografts. Brit. J. Ophth., 36 :S29, 1952. 2. Choyce, D. P.: Successful transplantation of human and cat corneal tissue into rabbit cerneas. Brit. ] . Ophth., 36:1952. 3. Maumenee, A. E.: The immune concept: Its relation to corneal homotransplantation. Atm. New York Acad. Sc., 59 :453, 1955. 4. Basu, P. K., and Ormsby, H. L.: Interlamellar frozen stored corneal homografts in rabbits. Am. J. Ophth., 42:71 (Part II) 1956.
T H E
BICARBONATE-ION
CONCENTRATION
BEFORE A N D AFTER DEATH HARRY GREEN,
Green, Sawyer, and Leopold* recently found that the bicarbonate-ion concentration of the rabbit vitreous humor from an eye removed immediately following death of the animal, and immediately frozen, was higher than that of the arterial blood plasma (25.3 m M / 1 compared with 19.4 m M / 1 ) . Analy sis of frozen sections of vitreous humor showed that the area lying between the cili ary body and the posterior half of the lens was higher (31.3 m M / 1 ) than the periph eral area (23.9 m M / 1 ) and the central core (24.8 m M / 1 ) . I n all three sections, therefore, the bicarbonate-ion concentration was definitely higher than the concentration in the arterial blood plasma. I n the above publication, reference was made to the work of Auricchio and A m b r o •From the Research Department, Wills Eye Hospital, and the Department of Physiological Chemistry, Graduate School of Medicine, Univer sity of Pennsylvania. This investigation was sup ported in part by a grant-in-aid (No. 189C) from the National Council to Combat Blindness.
RABBIT
VITREOUS*
OF T H E A N I M A L
P H . D . , A N D M A Y E R J.
Philadelphia,
OF
MANN,
A.B.
Pennsylvania
sio^ who reported the bicarbonate-ion con centration of the rabbit vitreous humor, re moved following death of the animal, to be lower than that of the arterial blood plasma (19.5 m M / 1 compared with 25.6 m M / 1 ) . T h e conditions under which the vitreous humor was removed from the eye were not described. I n two later publications'" * which came to our attention while our own paper was in press Auricchio reported that the bicarbon ate-ion concentration of the vitreous humor removed under in vivo conditions by cannulation through the sclera of the rabbit eye was higher than that of the blood plasma. H i s data a r e : vitreous humor 28.3 m M / 1 and venous plasma 19.6 m M / 1 , averages of seven samples. T h e previously recorded lower value of the vitreous humor compared to that of a r terial blood plasma was attributed by Auric chio to the fact that the vitreous humor was removed following death of the animal. U n d e r such conditions it was presumed that