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Protection of Penetrating Corneal Allografts from Immune Rejection
PROTECTION O F P E N E T R A T I N G CORNEAL ALLOGRAFTS FROM I M M U N E REJECTION JOEL M. ENGELSTEIN, M.D.,
RONALD B. HERBERMAN, M.D.,
STEPHEN R. WALTMAN,
AND
M.D.
Bethesda, Maryland
In clinical and experimental situations, penetrating corneal allografts are successful in subjects who reject other transplanted or gans. The protection of the donor cornea from the immune response of the recipient is generally considered to be related to the lack of blood and lymph vessels in the cornea and the anatomic barrier which this provides against the effectors of the immune re sponse, circulating antibody, and sensitized lymphocytes. The immune system can be schematically divided into three compart ments: (1) an afferent arc which serves to bring foreign antigens to the central immune mechanism; (2) a central mechanism, en compassing the cells and organs which pro cess antigens, become specifically sensitized, and produce the circulating antibody or sensi tized lymphocytes which are the effectors of the immune response; and (3) the efferent arc, which allows these effectors to come into contact with the specific foreign antigens, usually resulting in a sequence of events which terminates in the destruction of the offending substance. When viewing the im mune system in this manner it is generally thought that it is the last of these compart ments, the effector arc, which is not opera tive in corneal transplants, thereby account ing for the unusual protection of corneal grafts from immune attack. The purpose of this study is to examine both the afferent and efferent arcs of the immune system in rela tion to corneal transplants, and to indicate the relative part that each plays in protecting the corneal graft from rejection. From the Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland. Reprint requests to Joel Engelstein, M.D., De partment of Ophthalmology, University of Florida School of Medicine, Gainesville, Florida 32601.
MATERIALS AND METHODS
Virgin New Zealand White rabbits weigh ing 2.5 to 3.5 kg were obtained from a com mercial rabbitry (B&H Rabbitry, Rockville, Maryland) without regard to sex. The rab bits were randomly paired and 6.5 mm pene trating corneal allografts were exchanged between partners on day zero. At the same time 6.5 mm corneal autografts were per formed on the opposite eye by trephining a corneal button, rotating it 180 degrees, and suturing it in place using continuous 8-0 black silk suture, as previously described.1 The technique was clean but not sterile, and was identical for allografts and autografts. After the procedure 10% neosynephrine drops, 1% atropine ointment and Neosporin ointment were instilled into the conjunctival sac daily for 10 days. On the 10th postopera tive day the sutures were removed. The grafts were then examined three times a week, and scored for limbal flush, corneal vascularization, host corneal edema, and de gree of graft clouding. Observations were made without reference to notes indicating which eye was the allograft and which the autograft. The first day of corneal graft re jection was scored as the day on which 1 + flush occurred and progressed to graft cloud ing. The rejection phenomenon was consid ered complete when 3 + to 4 + graft cloudi ness had developed and the flush was past its peak and subsiding. Skin grafts were exchanged between part ners from seven to 10 weeks following cor neal grafting, and also in a control group which had not had keratoplasties. The skin graft was performed on the rabbit's ear ac cording to the technique of Warwick, 2 with a few modifications. The grafts were 12 mm in diameter. They were put in a sterile Petri
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TABLE 1 CORNEAL GRAFT TECHNICAL FAILURES
Total grafts Dehiscence Buphthalmos Dehiscence and buphthalmos Unknown Total failures
AUografts
Autografts
Total
44 1(2.3*) 1 (2.3) 2(4.5) 0(0)
44 2(4.5*) 1 (2.3) 0(0) 1(2.3)
88 3(3.4*) 2 (2.3) 2(2.3) 1(1.1)
4(9.1)
4(9.1)
8(9.1)
* Percent of total is given in parentheses.
dish containing minimum essential media with penicillin and streptomycin before be ing placed on the prepared graft bed. Once in place on the recipient they were sprayed with antiseptic (MediQuik) and then lightly sprayed with dressing (Aeroplast). Rabbits received 2 ml of an antibiotic (Bicillin) (300,000 units per ml) intramuscularly im mediately after the procedure and another 1 ml two to four days later. Grafts were ex amined daily and viability was judged on the basis of thickening, softness, color, and plia bility. The day of rejection was scored as that day when less than 10% of the graft re mained viable. Skin grafts were examined be fore corneal grafts and without reference to notes from previous examinations. For histologie study, the allografted eyes of two rabbits, number 104 and number 140, were enucleated five and seven days after the beginning of corneal rejection, respectively, and fixed in 10% formalin for two weeks. Then they were sectioned and stained with hematoxylin and eosin. RESULTS
Forty-four rabbits (22 rabbit pairs) re ceived corneal allografts and autografts. Of these 88 grafts there were eight technical failures (9.1%), equally divided between the autografts and the allografts (Table 1). Only one rabbit suffered both allo- and autograft failure ; and in no case did graft fail ure occur in both members of a rabbit pair. Forty corneal autografts were technically acceptable (a quiet eye without graft
clouding). Eight of these rabbits died or were killed within two weeks of grafting ; all had clear autografts at the time of death. The remaining 32 autografts were observed for from 34 to 164 days, with the majority under observation for more than 100 days. No rejections occurred. Forty corneal autografts were technically acceptable. Seven of these rabbits died within two weeks of surgery, so that their graft survival could not be evaluated. An ad ditional five rabbits died with clear corneas at 34, 36, 70, 70, and 70 days after corneal grafting. One rabbit had not rejected skin or corneal allograft after four months, and studies on this animal will be published later. The remaining 27 rabbits can be divided into two groups: 12 rabbits who spontaneously rejected their allografted corneas before skin grafting (Table 2 ) , and 15 who rejected allocorneas after skin grafting (Table 3 ) . Hence 12/27, or 44% of allografted corneas were spontaneously rejected within three to seven weeks. Seven to 10 weeks after corneal grafting rabbits received skin grafts from their part ners, regardless of whether or not they had already rejected their corneas. Of the 15 rabbits who had not previously rejected their corneal grafts, 14 rejected their corneal allo grafts within two to four weeks after skin grafting (Table 3 ) . Among those rabbits who had not rejected their corneal allo grafts at the time of skin grafting, skin sur vival ranged from seven to 13 days with a mean of 8.75 days. However, those rabbits
CORNEAL GRAFT PROTECTION
VOL. 74, NO. 2 TABLE 2
SPONTANEOUS CORNEAL ALLOGRAFT REJECTION
Rabbit No.
Corneal Allograft Survival (in days)
164 130 118 103 174 188 192 134 133 136 173 102
48 28 40 23 27 35 40 30 35 33 34 49
Time Between Corneal Graft Skin Graft ing and Skin Survival* Grafting * (in days) (in days) 48 53 54 56 56 57 57 64 64 NDf ND ND
5 TFf 5 5 7 7 5 6 6 ND ND ND
* Mean 5.75 days, S.D. 0.89 days, S.E. 0.31 days. f N D = N o t done, TF=skin graft was technical failure.
who had spontaneously rejected their corneal grafts before skin grafting had a skin graft survival ranging from five to seven days with a mean of 5.75 days (Table 2 ) . Fourteen rabbits (seven rabbit pairs) who
313
had not had corneal grafts then had skin grafts exchanged in order to determine the normal skin graft survival period (Table 4 ) . One of these died with a viable graft at six days and there were three technical failures. The remaining 10 grafts survived from seven to 15 days with a mean survival time of 9.4 days. Statistical analysis of this data (Table 5) indicates that the normal skin graft survival data is not significantly differ ent (p >0.24) from the survival of skin grafts on rabbits who had not yet rejected their corneas, but is significantly different (p < 0.001) from the skin graft survival on rabbits who had already rejected corneas from the same donor. The accelerated rejec tion rate of skin grafts on rabbits who had already rejected corneas from the same do nor indicates that these rabbits were sensi tized by their corneal grafts. Recipients of corneas which were not spontaneously re jected, however, did not reject their skin grafts via a second-set reaction, and always rejected their corneal grafts significantly later than their skin grafts. The median de-
TABLE 3 CORNEAL ALLOGRAFT REJECTION POST-SKIN GRAFTING
Rabbit No.
Time Between Corneal and Skin Grafting* (in days)
185 176 131 140 145 199 107 149 142 186 106 191 104 159 157
48 48 53 54 54, 64* 56 56 56, 65* 56, 65* 57 57 57 57 60 69
Corneal Allograft Survival (in days)
Time Between Skin Grafting and Corneal Rejection (in days)
Skin Graft Survivait (in days)
63 71 80 77 77 67 70 67 71 70 71 71 71 75 >129
15 23 27 23 23, 13* 11 14 11, 2* 15, 6 13 14 14 16 15 Still surviving
7 12 13 9 TF 8 7 TF TF 9 7 9 9 7 8
Time Between Skin Graft Rejection and Corneal Rejection { (in days) 8 11 14 14
— 3 7
— —
4 7 5 7 8 >121
* TF=Skin graft was technical failure. When skin graft was a technical failure, a second skin graft was applied. t Mean 8.75 days, S.D. 1.96 days, S.E. 0.57 days. t Median delay in initiation of corneal rejection compared to completion of skin graft rejection was 7.5 days.
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THE AMERICAN JOURNAL OF OPHTHALMOLOGY TABLE 4 NORMAL SKIN GRAFT SURVIVAL
Rabbit No.
Skin Graft Survival* (in days)
195 132 158 161 187 110 155 117 194 171 163 105 197 112
7 8 8 8 8 9 9 10 12 15 TFf TF TF Î
* Mean 9.40, S.D. 2.41, S.E. 0.76. t T F = Technical failure. | Rabbit died at six days with viable graft.
lay in the initiation of corneal graft rejection compared to the completion of skin graft re jection was 7.5 days. One rabbit (No. 157) did not reject his cornea during the observa tion period. Histologie examination of rejected cor neas revealed a predominantly round cell in filtrate in the stroma with sparse endothelial cells. In many areas lymphocytes had re placed the endothelial cells on Descemet's membrane. This picture is typical for im mune rejection.
AUGUST, 1972
autografts. Histologie examination of re jecting corneas was consistent with immune rejection. Hence the degree of histoincompatability between these rabbits, as well as the degree of their immune reactivity, may provide a more appropriate explanation. These factors, however, did not seem to op erate in the case of the normal skin graft survival data, since our data in this respect is very similar to that published by Warwick. 2 The fact that we used larger corneal grafts than did the previous investigators (6.5 mm as compared to 4.5 mm) may be important, although Khodadoust5 reported a rejection rate of only 10% in 100 rabbits with 8 mm corneal allografts, and 5% in 20 rabbits with 7 mm grafts. It is true that his observation period is simply listed as four to six weeks. This experience is instructive in that it em phasizes the difficulty in evolving broad gen eralizations from the results of limited ex periments performed on a specific group of animals in a particular manner. The high spontaneous rejection rate of corneal allografts did make it possible to study the immune reaction to corneal allo grafts in otherwise unmanipulated animals. The fact that rabbits who spontaneously re ject corneal grafts are sensitized to the histocompatability antigens of the corneal donor TABLE 5 SUMMARY OF SKIN GRAFT SURVIVAL DATA
DISCUSSION
We recognize that the high spontaneous rejection rate of penetrating corneal allografts in the rabbits used in this experiment, and the rejection, following delayed skin grafting (seven to 10 weeks post-corneal grafting), of previously clear corneal grafts, is at variance with the reported results of both Maumenee3 and O'Gawa, Guyton, San ders, Inch, and Ellis.4 Technical and meta bolic factors are unlikely explanations of this discrepancy in light of the healthy ap pearance of corneal grafts until rejection be gins, the earliest rejection occurring at 23 days, and the continued survival of corneal
Mean S.D. S.E. (days) (days) (days) Skin graft survival : Group 1—Spontaneously rejecting corneas 5.75 0.89 0.31 Group 2—Corneas reject ing after skin grafting 8.75 1.96 0.57 Group 3—Normal 9.40 2.41 0.76 Significance test* results: Group 1 Mean significantly less than group 2 and group 3 mean (p<.001) Group 2 Mean is not significantly less than group 3 mean (p>.24) * Using student t-test, taking into account un equal variances (Aspen-Welch procedure).
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CORNEAL GRAFT PROTECTION
and therefore reject skin grafts from this same donor in an accelerated fashion was expected. However, rabbits who did not spontaneously reject corneal allografts re acted to skin grafts from the corneal donors as if they had never been in contact with these histocompatability antigens before, al lowing the skin grafts to survive for a nor mal period of time. These rabbits then went on to reject their corneal grafts after the sensitizing experience of the skin graft, indi cating that the central mechanism and effer ent limb of the immune system remained in tact. Hence the lack of systemic sensitization by corneal allografts, indicated by normal skin graft survival, is in effect a block on the afferent limb of the immune system, and may play a major role in protecting corneal grafts from immune destruction. The fact that the initiation of corneal rejection in each rabbit was later than the rejection of the skin graft indicates that some degree of efferent protection also exists. This efferent protec tion was complete only in one rabbit (No. 157), and was obviously ineffective in those rabbits who become systemically sensitized by their corneal grafts and then rejected them spontaneously. Hence, the protection of corneal allografts from immune rejection is dependant on interference with both the afferent and efferent limbs of the immune system. The afferent block seems more com plete, operates more frequently, and is prob ably of greater biological value. The degree of efferent block is variable, rarely complete and occurs less frequently. It is of interest to note that corneal rejec tion in originally unsensitized rabbits occur red as late as 80 days after corneal grafting, with a mean of 72.2 days. This demonstrates that at this time the corneal graft still con sists of donor cells which continue to ex press their own histocompatability antigens.
SUMMARY
Corneal allografts were exchanged in a group of rabbits, and 12 of 27 of these grafts were spontaneously rejected in three to seven weeks. Seven to 10 weeks after keratoplasty, the rabbits received skin allografts from the corneal donor, regardless of the state of their corneal allografts. Skin graft rejec tion was accelerated in those rabbits who had rejected their corneas, indicating that they were systemically sensitized to the donor's histocompatability antigens. Skin graft sur vival was normal in rabbits that had not re jected corneal grafts spontaneously. After skin grafting, 14 out of 15 rabbits with pre viously clear corneas went on to reject their corneal grafts, indicating that the cell type and antigen expression on the corneal grafts had remained stable, and that the central mechanism and efferent arc of the immune system were intact. Hence, the major mecha nism protecting these corneal grafts from immune rejection was a block in the afferent arc, preventing systemic sensitization. ACKNOWLEDGMENTS
We appreciate the help of Dr. Robert Connor in statistically evaluating our data, and the excellent technical assistance of Mr. David Hoffman and Mrs. Hilda Porter. REFERENCES
1. Waltman, S. R., Faulkner, H. W., and Bürde, R. M. : Use of antilymphocyte serum to prevent im mune rejection of penetrating corneal homografts. Invest. Ophth. 8:196, 1969. 2. Warwick, W. j . : A rapid, sutureless method for grafting skin in rabbits. Trans. Bull. 30:51, 1962. 3. Maumenee, A. E. : The influence of donor-re cipient sensitization on corneal grafts. Am. J. Ophth. 34 (Part 2) :142,1951. 4. O'Gawa, G. M., Guyton, J. S., Sanders, W. R., Inch, F. A. B., and Ellis, R. C. : Behavior of clear, penetrating corneal homografts in rabbits. Am. J. Ophth. 61:267, 1966. 5. Khodadoust, A.: Penetrating keratoplasty in rabbits. Am. J. Ophth. 66:899, 1968.
RONALD B. HERBERMAN, M.D.,
STEPHEN R. WALTMAN,
AND
M.D.
Bethesda, Maryland
In clinical and experimental situations, penetrating corneal allografts are successful in subjects who reject other transplanted or gans. The protection of the donor cornea from the immune response of the recipient is generally considered to be related to the lack of blood and lymph vessels in the cornea and the anatomic barrier which this provides against the effectors of the immune re sponse, circulating antibody, and sensitized lymphocytes. The immune system can be schematically divided into three compart ments: (1) an afferent arc which serves to bring foreign antigens to the central immune mechanism; (2) a central mechanism, en compassing the cells and organs which pro cess antigens, become specifically sensitized, and produce the circulating antibody or sensi tized lymphocytes which are the effectors of the immune response; and (3) the efferent arc, which allows these effectors to come into contact with the specific foreign antigens, usually resulting in a sequence of events which terminates in the destruction of the offending substance. When viewing the im mune system in this manner it is generally thought that it is the last of these compart ments, the effector arc, which is not opera tive in corneal transplants, thereby account ing for the unusual protection of corneal grafts from immune attack. The purpose of this study is to examine both the afferent and efferent arcs of the immune system in rela tion to corneal transplants, and to indicate the relative part that each plays in protecting the corneal graft from rejection. From the Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland. Reprint requests to Joel Engelstein, M.D., De partment of Ophthalmology, University of Florida School of Medicine, Gainesville, Florida 32601.
MATERIALS AND METHODS
Virgin New Zealand White rabbits weigh ing 2.5 to 3.5 kg were obtained from a com mercial rabbitry (B&H Rabbitry, Rockville, Maryland) without regard to sex. The rab bits were randomly paired and 6.5 mm pene trating corneal allografts were exchanged between partners on day zero. At the same time 6.5 mm corneal autografts were per formed on the opposite eye by trephining a corneal button, rotating it 180 degrees, and suturing it in place using continuous 8-0 black silk suture, as previously described.1 The technique was clean but not sterile, and was identical for allografts and autografts. After the procedure 10% neosynephrine drops, 1% atropine ointment and Neosporin ointment were instilled into the conjunctival sac daily for 10 days. On the 10th postopera tive day the sutures were removed. The grafts were then examined three times a week, and scored for limbal flush, corneal vascularization, host corneal edema, and de gree of graft clouding. Observations were made without reference to notes indicating which eye was the allograft and which the autograft. The first day of corneal graft re jection was scored as the day on which 1 + flush occurred and progressed to graft cloud ing. The rejection phenomenon was consid ered complete when 3 + to 4 + graft cloudi ness had developed and the flush was past its peak and subsiding. Skin grafts were exchanged between part ners from seven to 10 weeks following cor neal grafting, and also in a control group which had not had keratoplasties. The skin graft was performed on the rabbit's ear ac cording to the technique of Warwick, 2 with a few modifications. The grafts were 12 mm in diameter. They were put in a sterile Petri
311
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T H E AMERICAN JOURNAL OF OPHTHALMOLOGY
AUGUST, 1972
TABLE 1 CORNEAL GRAFT TECHNICAL FAILURES
Total grafts Dehiscence Buphthalmos Dehiscence and buphthalmos Unknown Total failures
AUografts
Autografts
Total
44 1(2.3*) 1 (2.3) 2(4.5) 0(0)
44 2(4.5*) 1 (2.3) 0(0) 1(2.3)
88 3(3.4*) 2 (2.3) 2(2.3) 1(1.1)
4(9.1)
4(9.1)
8(9.1)
* Percent of total is given in parentheses.
dish containing minimum essential media with penicillin and streptomycin before be ing placed on the prepared graft bed. Once in place on the recipient they were sprayed with antiseptic (MediQuik) and then lightly sprayed with dressing (Aeroplast). Rabbits received 2 ml of an antibiotic (Bicillin) (300,000 units per ml) intramuscularly im mediately after the procedure and another 1 ml two to four days later. Grafts were ex amined daily and viability was judged on the basis of thickening, softness, color, and plia bility. The day of rejection was scored as that day when less than 10% of the graft re mained viable. Skin grafts were examined be fore corneal grafts and without reference to notes from previous examinations. For histologie study, the allografted eyes of two rabbits, number 104 and number 140, were enucleated five and seven days after the beginning of corneal rejection, respectively, and fixed in 10% formalin for two weeks. Then they were sectioned and stained with hematoxylin and eosin. RESULTS
Forty-four rabbits (22 rabbit pairs) re ceived corneal allografts and autografts. Of these 88 grafts there were eight technical failures (9.1%), equally divided between the autografts and the allografts (Table 1). Only one rabbit suffered both allo- and autograft failure ; and in no case did graft fail ure occur in both members of a rabbit pair. Forty corneal autografts were technically acceptable (a quiet eye without graft
clouding). Eight of these rabbits died or were killed within two weeks of grafting ; all had clear autografts at the time of death. The remaining 32 autografts were observed for from 34 to 164 days, with the majority under observation for more than 100 days. No rejections occurred. Forty corneal autografts were technically acceptable. Seven of these rabbits died within two weeks of surgery, so that their graft survival could not be evaluated. An ad ditional five rabbits died with clear corneas at 34, 36, 70, 70, and 70 days after corneal grafting. One rabbit had not rejected skin or corneal allograft after four months, and studies on this animal will be published later. The remaining 27 rabbits can be divided into two groups: 12 rabbits who spontaneously rejected their allografted corneas before skin grafting (Table 2 ) , and 15 who rejected allocorneas after skin grafting (Table 3 ) . Hence 12/27, or 44% of allografted corneas were spontaneously rejected within three to seven weeks. Seven to 10 weeks after corneal grafting rabbits received skin grafts from their part ners, regardless of whether or not they had already rejected their corneas. Of the 15 rabbits who had not previously rejected their corneal grafts, 14 rejected their corneal allo grafts within two to four weeks after skin grafting (Table 3 ) . Among those rabbits who had not rejected their corneal allo grafts at the time of skin grafting, skin sur vival ranged from seven to 13 days with a mean of 8.75 days. However, those rabbits
CORNEAL GRAFT PROTECTION
VOL. 74, NO. 2 TABLE 2
SPONTANEOUS CORNEAL ALLOGRAFT REJECTION
Rabbit No.
Corneal Allograft Survival (in days)
164 130 118 103 174 188 192 134 133 136 173 102
48 28 40 23 27 35 40 30 35 33 34 49
Time Between Corneal Graft Skin Graft ing and Skin Survival* Grafting * (in days) (in days) 48 53 54 56 56 57 57 64 64 NDf ND ND
5 TFf 5 5 7 7 5 6 6 ND ND ND
* Mean 5.75 days, S.D. 0.89 days, S.E. 0.31 days. f N D = N o t done, TF=skin graft was technical failure.
who had spontaneously rejected their corneal grafts before skin grafting had a skin graft survival ranging from five to seven days with a mean of 5.75 days (Table 2 ) . Fourteen rabbits (seven rabbit pairs) who
313
had not had corneal grafts then had skin grafts exchanged in order to determine the normal skin graft survival period (Table 4 ) . One of these died with a viable graft at six days and there were three technical failures. The remaining 10 grafts survived from seven to 15 days with a mean survival time of 9.4 days. Statistical analysis of this data (Table 5) indicates that the normal skin graft survival data is not significantly differ ent (p >0.24) from the survival of skin grafts on rabbits who had not yet rejected their corneas, but is significantly different (p < 0.001) from the skin graft survival on rabbits who had already rejected corneas from the same donor. The accelerated rejec tion rate of skin grafts on rabbits who had already rejected corneas from the same do nor indicates that these rabbits were sensi tized by their corneal grafts. Recipients of corneas which were not spontaneously re jected, however, did not reject their skin grafts via a second-set reaction, and always rejected their corneal grafts significantly later than their skin grafts. The median de-
TABLE 3 CORNEAL ALLOGRAFT REJECTION POST-SKIN GRAFTING
Rabbit No.
Time Between Corneal and Skin Grafting* (in days)
185 176 131 140 145 199 107 149 142 186 106 191 104 159 157
48 48 53 54 54, 64* 56 56 56, 65* 56, 65* 57 57 57 57 60 69
Corneal Allograft Survival (in days)
Time Between Skin Grafting and Corneal Rejection (in days)
Skin Graft Survivait (in days)
63 71 80 77 77 67 70 67 71 70 71 71 71 75 >129
15 23 27 23 23, 13* 11 14 11, 2* 15, 6 13 14 14 16 15 Still surviving
7 12 13 9 TF 8 7 TF TF 9 7 9 9 7 8
Time Between Skin Graft Rejection and Corneal Rejection { (in days) 8 11 14 14
— 3 7
— —
4 7 5 7 8 >121
* TF=Skin graft was technical failure. When skin graft was a technical failure, a second skin graft was applied. t Mean 8.75 days, S.D. 1.96 days, S.E. 0.57 days. t Median delay in initiation of corneal rejection compared to completion of skin graft rejection was 7.5 days.
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THE AMERICAN JOURNAL OF OPHTHALMOLOGY TABLE 4 NORMAL SKIN GRAFT SURVIVAL
Rabbit No.
Skin Graft Survival* (in days)
195 132 158 161 187 110 155 117 194 171 163 105 197 112
7 8 8 8 8 9 9 10 12 15 TFf TF TF Î
* Mean 9.40, S.D. 2.41, S.E. 0.76. t T F = Technical failure. | Rabbit died at six days with viable graft.
lay in the initiation of corneal graft rejection compared to the completion of skin graft re jection was 7.5 days. One rabbit (No. 157) did not reject his cornea during the observa tion period. Histologie examination of rejected cor neas revealed a predominantly round cell in filtrate in the stroma with sparse endothelial cells. In many areas lymphocytes had re placed the endothelial cells on Descemet's membrane. This picture is typical for im mune rejection.
AUGUST, 1972
autografts. Histologie examination of re jecting corneas was consistent with immune rejection. Hence the degree of histoincompatability between these rabbits, as well as the degree of their immune reactivity, may provide a more appropriate explanation. These factors, however, did not seem to op erate in the case of the normal skin graft survival data, since our data in this respect is very similar to that published by Warwick. 2 The fact that we used larger corneal grafts than did the previous investigators (6.5 mm as compared to 4.5 mm) may be important, although Khodadoust5 reported a rejection rate of only 10% in 100 rabbits with 8 mm corneal allografts, and 5% in 20 rabbits with 7 mm grafts. It is true that his observation period is simply listed as four to six weeks. This experience is instructive in that it em phasizes the difficulty in evolving broad gen eralizations from the results of limited ex periments performed on a specific group of animals in a particular manner. The high spontaneous rejection rate of corneal allografts did make it possible to study the immune reaction to corneal allo grafts in otherwise unmanipulated animals. The fact that rabbits who spontaneously re ject corneal grafts are sensitized to the histocompatability antigens of the corneal donor TABLE 5 SUMMARY OF SKIN GRAFT SURVIVAL DATA
DISCUSSION
We recognize that the high spontaneous rejection rate of penetrating corneal allografts in the rabbits used in this experiment, and the rejection, following delayed skin grafting (seven to 10 weeks post-corneal grafting), of previously clear corneal grafts, is at variance with the reported results of both Maumenee3 and O'Gawa, Guyton, San ders, Inch, and Ellis.4 Technical and meta bolic factors are unlikely explanations of this discrepancy in light of the healthy ap pearance of corneal grafts until rejection be gins, the earliest rejection occurring at 23 days, and the continued survival of corneal
Mean S.D. S.E. (days) (days) (days) Skin graft survival : Group 1—Spontaneously rejecting corneas 5.75 0.89 0.31 Group 2—Corneas reject ing after skin grafting 8.75 1.96 0.57 Group 3—Normal 9.40 2.41 0.76 Significance test* results: Group 1 Mean significantly less than group 2 and group 3 mean (p<.001) Group 2 Mean is not significantly less than group 3 mean (p>.24) * Using student t-test, taking into account un equal variances (Aspen-Welch procedure).
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CORNEAL GRAFT PROTECTION
and therefore reject skin grafts from this same donor in an accelerated fashion was expected. However, rabbits who did not spontaneously reject corneal allografts re acted to skin grafts from the corneal donors as if they had never been in contact with these histocompatability antigens before, al lowing the skin grafts to survive for a nor mal period of time. These rabbits then went on to reject their corneal grafts after the sensitizing experience of the skin graft, indi cating that the central mechanism and effer ent limb of the immune system remained in tact. Hence the lack of systemic sensitization by corneal allografts, indicated by normal skin graft survival, is in effect a block on the afferent limb of the immune system, and may play a major role in protecting corneal grafts from immune destruction. The fact that the initiation of corneal rejection in each rabbit was later than the rejection of the skin graft indicates that some degree of efferent protection also exists. This efferent protec tion was complete only in one rabbit (No. 157), and was obviously ineffective in those rabbits who become systemically sensitized by their corneal grafts and then rejected them spontaneously. Hence, the protection of corneal allografts from immune rejection is dependant on interference with both the afferent and efferent limbs of the immune system. The afferent block seems more com plete, operates more frequently, and is prob ably of greater biological value. The degree of efferent block is variable, rarely complete and occurs less frequently. It is of interest to note that corneal rejec tion in originally unsensitized rabbits occur red as late as 80 days after corneal grafting, with a mean of 72.2 days. This demonstrates that at this time the corneal graft still con sists of donor cells which continue to ex press their own histocompatability antigens.
SUMMARY
Corneal allografts were exchanged in a group of rabbits, and 12 of 27 of these grafts were spontaneously rejected in three to seven weeks. Seven to 10 weeks after keratoplasty, the rabbits received skin allografts from the corneal donor, regardless of the state of their corneal allografts. Skin graft rejec tion was accelerated in those rabbits who had rejected their corneas, indicating that they were systemically sensitized to the donor's histocompatability antigens. Skin graft sur vival was normal in rabbits that had not re jected corneal grafts spontaneously. After skin grafting, 14 out of 15 rabbits with pre viously clear corneas went on to reject their corneal grafts, indicating that the cell type and antigen expression on the corneal grafts had remained stable, and that the central mechanism and efferent arc of the immune system were intact. Hence, the major mecha nism protecting these corneal grafts from immune rejection was a block in the afferent arc, preventing systemic sensitization. ACKNOWLEDGMENTS
We appreciate the help of Dr. Robert Connor in statistically evaluating our data, and the excellent technical assistance of Mr. David Hoffman and Mrs. Hilda Porter. REFERENCES
1. Waltman, S. R., Faulkner, H. W., and Bürde, R. M. : Use of antilymphocyte serum to prevent im mune rejection of penetrating corneal homografts. Invest. Ophth. 8:196, 1969. 2. Warwick, W. j . : A rapid, sutureless method for grafting skin in rabbits. Trans. Bull. 30:51, 1962. 3. Maumenee, A. E. : The influence of donor-re cipient sensitization on corneal grafts. Am. J. Ophth. 34 (Part 2) :142,1951. 4. O'Gawa, G. M., Guyton, J. S., Sanders, W. R., Inch, F. A. B., and Ellis, R. C. : Behavior of clear, penetrating corneal homografts in rabbits. Am. J. Ophth. 61:267, 1966. 5. Khodadoust, A.: Penetrating keratoplasty in rabbits. Am. J. Ophth. 66:899, 1968.