Ophthalmologica, Basel 176: 45 51 (1978)
Failure of Inhibiting Corneal Graft Rejection in Rabbits by 5 a-Androstane-3,17-dione Werner Niederer and Heinz Eltz ' Department of Ophthalmology, University of Basle (Chairman: Prof. B. Gloor), Basle
Rembiesa et al. [1974] have reported that 5-«-androstane-3,17-dione («A) sup presses delayed type hypersensitivity (p < 0 .0 1 ) when given systematically to mice during immunization. They isolated on this compound when they analyzed placental steroids in order to find a local factor re sponsible for the suppression of immuno logical chorion rejection. The fact that the placenta produces aA together with the chemical structure of this steroid suggests that it is not antimetabolic. In human corneal grafts, rejection is usu ally inhibited by topical cortisol application. This treatment, however, may be hazardous because of induction of glaucoma. Another potential danger of cortisol treatment exists in patients with herpes corneae, since corti sol is not only immunosuppressive but also antimetabolic. This latter effect reduces mu copolysaccharide synthesis in the corneal stroma \Bleckmann and Wollensak, 1975] thereby leading to an increased permeability 1 We thank Prof. Gloor for valuable discussion and Mrs. Wieser for technical assistance.
of the stroma to particles of the size of vi rions [Robbins and Galin, 1975]. It would therefore be an advantage to use an immu nosuppressive drug without antimetabolic effect for preventing corneal graft rejection. These considerations prompted us to test the possibility whether a A could inhibit cor neal graft rejection in rabbits.
Methods Transplantation 3-4 months old New Zeeland rabbits were used. We performed corneal transplantation on both eyes in one session using grafts from the same donor (pigmented rabbits). For general an esthesia we injected 5 ml/kg urethan 20% in dis tilled water sterilized by filtration through millipore filters. The urethan was given 1 h before operation together with 0.2 mg/kg atropin. Im mediately before operation 20 mg/kg ketamin (Ketalar) was injected intramuscularly. The ketamin injection was repeated after I h. The operation field was covered by a sterile plastic drape with a slit and all instruments were sterilized. The use of an operating microscope is indispensable. We exposed the cornea by 3 or 4 holding threads (4/0-silk) which were fixed to the limbus by 10/0-Barraquer silk sutures. Just before trepanation 2,500 U of heparin were injected intra
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 5:30:29 AM
Introduction
Niederer/Eltz
46
Immunization The animals were immunized with residual cor neal tissue of the donor eyes. In the first 6 rabbits this tissue was implanted subcutaneously through a small skin incision on the back. In the following experiments the tissue was minced and homoge nized in 1 ml of saline. The suspension was then mixed with 1 ml of complete Freund’s adjuvant (Difco) to give a water in oil emulsion which was injected intracutaneously at two sites. Topical Treatment The eye ointments were applied three times daily. In addition to 2% chloramphenicol the oint ments contained in group A (6 rabbits): for the right eye no steroid; for the left eye 2% aA, and in group B (13 rabbits): for the right eye 2% corti sol; for the left eye 2% «A. Evaluation of the Treatment Only those animals which had clear grafts during the first postoperative week were kept alive. We photographed the eyes several times during the observation period (1-3 months). The pictures were judged in randomized order by 16 colleagues. As an index for graft rejection we took the vas cularization of the graft. Other rejection features were too difficult to judge from photographs. However, the vascularization corresponded well with other clinical criteria of graft rejection. In tables I and II we indicate whether there was a significant difference (p < 0.05) of graft rejection between the two eyes of an animal when the col leagues judgements were analyzed by the paired t test (the two judgements made by one colleague forming a pair).
Results
From the relatively small number of ex periments in table I it is not possible to deeide whether aA has any effect at all. Table II, however, convincingly demonstrates that
aA exerts little or no inhibition of the graft rejection as compared to the effect of corti sol (p < 0.1). 5 eyes treated with cortisol, 5 eyes treat ed with aA and 5 eyes which received chlor amphenicol only were examined histologi cally. Table III shows that we found less retrocorneal membranes in the cortisoltreated group. Our study gave opportunity to correlate the distinct clinical features of epithelial, stromal and endothelial graft rejection with their histological characteristics. Our find ings (fig. 1) correspond well with those de scribed by Khodadoust and Silverstein [1969], Polack [1972] and Maumenee [1973]. We found cells which obviously migrated from vessels and invaded the stroma form ing epitheloid cell rows in several grafts (fig- 2). On some microscopic slides we observed retrocorneal membranes. These membranes may consist of fibroblast-like cells or of endotheloid cells. Figure 3 shows a retro corneal membrane which is made up of both cell types. At the edge of the graft's Descemet’s membrane, but also in other parts of retrocorneal membranes, we often noticed splitting of newly formed Descemet's mem brane, which radiates between the cells (fig- 4).
Discussion
Immunosuppression by aA Front the work of Rembiesa el al. [1974], it was shown that aA suppresses the im mune reaction. However, it was not shown whether it was the afferent limb, the efferent limb or both that is suppressed. In our ex-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 5:30:29 AM
venously. We chose the graft diameter 0.5 mm larger than the trepanation (7-8 mm). The corneal button was first fixed with four 10/0-Barraquer silk sutures and then with a continuous 10/0 nylon monofilament suture.
Graft Rejection in Rabbits
47
Table I. Group A : estimated percent of vascularization of corneal grafts in comparison to a given standard
Rabbit No.
«A-treated eye Chloramphenicol-treated eye
1
2
3
4
5
6
38.4 35.0
9.6 18.6
23.1 25.0
38.4 35.0
7.5 10.0
17.7 21.5
t paired significance of the difference of the mean values (0.2
Failure of Inhibiting Corneal Graft Rejection in Rabbits by 5 a-Androstane-3,17-dione Werner Niederer and Heinz Eltz ' Department of Ophthalmology, University of Basle (Chairman: Prof. B. Gloor), Basle
Rembiesa et al. [1974] have reported that 5-«-androstane-3,17-dione («A) sup presses delayed type hypersensitivity (p < 0 .0 1 ) when given systematically to mice during immunization. They isolated on this compound when they analyzed placental steroids in order to find a local factor re sponsible for the suppression of immuno logical chorion rejection. The fact that the placenta produces aA together with the chemical structure of this steroid suggests that it is not antimetabolic. In human corneal grafts, rejection is usu ally inhibited by topical cortisol application. This treatment, however, may be hazardous because of induction of glaucoma. Another potential danger of cortisol treatment exists in patients with herpes corneae, since corti sol is not only immunosuppressive but also antimetabolic. This latter effect reduces mu copolysaccharide synthesis in the corneal stroma \Bleckmann and Wollensak, 1975] thereby leading to an increased permeability 1 We thank Prof. Gloor for valuable discussion and Mrs. Wieser for technical assistance.
of the stroma to particles of the size of vi rions [Robbins and Galin, 1975]. It would therefore be an advantage to use an immu nosuppressive drug without antimetabolic effect for preventing corneal graft rejection. These considerations prompted us to test the possibility whether a A could inhibit cor neal graft rejection in rabbits.
Methods Transplantation 3-4 months old New Zeeland rabbits were used. We performed corneal transplantation on both eyes in one session using grafts from the same donor (pigmented rabbits). For general an esthesia we injected 5 ml/kg urethan 20% in dis tilled water sterilized by filtration through millipore filters. The urethan was given 1 h before operation together with 0.2 mg/kg atropin. Im mediately before operation 20 mg/kg ketamin (Ketalar) was injected intramuscularly. The ketamin injection was repeated after I h. The operation field was covered by a sterile plastic drape with a slit and all instruments were sterilized. The use of an operating microscope is indispensable. We exposed the cornea by 3 or 4 holding threads (4/0-silk) which were fixed to the limbus by 10/0-Barraquer silk sutures. Just before trepanation 2,500 U of heparin were injected intra
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 5:30:29 AM
Introduction
Niederer/Eltz
46
Immunization The animals were immunized with residual cor neal tissue of the donor eyes. In the first 6 rabbits this tissue was implanted subcutaneously through a small skin incision on the back. In the following experiments the tissue was minced and homoge nized in 1 ml of saline. The suspension was then mixed with 1 ml of complete Freund’s adjuvant (Difco) to give a water in oil emulsion which was injected intracutaneously at two sites. Topical Treatment The eye ointments were applied three times daily. In addition to 2% chloramphenicol the oint ments contained in group A (6 rabbits): for the right eye no steroid; for the left eye 2% aA, and in group B (13 rabbits): for the right eye 2% corti sol; for the left eye 2% «A. Evaluation of the Treatment Only those animals which had clear grafts during the first postoperative week were kept alive. We photographed the eyes several times during the observation period (1-3 months). The pictures were judged in randomized order by 16 colleagues. As an index for graft rejection we took the vas cularization of the graft. Other rejection features were too difficult to judge from photographs. However, the vascularization corresponded well with other clinical criteria of graft rejection. In tables I and II we indicate whether there was a significant difference (p < 0.05) of graft rejection between the two eyes of an animal when the col leagues judgements were analyzed by the paired t test (the two judgements made by one colleague forming a pair).
Results
From the relatively small number of ex periments in table I it is not possible to deeide whether aA has any effect at all. Table II, however, convincingly demonstrates that
aA exerts little or no inhibition of the graft rejection as compared to the effect of corti sol (p < 0.1). 5 eyes treated with cortisol, 5 eyes treat ed with aA and 5 eyes which received chlor amphenicol only were examined histologi cally. Table III shows that we found less retrocorneal membranes in the cortisoltreated group. Our study gave opportunity to correlate the distinct clinical features of epithelial, stromal and endothelial graft rejection with their histological characteristics. Our find ings (fig. 1) correspond well with those de scribed by Khodadoust and Silverstein [1969], Polack [1972] and Maumenee [1973]. We found cells which obviously migrated from vessels and invaded the stroma form ing epitheloid cell rows in several grafts (fig- 2). On some microscopic slides we observed retrocorneal membranes. These membranes may consist of fibroblast-like cells or of endotheloid cells. Figure 3 shows a retro corneal membrane which is made up of both cell types. At the edge of the graft's Descemet’s membrane, but also in other parts of retrocorneal membranes, we often noticed splitting of newly formed Descemet's mem brane, which radiates between the cells (fig- 4).
Discussion
Immunosuppression by aA Front the work of Rembiesa el al. [1974], it was shown that aA suppresses the im mune reaction. However, it was not shown whether it was the afferent limb, the efferent limb or both that is suppressed. In our ex-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 5:30:29 AM
venously. We chose the graft diameter 0.5 mm larger than the trepanation (7-8 mm). The corneal button was first fixed with four 10/0-Barraquer silk sutures and then with a continuous 10/0 nylon monofilament suture.
Graft Rejection in Rabbits
47
Table I. Group A : estimated percent of vascularization of corneal grafts in comparison to a given standard
Rabbit No.
«A-treated eye Chloramphenicol-treated eye
1
2
3
4
5
6
38.4 35.0
9.6 18.6
23.1 25.0
38.4 35.0
7.5 10.0
17.7 21.5
t paired significance of the difference of the mean values (0.2
