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A Simplified Technique for Experimental Keratoplasty*
894
RICHARD II. KEATES AND T. CVINTAL
and clinical examinations in all three did not show findings consistent with the forme fruste type of Hurler's disease. This condition described herein should be ruled out
when the diagnosis of congenital glaucoma is considered. O n e successful penetrating keratoplasty has also been reported. 410 West 10th Avenue (43210).
REFERENCES
1. Maumenee, A. E. : Congenital hereditary corneal dystrophy. Am. J. Ophth., 50:1114-1124, 1960. 2. Scheie, H. G., Hambrick, G. W., Jr., and Barness, L. A.: A newly recognized forme fruste of Hurler's disease (gargoylism) : The Sanford R. Gifford Lecture. Am. J. Ophth., 53:753-769, 1962.
A SIMPLIFIED TECHNIQUE FOR EXPERIMENTAL GILBERT W.
KERATOPLASTY*
CLEASBY, M.D.,
S A M U E L S.
BYLAND,
A N D ROBERT G.
WEBSTER,
M.D.
M.D.,
San Francisco, California Experimental keratoplasty has been performed on laboratory animals for a wide variety of purposes. These studies have included corneal wound healing, investigation of the fate of grafted tissues and development of surgical technique. I n recent years much attention has been given to the homograft rejection reaction as seen in corneal transplantation. Current studies in our laboratory are concerned with methods for modification of the homograft rejection reaction by reducing body defenses against foreign tissue through use of various antimetabolic chemical agents. 1 It became necessary to develop reliable, easy and safe methods for homotransplantation on laboratory animals prior to carrying out this work. T h e albino rabbit was chosen because it is docile and inexpensive and because of the large amount of experimental corneal work previously done with this animal. Many technical problems were encountered, as was the case in other reported studies. 2 " 5 Over a long period of time we developed a technique which is simple to perform, requires minimum time and assistance and produces a high proportion of technically successful grafts. * From, the Presbyterian Medical Center. These studies were supported by grant No. NB-04684-02 from the National Institutes of Health.
M A T E R I A L S AND METHODS ANIMALS
These studies were carried out on young adult New Zealand white rabbits weighing 2 to 3 kg. T h e rabbit cornea averages 15 mm in diameter horizontally and 13.8 m m vertically. I t is fairly uniform in thickness, being about 0.37 mm centrally and 0.45 mm at the limbus. 6 These large, thin and soft corneas are more difficult to work with than the rigid, thicker human corneas. PREPARATION
U p o n the animal's arrival in the laboratory, the periorbital hair is trimmed with an electric razor. This facilitates care of the eyes in the pre- and postoperative periods. Each animal is kept in an individual cage and observed daily during the weeks necessary to produce heavily vascularized corneal scars. T h e scars are created by repeated cauterization with a small soldering iron (fig. 1 ) . This can be done quite easily under topical anesthesia. Initial cauterization is carried out on both sides. T h e eyes are inspected daily for any evidence of infection, perforation or other complication. Infections are treated with antibiotic ointment. If there is rapid corneal clearing, repeat cautery is carried out in one week.
EXPERIMENTAL KF.RATOPI.ASTY
895
Initially, there was a tendency to cauterize too heavily with resulting loss of many eyes. It was found preferable to err on the side of treating too lightly, since satisfactory scars can usually be obtained by three or four treatments (fig. 2 ) . Keratoplasty is not carried out until adequate opacities are present in both eyes. It is advisable to allow two weeks for clearing of postcautery reaction before performing surgery. P R E M E D I C A T I O N AND A N E S T H E S I A
A single intramuscular injection containing 1 cc of procaine penicillin-streptomycin sulfate (Combiotic) and benzathine penicillin G (Bicillin) is given. T h e pupil is widely dilated by multiple instillations of 5 % homatropine and 10% phenylephrine ophthalmic solutions. Topical anesthesia is obtained with 0 . 5 % tetracaine. T h e marginal ear vein is injected with 0.5 cc chlorpromazine (Thorazine) (25 m g / c c ) and 0.5 cc of veterinary pentobarbital sodium (60 m g / c c ) through a 27-gauge needle. T h e vein becomes quite prominent following application of xylene. Administration is further facilitated by draping the ear firmly over a large flashlight. A retrobulbar injection of 2 cc of lidocaine (Xylocaine) containing 1:100,000 epinephrine is administered with a 0.5-inch 25-gauge needle via the superior fornix. Careful aspiration is advisable to insure that
Fig. 1 (Cleasby, Byland and Webster). Corneal scars are created by multiple applications of the tip of a heated soldering iron to the entire surface of the cornea.
the injection is not given in one of the large orbital veins. This mishap can cause prompt death of the animal. T h e described technique usually provides adequate anesthesia with minimum mortality. T h e response is somewhat variable but it is easy to supplement the anesthesia with small injections of pentobarbital sodium (0.2-0.4 c c ) . This is frequently necessary prior to performing surgery on the second eye. P R E P A R A T I O N OF T H E DONOR
T h e donor animal is anesthetized in identical fashion. It is prepared and draped at a separate table and the donor eye is enucleated. T h e eye is wrapped firmly in a moist gauze strip. A small amount of antibiotic ointment is instilled in the orbit and the socket packed with gauze. If a bilateral operation is to be performed, both eyes are removed. A 5-mm hand-held corneal trephine is positioned over the center of the donor eye and set for a depth of 0.3 mm. T h e trephine is rotated firmly and evenly until penetration is achieved. This usually provides a deep, uniform cut down to Descemet's membrane with an opening extending for about half the circumference.
896
G. W. CLEASBY, S. S. B Y L A N D A N D R. G. W E B S T E R
and the adnexal hairs trimmed prior to draping with a sterile eye sheet. FIXATION
Fig. 3 (Cleasby, Byland and Webster). The corneal button is excised from the donor eye with fine scissors.
The eye is proptosed by gentle pressure on the lids with applicators and lock fixation forceps applied to opposing rectus muscles at the level of the equator. This provides excellent proptosis and stability. The forceps need not be held by an assistant. At this point final preoperative photographs are taken and any subconjunctival edema from the retrobulbar injection is milked posteriorly with applicators. SUTURING
Four equidistant points at the limbus are located by creating horizontal and vertical lines passing through the center of the cornea by impression with a 6-0 silk suture (fig. 4). These four points and the central point of the cornea are marked with gentian violet. Black silk 6-0 sutures (Ethicon No. 780) are used. Using the limbal marks as a guide, two double-armed indirect overlying cross sutures are inserted so that the eight points of attachment at the limbus are equidistant (fig. 5). The sutures cross over the center of the cornea. Bites are taken through the superficial sciera immediately Fig. 4 (Cleasby, Byland and Webster). Four equidistant points are located at the limbus of the recipient eye by impression with a 6-0 silk suture.
Excision is completed with fine corneal scissors, the blades being held to create a bevel with a slight relative excess of tissue along the posterior margin of the button (fig. 3). SURGICAL TECHNIQUE
After obtaining adequate anesthesia, the recipient animal is placed on the operating platform. A small wooden block measuring 6 by 4 by 1 inches is placed under the head to elevate the eye into a more horizontal position. The lids are cleansed with pHisoHex®
Fig. 5 (Cleasby, Byland and Webster). The overlying preplaced sutures are attached at eight equidistant points around the limbus of the recipient eye.
EXPERIMENTAL KERATOPLASTY
897
adjacent to the limbus. T h e sutures are placed so that the knots will be tied superiorly and temporally away from the nictitating membrane. This facilitates later removal. The sutures are then loosened and pushed aside. TREPHINATION
T h e 5-mm corneal trephine is set for a depth of 0.4 mm and the cutting edge touched lightly with genatin violet to produce a mark on the cornea as an aid in centering. Trephining is carried out gently until penetration is achieved. T h e trephine is lifted quickly to avoid damage to the lens. T h e button is excised in a manner similar to that used on the donor, except that the bevel is reversed to leave a slight excess posteriorly on the recipient side. GRAFT
PLACEMENT
T h e donor corneal button is transferred to the recipient on a spatula. If the button is dropped, careful inspection with the operating microscope usually will determine which is anterior and posterior. T h e overlying sutures are repositioned and tied snugly (fig. 6 ) . Steroid-antibiotic and atropine ophthalmic ointments are applied. T h e lids are not sutured.
Fig. 7 (Cleasby, Byland and Webster). Postoperative examination is facilitated by a small platform attached to the slitlamp. SECOND EYE
W h e n surgery is to be performed on the second eye under the same anesthetic, the drapes are lifted and the rabbit turned over. P r o p e r positioning of the head on the wooden block avoids pressure or trauma to the first eye. It is advisable to withhold the retrobulbar injection until the operation is to begin. It is usually necessary to give a supplemental injection of 0.2-0.4 cc pentobarbital sodium at this point. Surgery is performed with the use of the Zeiss operating microscope set at a magnification of X 1 0 . POSTOPERATIVE
Fig. 6 (Cleasby, Byland and Webster). The donor button is secured in place with the overlying sutures.
CARE
Daily rounds are made for careful inspection of operated eyes. A n y wound disruption, suture displacement, infection, etc., is noted on the record. T h e eyes are studied frequently by slitlamp. A small platform has been devised to hold the animals in the proper position for examination (fig. 7 ) .
G. W. CT.EASRY, S. S. RYLAND AND R. G. WEBSTER
898
TABLE 1 SUMMARY OF TECHNICAL COMPLICATIONS Type of Complication
No. of Cases
Lens trauma
5
Loss of intraocular contents a t surgery
2
Suture displacement
1
Wound disruption
5 13
TOTAL
tained a technical success of 7 0 % in a smaller series of homografts placed in vascularized recipient corneas. W e did not find a significant difference in the incidence of technical success between vascularized and unvascularized corneas. DISCUSSION A N E S T H E S I A AND PREMEDICATION
RESULTS
A single injection of procaine penicillin, streptomycin and benzathine penicillin at the time of surgery appears to provide adequate prophylaxis against infection. Both mydriatics and miotics have been used to keep the iris away from the wound. Dilatation of the pupil is preferred in our cases because of the small grafts performed. This aids in prevention of damage to the iris at the time of surgery and in the formation of synechiae postoperatively.
A graft is felt to be technically acceptable if there are no operative or postoperative complications and the incision is well healed at the end of the second postoperative week. Clarity is not included in the criteria for technical success. Cases are eliminated as technical failures in instances of such obvious complications as damage to the lens and loss of vitreous during surgery (usually due to inadequate depth of anesthesia) and to such postoperative complications as infection, suture displacement and wound disruption. These are summarized in Table 1.
It has been felt by many workers 2 ' 4 ' 5 that the high fibrinogen content of rabbit aqueous results in operative and postoperative problems. Heparin has been administered intravenously and sodium citrate has been given topically to counteract the fibrinogen. W e used these agents in our early cases but encountered troublesome hemorrhage from corneal vessels during surgery. Subsequently it was found that the fibrinogen does not appear to be the cause of any difficulties. In fact, it is possible that it aids in sealing the wound.
A trial series of homografts was performed initially in clear recipients after development of the definitive surgical technique. Five out of six ( 8 3 . 3 % ) were technically successful. After the study was begun, 80 grafts were performed in rabbits with scarred, vascularized corneas. A technically successful result was obtained in 68 cases (85%). I n one reported series 7 there was 3 5 % technical success in a group of 40 autografts and homografts placed in clear, avascular recipient corneas. T h e same workers ob-
O u r technique for anesthesia is quite simple and provides a very low mortality. Rabbits are notoriously easy to kill with most anesthetic agents. T h e chlorpromazine must be given in a separate syringe, since it forms a coagulum when mixed with other agents. T h e marked dilatation of the ear vein with the help of xylene facilitates the injection and allows ready supplementation during the operation. T h e retrobulbar injection renders the animal pain-free and permits use of lesser amounts of the other agents.
Sutures are removed about the seventh postoperative day, at which time steroid-antibiotic and atropine ophthalmic ointments are instilled. T h e only other medications given during the postoperative period are chemicals for modification of the homograft reaction.
899
EXPERIMENTAL KERATOPLASTY SURGICAL
TECHNIQUE
Initially the rabbits were immobilized by taping and tying the legs and body to the operating table. However, this is cumbersome and is not necessary if adequate depth of anesthesia is maintained with supplementary injections of pentobarbital sodium. T h e technique for fixation already described frees the hands of the operator and avoids the need for an assistant. A 5-mm penetrating graft proved to be the optimum size. Because of the architectural qualities of the rabbit cornea, both larger and smaller grafts resulted in technical problems. Overlying sutures anchored at eight equidistant points around the limbus proved to be the most satisfactory. Direct sutures are difficult to insert, difficult to remove and result in damage to the donor cornea. Abrahamson 2 found that insertion of direct sutures often exposes the graft to endothelial damage. A n intact endothelium is known to be essential for clarity of a penetrating graft. 3 Abrahamson described a procedure in which the graft is held in place by a plastic contact lens. W e tried various techniques for graft fixation with plastic materials and found them to be cumbersome and less reliable than overlying sutures. Initially, we used an air injection at the termination of the operation to facilitate re-
formation of the anterior chamber. 5 This occasionally caused operative complications and did not appear to be of significant help, so has been abandoned. T h e lids were closed with sutures at the end of the operation in our early cases. However, we encountered a high incidence of postoperative lid infection. T h e number of lid infections has been reduced markedly since discontinuing this practice and there has not been an increase in the number of cases of self-inflicted damage to the graft. Operating on both eyes under the same anesthesia has proved to be quite simple and has permitted the performance of a greater number of grafts in a shorter period of time. SUMMARY
A simplified technique for the performance of technically successful penetrating keratoplasty in rabbits is described. This method provides technical simplicity and ease of performance, requires minimum pre- and postoperative care, can be performed by a single operator and provides a high yield of technically successful grafts. Clay and Wehster Streets (94115). ADDENDUM
Since this paper was prepared, an additional 158 operations have been performed with an incidence of 12% technically unsuccessful grafts.
REFERENCES
1. Cleasby, G. W., and Byland, S. S. : Modification of the homograft reaction in corneal transplantation, Ann. N.Y. Acad. Sei., 120:802, 1964. 2. Abrahamson, I. A., Jr., Brumen, L. P. and Abrahamson, I. A., Sr. : A simplified technique for corneal transplantation. Am. J. Ophth., 41:79, 1956. 3. Maumenee, A. E. : The immune reaction to corneal homograf ts. In The Transparency of the Cornea : A Symposium (edited by Sir S. Duke Elder and E. S. Perkins). Oxford, Blackwell Scientific Publications, 1960. 4. Hanna, C. and Irwin, E. S. : Fate of cells in the corneal graft. Arch. Ophth., 68 :810-817 (Dec.) 1962. 5. Stocker, F. W., Matton, M. T., Eiring, A., Georgiade, R., and Georgiade, N. : Long-term preservation of donor tissues for corneal grafting. Am. J. Ophth., 49:729, 1960. 6. Prince, J. H., Diesem, C. D., Eglits, I., and Ruskell, G. L. : Anatomy and Histology of the Eye and Orbit in Domestic Animals. Springfield, 111., Thomas, 1960. 7. Nelken, E., Nelken, D., Michaelson, I. C. and Gurevitch, J. : Late clouding of experimental corneal grafts. Arch. Ophth., 65:584-590 (Apr.) 196!. 8. Kornblueth, W., and Nelken, E. : A study of donor-recipient sensitization. Am. J. Ophth., 45:843 (June) 1958.
RICHARD II. KEATES AND T. CVINTAL
and clinical examinations in all three did not show findings consistent with the forme fruste type of Hurler's disease. This condition described herein should be ruled out
when the diagnosis of congenital glaucoma is considered. O n e successful penetrating keratoplasty has also been reported. 410 West 10th Avenue (43210).
REFERENCES
1. Maumenee, A. E. : Congenital hereditary corneal dystrophy. Am. J. Ophth., 50:1114-1124, 1960. 2. Scheie, H. G., Hambrick, G. W., Jr., and Barness, L. A.: A newly recognized forme fruste of Hurler's disease (gargoylism) : The Sanford R. Gifford Lecture. Am. J. Ophth., 53:753-769, 1962.
A SIMPLIFIED TECHNIQUE FOR EXPERIMENTAL GILBERT W.
KERATOPLASTY*
CLEASBY, M.D.,
S A M U E L S.
BYLAND,
A N D ROBERT G.
WEBSTER,
M.D.
M.D.,
San Francisco, California Experimental keratoplasty has been performed on laboratory animals for a wide variety of purposes. These studies have included corneal wound healing, investigation of the fate of grafted tissues and development of surgical technique. I n recent years much attention has been given to the homograft rejection reaction as seen in corneal transplantation. Current studies in our laboratory are concerned with methods for modification of the homograft rejection reaction by reducing body defenses against foreign tissue through use of various antimetabolic chemical agents. 1 It became necessary to develop reliable, easy and safe methods for homotransplantation on laboratory animals prior to carrying out this work. T h e albino rabbit was chosen because it is docile and inexpensive and because of the large amount of experimental corneal work previously done with this animal. Many technical problems were encountered, as was the case in other reported studies. 2 " 5 Over a long period of time we developed a technique which is simple to perform, requires minimum time and assistance and produces a high proportion of technically successful grafts. * From, the Presbyterian Medical Center. These studies were supported by grant No. NB-04684-02 from the National Institutes of Health.
M A T E R I A L S AND METHODS ANIMALS
These studies were carried out on young adult New Zealand white rabbits weighing 2 to 3 kg. T h e rabbit cornea averages 15 mm in diameter horizontally and 13.8 m m vertically. I t is fairly uniform in thickness, being about 0.37 mm centrally and 0.45 mm at the limbus. 6 These large, thin and soft corneas are more difficult to work with than the rigid, thicker human corneas. PREPARATION
U p o n the animal's arrival in the laboratory, the periorbital hair is trimmed with an electric razor. This facilitates care of the eyes in the pre- and postoperative periods. Each animal is kept in an individual cage and observed daily during the weeks necessary to produce heavily vascularized corneal scars. T h e scars are created by repeated cauterization with a small soldering iron (fig. 1 ) . This can be done quite easily under topical anesthesia. Initial cauterization is carried out on both sides. T h e eyes are inspected daily for any evidence of infection, perforation or other complication. Infections are treated with antibiotic ointment. If there is rapid corneal clearing, repeat cautery is carried out in one week.
EXPERIMENTAL KF.RATOPI.ASTY
895
Initially, there was a tendency to cauterize too heavily with resulting loss of many eyes. It was found preferable to err on the side of treating too lightly, since satisfactory scars can usually be obtained by three or four treatments (fig. 2 ) . Keratoplasty is not carried out until adequate opacities are present in both eyes. It is advisable to allow two weeks for clearing of postcautery reaction before performing surgery. P R E M E D I C A T I O N AND A N E S T H E S I A
A single intramuscular injection containing 1 cc of procaine penicillin-streptomycin sulfate (Combiotic) and benzathine penicillin G (Bicillin) is given. T h e pupil is widely dilated by multiple instillations of 5 % homatropine and 10% phenylephrine ophthalmic solutions. Topical anesthesia is obtained with 0 . 5 % tetracaine. T h e marginal ear vein is injected with 0.5 cc chlorpromazine (Thorazine) (25 m g / c c ) and 0.5 cc of veterinary pentobarbital sodium (60 m g / c c ) through a 27-gauge needle. T h e vein becomes quite prominent following application of xylene. Administration is further facilitated by draping the ear firmly over a large flashlight. A retrobulbar injection of 2 cc of lidocaine (Xylocaine) containing 1:100,000 epinephrine is administered with a 0.5-inch 25-gauge needle via the superior fornix. Careful aspiration is advisable to insure that
Fig. 1 (Cleasby, Byland and Webster). Corneal scars are created by multiple applications of the tip of a heated soldering iron to the entire surface of the cornea.
the injection is not given in one of the large orbital veins. This mishap can cause prompt death of the animal. T h e described technique usually provides adequate anesthesia with minimum mortality. T h e response is somewhat variable but it is easy to supplement the anesthesia with small injections of pentobarbital sodium (0.2-0.4 c c ) . This is frequently necessary prior to performing surgery on the second eye. P R E P A R A T I O N OF T H E DONOR
T h e donor animal is anesthetized in identical fashion. It is prepared and draped at a separate table and the donor eye is enucleated. T h e eye is wrapped firmly in a moist gauze strip. A small amount of antibiotic ointment is instilled in the orbit and the socket packed with gauze. If a bilateral operation is to be performed, both eyes are removed. A 5-mm hand-held corneal trephine is positioned over the center of the donor eye and set for a depth of 0.3 mm. T h e trephine is rotated firmly and evenly until penetration is achieved. This usually provides a deep, uniform cut down to Descemet's membrane with an opening extending for about half the circumference.
896
G. W. CLEASBY, S. S. B Y L A N D A N D R. G. W E B S T E R
and the adnexal hairs trimmed prior to draping with a sterile eye sheet. FIXATION
Fig. 3 (Cleasby, Byland and Webster). The corneal button is excised from the donor eye with fine scissors.
The eye is proptosed by gentle pressure on the lids with applicators and lock fixation forceps applied to opposing rectus muscles at the level of the equator. This provides excellent proptosis and stability. The forceps need not be held by an assistant. At this point final preoperative photographs are taken and any subconjunctival edema from the retrobulbar injection is milked posteriorly with applicators. SUTURING
Four equidistant points at the limbus are located by creating horizontal and vertical lines passing through the center of the cornea by impression with a 6-0 silk suture (fig. 4). These four points and the central point of the cornea are marked with gentian violet. Black silk 6-0 sutures (Ethicon No. 780) are used. Using the limbal marks as a guide, two double-armed indirect overlying cross sutures are inserted so that the eight points of attachment at the limbus are equidistant (fig. 5). The sutures cross over the center of the cornea. Bites are taken through the superficial sciera immediately Fig. 4 (Cleasby, Byland and Webster). Four equidistant points are located at the limbus of the recipient eye by impression with a 6-0 silk suture.
Excision is completed with fine corneal scissors, the blades being held to create a bevel with a slight relative excess of tissue along the posterior margin of the button (fig. 3). SURGICAL TECHNIQUE
After obtaining adequate anesthesia, the recipient animal is placed on the operating platform. A small wooden block measuring 6 by 4 by 1 inches is placed under the head to elevate the eye into a more horizontal position. The lids are cleansed with pHisoHex®
Fig. 5 (Cleasby, Byland and Webster). The overlying preplaced sutures are attached at eight equidistant points around the limbus of the recipient eye.
EXPERIMENTAL KERATOPLASTY
897
adjacent to the limbus. T h e sutures are placed so that the knots will be tied superiorly and temporally away from the nictitating membrane. This facilitates later removal. The sutures are then loosened and pushed aside. TREPHINATION
T h e 5-mm corneal trephine is set for a depth of 0.4 mm and the cutting edge touched lightly with genatin violet to produce a mark on the cornea as an aid in centering. Trephining is carried out gently until penetration is achieved. T h e trephine is lifted quickly to avoid damage to the lens. T h e button is excised in a manner similar to that used on the donor, except that the bevel is reversed to leave a slight excess posteriorly on the recipient side. GRAFT
PLACEMENT
T h e donor corneal button is transferred to the recipient on a spatula. If the button is dropped, careful inspection with the operating microscope usually will determine which is anterior and posterior. T h e overlying sutures are repositioned and tied snugly (fig. 6 ) . Steroid-antibiotic and atropine ophthalmic ointments are applied. T h e lids are not sutured.
Fig. 7 (Cleasby, Byland and Webster). Postoperative examination is facilitated by a small platform attached to the slitlamp. SECOND EYE
W h e n surgery is to be performed on the second eye under the same anesthetic, the drapes are lifted and the rabbit turned over. P r o p e r positioning of the head on the wooden block avoids pressure or trauma to the first eye. It is advisable to withhold the retrobulbar injection until the operation is to begin. It is usually necessary to give a supplemental injection of 0.2-0.4 cc pentobarbital sodium at this point. Surgery is performed with the use of the Zeiss operating microscope set at a magnification of X 1 0 . POSTOPERATIVE
Fig. 6 (Cleasby, Byland and Webster). The donor button is secured in place with the overlying sutures.
CARE
Daily rounds are made for careful inspection of operated eyes. A n y wound disruption, suture displacement, infection, etc., is noted on the record. T h e eyes are studied frequently by slitlamp. A small platform has been devised to hold the animals in the proper position for examination (fig. 7 ) .
G. W. CT.EASRY, S. S. RYLAND AND R. G. WEBSTER
898
TABLE 1 SUMMARY OF TECHNICAL COMPLICATIONS Type of Complication
No. of Cases
Lens trauma
5
Loss of intraocular contents a t surgery
2
Suture displacement
1
Wound disruption
5 13
TOTAL
tained a technical success of 7 0 % in a smaller series of homografts placed in vascularized recipient corneas. W e did not find a significant difference in the incidence of technical success between vascularized and unvascularized corneas. DISCUSSION A N E S T H E S I A AND PREMEDICATION
RESULTS
A single injection of procaine penicillin, streptomycin and benzathine penicillin at the time of surgery appears to provide adequate prophylaxis against infection. Both mydriatics and miotics have been used to keep the iris away from the wound. Dilatation of the pupil is preferred in our cases because of the small grafts performed. This aids in prevention of damage to the iris at the time of surgery and in the formation of synechiae postoperatively.
A graft is felt to be technically acceptable if there are no operative or postoperative complications and the incision is well healed at the end of the second postoperative week. Clarity is not included in the criteria for technical success. Cases are eliminated as technical failures in instances of such obvious complications as damage to the lens and loss of vitreous during surgery (usually due to inadequate depth of anesthesia) and to such postoperative complications as infection, suture displacement and wound disruption. These are summarized in Table 1.
It has been felt by many workers 2 ' 4 ' 5 that the high fibrinogen content of rabbit aqueous results in operative and postoperative problems. Heparin has been administered intravenously and sodium citrate has been given topically to counteract the fibrinogen. W e used these agents in our early cases but encountered troublesome hemorrhage from corneal vessels during surgery. Subsequently it was found that the fibrinogen does not appear to be the cause of any difficulties. In fact, it is possible that it aids in sealing the wound.
A trial series of homografts was performed initially in clear recipients after development of the definitive surgical technique. Five out of six ( 8 3 . 3 % ) were technically successful. After the study was begun, 80 grafts were performed in rabbits with scarred, vascularized corneas. A technically successful result was obtained in 68 cases (85%). I n one reported series 7 there was 3 5 % technical success in a group of 40 autografts and homografts placed in clear, avascular recipient corneas. T h e same workers ob-
O u r technique for anesthesia is quite simple and provides a very low mortality. Rabbits are notoriously easy to kill with most anesthetic agents. T h e chlorpromazine must be given in a separate syringe, since it forms a coagulum when mixed with other agents. T h e marked dilatation of the ear vein with the help of xylene facilitates the injection and allows ready supplementation during the operation. T h e retrobulbar injection renders the animal pain-free and permits use of lesser amounts of the other agents.
Sutures are removed about the seventh postoperative day, at which time steroid-antibiotic and atropine ophthalmic ointments are instilled. T h e only other medications given during the postoperative period are chemicals for modification of the homograft reaction.
899
EXPERIMENTAL KERATOPLASTY SURGICAL
TECHNIQUE
Initially the rabbits were immobilized by taping and tying the legs and body to the operating table. However, this is cumbersome and is not necessary if adequate depth of anesthesia is maintained with supplementary injections of pentobarbital sodium. T h e technique for fixation already described frees the hands of the operator and avoids the need for an assistant. A 5-mm penetrating graft proved to be the optimum size. Because of the architectural qualities of the rabbit cornea, both larger and smaller grafts resulted in technical problems. Overlying sutures anchored at eight equidistant points around the limbus proved to be the most satisfactory. Direct sutures are difficult to insert, difficult to remove and result in damage to the donor cornea. Abrahamson 2 found that insertion of direct sutures often exposes the graft to endothelial damage. A n intact endothelium is known to be essential for clarity of a penetrating graft. 3 Abrahamson described a procedure in which the graft is held in place by a plastic contact lens. W e tried various techniques for graft fixation with plastic materials and found them to be cumbersome and less reliable than overlying sutures. Initially, we used an air injection at the termination of the operation to facilitate re-
formation of the anterior chamber. 5 This occasionally caused operative complications and did not appear to be of significant help, so has been abandoned. T h e lids were closed with sutures at the end of the operation in our early cases. However, we encountered a high incidence of postoperative lid infection. T h e number of lid infections has been reduced markedly since discontinuing this practice and there has not been an increase in the number of cases of self-inflicted damage to the graft. Operating on both eyes under the same anesthesia has proved to be quite simple and has permitted the performance of a greater number of grafts in a shorter period of time. SUMMARY
A simplified technique for the performance of technically successful penetrating keratoplasty in rabbits is described. This method provides technical simplicity and ease of performance, requires minimum pre- and postoperative care, can be performed by a single operator and provides a high yield of technically successful grafts. Clay and Wehster Streets (94115). ADDENDUM
Since this paper was prepared, an additional 158 operations have been performed with an incidence of 12% technically unsuccessful grafts.
REFERENCES
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