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Lamellar Corneal Transplantation in the Rabbit
L A M E L L A R C O R N E A L T R A N S P L A N T A T I O N IN T H E RABBIT A L I A . KHOD. UXJUST, M . D . Baltimore, Maryland
Lamellar corneal transplantation was introduced by von Hippel almost 80 years ago, and has since become a generally accepted technique in the armamentarium of the ocular surgeon. However, lamellar transplants have not been utilized as widely as penetrating grafts in experimental studies of the immunologic aspects of corneal transplantation. Few descriptions of the technique of lamellar keratoplasty in experimental animals appear in the literature. Shafer reported difficulty in preventing the ingrowth of epithelium between the recipient stroma and 5-mm lamellar grafts in rabbits. He concluded that an edge-to-edge interrupted suture did not produce a sufficiently firm wound closure in the thin and flexible rabbit cornea, thus permitting epithelial downgrowth. In 1958, Kornblueth and Nelken described a technique for partial lamellar corneal grafts in rabbits. They used a criss-cross suture over a 7.1-mm lamellar disc in 146 eyes. In the first 94 operations in their series, clear grafts were obtained in 21% of the eyes, and with improvement in techniques, in 55,% of their next 52 operations.
MATERIALS AND METHODS
1
2
3
This paper describes a technique for the transplantation of deep lamellar corneal grafts in rabbits, which we developed for experimental purposes and for the training of ocular surgeons. With this technique, clear grafts were obtained in 98 of 100 consecutive operations. This paper describes the technical aspects of the approach, as well as the gross and histologic observations made on these cases. The results of the application of this technique to the study of corneal allograft rejection will be presented in subsequent reports. From the Wilmer Institute, the Johns Hopkins University School of Medicine. Supported in part by U S P H S Grant No. N B 03040 from the National Institute of Neurological Diseases and Blindness, and by an unrestricted gift from the Alcon Laboratories, Inc.
ANIMALS
All experiments were performed on young adult New Zealand Giant albino rabbits, weighing 2 to 3 kg. The corneal thickness in these animals is reported to average 0.45 mm near the corneoscleral limbus and 0.37 mm centrally, with an overall average value of 0.4 mm. 4
5
ANESTHESIA
Thirty to 45 minutes before surgery, each animal received 25 mg of promazine HCl (Sparine) by subcutaneous or intramuscular injection. Just prior to surgery, each rabbit was given 60 to 70 mg of pentobarbital sodium (Nembutal) intravenously in an ear vein. Since rabbits vary considerably in the amount of barbiturate needed to produce satisfactory anesthesia, the pentobarbital sodium was given slowly, and the animal was tested for depth of anesthesia by exerting pressure on the foot. Only a slight withdrawal reflex should occur if the plane of anesthesia is satisfactory, and the amount of pentobarbital sodium given can be titrated according to this response. With this technique, not a single rabbit was lost in 100 consecutive surgical procedures. Topical anesthesia of the eye was achieved by the repeated instillation of proparacaine HCl (Ophthaine) into the conjunctival sac before and during surgery. No retrobulbar injections were used. PREPARATION AND DRAPE
All the rabbits were operated upon in pairs. The periorbital hair was trimmed with scissors, and the lids of the nonoperated eye were held closed with a small piece of adhesive tape to prevent drying of the cornea during the procedure. The rabbits were then positioned side by side on the operating table and the conjunctival sacs irrigated with ster-
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AMERICAN JOURNAL OF OPHTHALMOLOGY
ile saline, followed by another drop of Ophthaine. A mask and sterile gloves were used to maintain aseptic technique during the remainder .of the procedure. Each rabbit was draped with a self-adhering plastic sheet (Steri-Drape No. 1040, Minnesota Mining & Manufacturing Co.) with the eye positioned in the center of the adhesive area, and the drape was pressed down along the lid margins. A 10- to 12-mm-incision was made through the drape over the center of the cornea and parallel to the eyelid margins. The eye was then proptosed through this hole in the drape ; being somewhat elastic, the drape automatically retracts behind the globe and thereby keeps all skin and hair out of the operative field. An excellent exposure with moderately tight positioning of the eyeball resulted, eliminating the necessity for a lid speculum and traction sutures. The size of the opening in the drape was critical, since too small an opening may embarrass the blood supply, whereas too large an opening will fail to hold the globe in proptosis. Drying of the bulbar conjunctiva and the sclera was prevented by a narrow piece of gauze which was wrapped around the globe and kept moist with saline. The cornea was moistened throughout the procedure by frequent applications of sterile saline.
D E C E M B E R , 1968
secting microscope. The corneal disc was then separated from the deep stromal fibers by holding the edge of the disc with fine tooth forceps and passing the blade of a Gill knife between the lamellae (fig. 1 ) . In order to avoid unintentional perforation into the anterior chamber, the knife blade should be kept parallel to (and thus follow) the curvature of the cornea. With a gentle to-and-fro motion of the knife, the dissection was extended laterally just beyond the limits of the outline made by the corneal trephine. The excision of the corneal button was then completed by using curved corneal scissors. Just before the graft was completely freed with the scissors, a single 8-0 virgin silk suture was placed partially through the lamellar button. This suture facilitates the transfer of the graft from one rabbit to the other, and serves as the first suture for attachment to the recipient bed. The freed graft was left on the keratectomy bed and the dissecting microscope was then positioned over the eye of the other rabbit, from which an identical corneal disc was removed by the same technique.
SURGICAL TECHNIQUE
An 8-mm corneal trephine was used to outline the lamellar button. This trephine incision may extend in depth to two-thirds of the corneal thickness. (The commercially available trephines are specifically designed for the curvature of the human cornea, and their depth setting is not reliable for incising the rabbit cornea; success in this procedure therefore takes practice. While gaining this experience, the beginner is advised to make a relatively shallow incision with the trephine.) It is possible to increase the depth of the incision on one side of the corneal button by using the fine blade of a Gill knife. This incision with the knife, and the remainder of the fine work, were done under a 16 X dis-
Fig 1 (Khodadou-.t). After outlining the lamellar button to an appropriate depth with an 8-mm trephine, the corneal disc is separated from the donor stroma by a to-and-fro motion of the Gill knife. Note the narrow strip of saline-moistened gauze which is wrapped around the globe to prevent drying of the bulbar conjunctiva.
V O L . 66, N O . 6
LAMELLAR CORNEAL TRANSPLANTATION
The grafts were exchanged immediately after the disc was freed from the second rabbit. Each graft was sutured into the recipient cornea using interrupted sutures of 8-0 virgin silk on a G-7 needle or fine black nylon thread of 30¡x diameter on a 4-mm Grieshaber corneal needle. Twelve sutures were usually used for each graft (fig. 2 ) . A continuous suture has also been employed to good advantage in this procedure (fig. 3 ) , and has the added advantage of reducing the irritation arising from the use of multiple knots. Following completion of surgery, the incision in the plastic drape was enlarged and the proptosed globe then retracted into the orbit. Antibiotic ointment (polymyxin Bneomycin-bacitracin) was then placed in the conjunctival sac. The adhesive tape was removed from the nonoperated eye, and the rabbits transferred to their cages. No eyelid sutures were used. POSTOPERATIVE CARE
Gross and slitlamp examinations were performed daily. Antibiotic ointment was used only when indicated after the day of operation. The sutures were removed under the dissecting microscope on the fifth to seventh postoperative days. Prior to suture removal, the rabbits were anesthetized with 60 mg of intravenous pentobarbital sodium, and
Fig. 2 (Khodadoust). The deep lamellar graft is attached to the recipient corneal bed, using interrupted sutures of 8-0 virgin silk.
1113
Fig. 3 (Khodadoust). Good results have also been obtained employing a continuous suture of 8-0 virgin silk.
proparacaine HCl was instilled into the conjunctival sac. RESULTS
The foregoing technique of lamellar keratoplasty was used in 100 consecutive eyes in 100 rabbits. There were no complications at the time of surgery in 90 eyes. In these, a mild transient edema of the graft developed which could be visualized only with the slitlamp. Four days after surgery, this edema had completely disappeared. In these 90 eyes, the cornea remained free of blood vessels and the grafts remained clear during the 8- to 24-week period before the animals were used for other investigations. The anterior chamber was unintentionally entered in 10 eyes. In each of these, the operation was completed after two to three drops of 1:1,000 aqueous sodium heparin were placed on the cornea in order to prevent clotting of the fluid from the anterior chamber. In eight of these 10 animals, the size of the corneal rupture was less than 2 mm, and the anterior chamber had reformed by the end of the operation. In three of these eight animals the corneal perforation was located peripherally, and iris was incarcerated in the wound. In each instance the iris was freed after the completion of the transplantation with the use of a 25-gauge needle obliquely inserted into the anterior chamber from the corneoscleral limbus. The
1114
AMERICAN JOURNAL OF OPHTHALMOLOGY
D E C E M B E R , 1968
Fig. 4 (Khodadoust). ( A ) A t 24 hours after corneal transplantation, stromal edema is limited to the margins of the graft. (Hematoxylin-eosin, X l 2 . ) ( B ) A t higher magnification, the epithelium has already bridged the gap between donor and recipient tissues, resulting in a thinning of both donor and recipient epithelium adjacent to the wound margin. There is a relatively acellular area near the wound edges. Note depth of the graft, as indicated by the arrows marking the donor-recipient interface. (Hematoxylin-eosin, Xl2S.)
postoperative course in the eight eyes with small perforations was benign. There was edema of the graft localized in an area 1 to 2 mm around the penetration site, and lasting from nine to 13 days. When the edema had cleared, slitlamp examination revealed an intact endothelium covering the defect in Descemet's membrane at the site of perforation. In six of the eight eyes with small anterior chamber perforations, a few fine vessels extended from the corneoscleral limbus to the edge of the graft ; in the other two eyes, the vessels penetrated 1 to 2 mm into the graft. In two eyes, the size of the corneal perforation was larger than 2 mm (3mm in one and 4 mm in the other). Both grafts became partially cloudy and vascularized. The eye with the 3-mm perforation developed permanent cloudiness and vascularization in onethird of the graft area. The eye with the 4-mm perforation also had one-half of the graft bed denuded of Descemet's membrane and endothelium, and this graft became cloudy and vascularized over more than twothirds its area.
In the 100 operated eyes, not a single instance of infection was noted, nor was there any sloughing of the graft or opening of the wound. Successful grafts were obtained in 98% of the operated eyes. These grafts remained clear during the 8- to 24-week observation period. HISTOLOGIC OBSERVATIONS
An extensive histologic description of the sequence of changes in the graft and the recipient cornea will not be given, since the process of corneal wound healing has already been studied and discussed extensively. However, for completeness of the present study, histologic observations were made on the corneas of eyes enucleated 24 hours, 3 days, 10 days, 2 weeks, and 8 months after lamellar corneal transplantation, and these findings will be outlined briefly. At 24 hours following the grafting procedure, the epithelium had already bridged the gap between the donor and recipient stroma, and there was slight stromal edema near the 9-9
V O L . 66, N O . 6
LAMELLAR
CORNEAL
TRANSPLANTATION
IIIS
Fig. S (Khodadoust). Three days after transplantation, a mild infiltrate of the stroma at the graft margins signals the active repair process which is taking place. The epithelium has almost completely filled in the surface defect, although a discrete basal epithelial layer has not yet formed at the repair site. The arrows indicate the donor-recipient junction. (Hematoxylin-eosin, X l 5 0 . )
w o u n d edges as well as a relatively acellular
gained their normal compactness
area (fig. 4 - A ) . Both the donor and recipient
T h e donor-recipient interface could hardly be
stroma were covered with a normal thickness
recognized at the base of the graft, and the
of
cellular infiltration at the w o u n d margins was
epithelium except near the w o u n d m a r -
fibroblasts
(fig. 6 ) .
gins, where the epithelial thickness was re-
reduced to a f e w
duced to a layer two to three cells deep (fig.
round cells (fig. 6 - B ) . T h e epithelium over
and occasional
4 - B ) . T h i s epithelial thinning was due to the
the w o u n d n o w had a well-developed basal
sliding of those cells involved in the repair
layer. T h e leveling tendency of the epithe-
and closure of the adjacent corneal wound.
lium at the junction of the donor and recipi-
three days after transplantation, the
ent stroma was apparent, with the epithelium
relatively acellular area of the graft and re-
increasing its thickness in order to restore
cipient stroma described above was populated
the original smooth corneal surface.
At
with a sparse infiltrate of polymorphonuclear
At eight weeks, the histologic picture of
leukocytes, fibroblasts, and occasional m o n o -
the grafted cornea was the same centrally as
cytes (fig. 5 ) . T h e r e w a s less stromal edema.
that of a normal ungrafted cornea. A small,
T h e epithelium over the donor and recipient
well-healed scar lay at the junction of donor
stroma adjacent to the w o u n d was three to
and recipient tissues at the periphery.
four cells thick and mitotic activity could be COMMENT
seen in the basal layer of epithelium on either side, although a discrete basal layer had
1. T h e usefulness of lamellar keratoplasty
not yet formed at the repair site. Elsewhere,
in
the
normal
volved in allograft rejection depends upon
thickness, with n o evidence of destruction or
the ability to p e r f o r m reproducibly success-
donor
epithelium
retained
its
abnormal histologic change.
studying
the
immunologic
factors
in-
ful corneal grafts whose clarity (prior to the
At ten days after transplantation, the stro-
onset o f graft rejection)
can be depended
mal edema near the w o u n d edge had disap-
upon. W e report here a technique permitting
peared,
successful
and the
stromal lamellae
had
re-
deep-lamellar
8-mm
corneal
1116
A M E R I C A N JOURNAL OF O P H T H A L M O L O G Y
D E C E M B E R , 1968
Fig. 6 (Khodadoust). ( A ) Ten days after transplantation, stromal edema has disappeared and the donor-recipient interface can scarcely be recognized at the base of the graft. (Hematoxylin-eosin, X l 2 . ) ( B ) A t this time, the cellular infiltrate at the wound margins is reduced to a few fibroblasts and occasional round cells. The epithelium over the wound has completed the leveling process, and now has a well developed basal layer. (Hematoxylin-eosin, X12S.)
grafts in the rabbit in 98 of 100 consecutive procedures. 2. W e were impressed by the minimal reaction and relative clarity of these lamellar grafts in the early postoperative period. The technique used in separating the lamellar corneal disc from the deep stromal fibers causes minimal embarrassment of the tissue ; there is no pulling or folding of the disc and the overlying epithelium remains intact. A c cidental perforation into the anterior chamber during deep dissection does not necessarily damage the graft. If the perforation is less than 2 mm in size, the resultant localized edema at the perforation site will clear within two weeks with healing of the endothelium. A break larger than 2 mm in Descemet's membrane and in the endothelium causes cloudiness and subsequent vascularization at the site of perforation. This is probably due to curling of Descemet's membrane and slow endothelial repair, resulting
in persistent edema, vascularization, and scar tissue formation. 3. Good suturing technique is important for successful grafting. The use of a dissecting microscope is essential for correct suture placement and tension, and we customarily use a 16X magnification. Using the microscope, we have not had any difficulty with the wound notching described by Shafer. Although we routinely use 8-0 virgin silk suture, we have also used two other suture materials to secure the graft into the corneal bed. Very good results were obtained earlier with an experimental 7-0 absorbable collagen suture, but this material is no longer available. W e have had better technical results with fine black nylon thread of 30p. diameter than with 7-0 collagen suture. 2
4. The timing of suture removal also affects the outcome of the graft procedure. The longer the sutures are left in place, the greater will be the growth of vessels through
V O L . 66, N O . 6
LAMELLAR CORNEAL T R A N S P L A N T A T I O N
the recipient cornea toward the grafted corneal disc. In some experimental situations, heavy vascularization is desirable. This may be achieved by leaving the sutures in place beyond seven days. Eccentric placement of the graft has also been employed intentionally in order to have more vessels clustered at one edge of the graft. The vessels growing in from the corneoscleral limbus will first reach that portion of the graft which is nearest the limbus. But beyond this, placement of the graft near the limbus seems to incite a higher degree of vascularization of the graft than can be accounted for by the simple explanation that the vessels have a shorter distance to grow to reach the wound edge. This phenomenon is not well understood. 5. Fortunately, postoperative infection has not been a problem in this series of operations on rabbits. We attribute this lack of complications to a variety of factors. Irrigation of the conjunctival sac prior to surgery and use of the sterile plastic drape minimize contamination of the eye during surgery. Careful apposition of the corneal wound edges, the careful handling of the tissue so that a minimal amount of epithelium is destroyed, and the routine use of antibiotic ointment after surgery minimize the growth of organisms postoperatively. W e do not suture the eyelids closed at the end of the surgical procedure. It is possible that leaving the lids free permits a more physiologic distribution of the tear film over the corneal graft and that the beneficial properties of the tears are important postoperatively. 6. A combination of promazine HCl and pentobarbital sodium provides satisfactory anesthesia throughout the surgical procedure. The technique is simple to perform, and the mortality rate in more than 500 operations done during the past two years has
1117
been less than 1%. On rare occasions, a rabbit will develop respiratory arrest early in the operative procedure. Ventilation can be restarted easily by placing a rubber tube in the mouth of the rabbit, occluding its nostrils, and then blowing gently on the other end of the tube to inflate the lungs. W e sometimes use a tube under the plastic drape to circulate a small flow of oxygen to the animal in order to prevent rebreathing of expired air. SUMMARY
A technique for deep lamellar corneal transplantation in the rabbit is described. Typical gross and histologic results are illustrated. With this technique, clear grafts were obtained in 98% of the operated eyes in a consecutive series of 100 corneal allograft transplants. 601 North Broadway (21205) REFERENCES 1. von Hippel, A. : Eine neue Methode der Hornhauttransplantation. Arch. f. Ophth. 34(Abt. 1) :108, 1888. 2. Shafer, D. M. : Experimental corneal grafts of the lamellar partial type. Am. J. Ophth. 33 :26, 1950. 3. Kornblueth, W . and Nelken, E. : Partial lamellar corneal grafts in rabbits. Histologic observations. Am. J. Ophth. 45:242, 1958. 4. Davis, F. A. : The anatomy and histology of the eye and orbit of the rabbit. Tr. Am. Ophth. Soc. 27:401, 1929. 5. von Bahr, G. : Corneal thickness : Its measurement and changes. Am. J. Ophth. 42:251, 1956. 6. Bonnefon, G. and Lacoste, A . : Recherches histologiques sur la greffe cornéenne autoplastique. Arch. d'Ophtal. 33 :206, 1913. 7. Weimar, V . L. : The sources of fibroblasts in corneal wound repair: A quantitative analysis. Arch. Ophth. 60:93, 1958. 8. Robb, R. M . and Kuwabara, T. : Corneal wound healing. II. An autoradiographic study of the cellular components. Arch. Ophth. 72 :401, 1964. 9. Kitano, S. and Goldman, J. N. : Cytologic and histochemical changes in corneal wound repair. Arch. Ophth. 76:345, 1966.
Lamellar corneal transplantation was introduced by von Hippel almost 80 years ago, and has since become a generally accepted technique in the armamentarium of the ocular surgeon. However, lamellar transplants have not been utilized as widely as penetrating grafts in experimental studies of the immunologic aspects of corneal transplantation. Few descriptions of the technique of lamellar keratoplasty in experimental animals appear in the literature. Shafer reported difficulty in preventing the ingrowth of epithelium between the recipient stroma and 5-mm lamellar grafts in rabbits. He concluded that an edge-to-edge interrupted suture did not produce a sufficiently firm wound closure in the thin and flexible rabbit cornea, thus permitting epithelial downgrowth. In 1958, Kornblueth and Nelken described a technique for partial lamellar corneal grafts in rabbits. They used a criss-cross suture over a 7.1-mm lamellar disc in 146 eyes. In the first 94 operations in their series, clear grafts were obtained in 21% of the eyes, and with improvement in techniques, in 55,% of their next 52 operations.
MATERIALS AND METHODS
1
2
3
This paper describes a technique for the transplantation of deep lamellar corneal grafts in rabbits, which we developed for experimental purposes and for the training of ocular surgeons. With this technique, clear grafts were obtained in 98 of 100 consecutive operations. This paper describes the technical aspects of the approach, as well as the gross and histologic observations made on these cases. The results of the application of this technique to the study of corneal allograft rejection will be presented in subsequent reports. From the Wilmer Institute, the Johns Hopkins University School of Medicine. Supported in part by U S P H S Grant No. N B 03040 from the National Institute of Neurological Diseases and Blindness, and by an unrestricted gift from the Alcon Laboratories, Inc.
ANIMALS
All experiments were performed on young adult New Zealand Giant albino rabbits, weighing 2 to 3 kg. The corneal thickness in these animals is reported to average 0.45 mm near the corneoscleral limbus and 0.37 mm centrally, with an overall average value of 0.4 mm. 4
5
ANESTHESIA
Thirty to 45 minutes before surgery, each animal received 25 mg of promazine HCl (Sparine) by subcutaneous or intramuscular injection. Just prior to surgery, each rabbit was given 60 to 70 mg of pentobarbital sodium (Nembutal) intravenously in an ear vein. Since rabbits vary considerably in the amount of barbiturate needed to produce satisfactory anesthesia, the pentobarbital sodium was given slowly, and the animal was tested for depth of anesthesia by exerting pressure on the foot. Only a slight withdrawal reflex should occur if the plane of anesthesia is satisfactory, and the amount of pentobarbital sodium given can be titrated according to this response. With this technique, not a single rabbit was lost in 100 consecutive surgical procedures. Topical anesthesia of the eye was achieved by the repeated instillation of proparacaine HCl (Ophthaine) into the conjunctival sac before and during surgery. No retrobulbar injections were used. PREPARATION AND DRAPE
All the rabbits were operated upon in pairs. The periorbital hair was trimmed with scissors, and the lids of the nonoperated eye were held closed with a small piece of adhesive tape to prevent drying of the cornea during the procedure. The rabbits were then positioned side by side on the operating table and the conjunctival sacs irrigated with ster-
1112
AMERICAN JOURNAL OF OPHTHALMOLOGY
ile saline, followed by another drop of Ophthaine. A mask and sterile gloves were used to maintain aseptic technique during the remainder .of the procedure. Each rabbit was draped with a self-adhering plastic sheet (Steri-Drape No. 1040, Minnesota Mining & Manufacturing Co.) with the eye positioned in the center of the adhesive area, and the drape was pressed down along the lid margins. A 10- to 12-mm-incision was made through the drape over the center of the cornea and parallel to the eyelid margins. The eye was then proptosed through this hole in the drape ; being somewhat elastic, the drape automatically retracts behind the globe and thereby keeps all skin and hair out of the operative field. An excellent exposure with moderately tight positioning of the eyeball resulted, eliminating the necessity for a lid speculum and traction sutures. The size of the opening in the drape was critical, since too small an opening may embarrass the blood supply, whereas too large an opening will fail to hold the globe in proptosis. Drying of the bulbar conjunctiva and the sclera was prevented by a narrow piece of gauze which was wrapped around the globe and kept moist with saline. The cornea was moistened throughout the procedure by frequent applications of sterile saline.
D E C E M B E R , 1968
secting microscope. The corneal disc was then separated from the deep stromal fibers by holding the edge of the disc with fine tooth forceps and passing the blade of a Gill knife between the lamellae (fig. 1 ) . In order to avoid unintentional perforation into the anterior chamber, the knife blade should be kept parallel to (and thus follow) the curvature of the cornea. With a gentle to-and-fro motion of the knife, the dissection was extended laterally just beyond the limits of the outline made by the corneal trephine. The excision of the corneal button was then completed by using curved corneal scissors. Just before the graft was completely freed with the scissors, a single 8-0 virgin silk suture was placed partially through the lamellar button. This suture facilitates the transfer of the graft from one rabbit to the other, and serves as the first suture for attachment to the recipient bed. The freed graft was left on the keratectomy bed and the dissecting microscope was then positioned over the eye of the other rabbit, from which an identical corneal disc was removed by the same technique.
SURGICAL TECHNIQUE
An 8-mm corneal trephine was used to outline the lamellar button. This trephine incision may extend in depth to two-thirds of the corneal thickness. (The commercially available trephines are specifically designed for the curvature of the human cornea, and their depth setting is not reliable for incising the rabbit cornea; success in this procedure therefore takes practice. While gaining this experience, the beginner is advised to make a relatively shallow incision with the trephine.) It is possible to increase the depth of the incision on one side of the corneal button by using the fine blade of a Gill knife. This incision with the knife, and the remainder of the fine work, were done under a 16 X dis-
Fig 1 (Khodadou-.t). After outlining the lamellar button to an appropriate depth with an 8-mm trephine, the corneal disc is separated from the donor stroma by a to-and-fro motion of the Gill knife. Note the narrow strip of saline-moistened gauze which is wrapped around the globe to prevent drying of the bulbar conjunctiva.
V O L . 66, N O . 6
LAMELLAR CORNEAL TRANSPLANTATION
The grafts were exchanged immediately after the disc was freed from the second rabbit. Each graft was sutured into the recipient cornea using interrupted sutures of 8-0 virgin silk on a G-7 needle or fine black nylon thread of 30¡x diameter on a 4-mm Grieshaber corneal needle. Twelve sutures were usually used for each graft (fig. 2 ) . A continuous suture has also been employed to good advantage in this procedure (fig. 3 ) , and has the added advantage of reducing the irritation arising from the use of multiple knots. Following completion of surgery, the incision in the plastic drape was enlarged and the proptosed globe then retracted into the orbit. Antibiotic ointment (polymyxin Bneomycin-bacitracin) was then placed in the conjunctival sac. The adhesive tape was removed from the nonoperated eye, and the rabbits transferred to their cages. No eyelid sutures were used. POSTOPERATIVE CARE
Gross and slitlamp examinations were performed daily. Antibiotic ointment was used only when indicated after the day of operation. The sutures were removed under the dissecting microscope on the fifth to seventh postoperative days. Prior to suture removal, the rabbits were anesthetized with 60 mg of intravenous pentobarbital sodium, and
Fig. 2 (Khodadoust). The deep lamellar graft is attached to the recipient corneal bed, using interrupted sutures of 8-0 virgin silk.
1113
Fig. 3 (Khodadoust). Good results have also been obtained employing a continuous suture of 8-0 virgin silk.
proparacaine HCl was instilled into the conjunctival sac. RESULTS
The foregoing technique of lamellar keratoplasty was used in 100 consecutive eyes in 100 rabbits. There were no complications at the time of surgery in 90 eyes. In these, a mild transient edema of the graft developed which could be visualized only with the slitlamp. Four days after surgery, this edema had completely disappeared. In these 90 eyes, the cornea remained free of blood vessels and the grafts remained clear during the 8- to 24-week period before the animals were used for other investigations. The anterior chamber was unintentionally entered in 10 eyes. In each of these, the operation was completed after two to three drops of 1:1,000 aqueous sodium heparin were placed on the cornea in order to prevent clotting of the fluid from the anterior chamber. In eight of these 10 animals, the size of the corneal rupture was less than 2 mm, and the anterior chamber had reformed by the end of the operation. In three of these eight animals the corneal perforation was located peripherally, and iris was incarcerated in the wound. In each instance the iris was freed after the completion of the transplantation with the use of a 25-gauge needle obliquely inserted into the anterior chamber from the corneoscleral limbus. The
1114
AMERICAN JOURNAL OF OPHTHALMOLOGY
D E C E M B E R , 1968
Fig. 4 (Khodadoust). ( A ) A t 24 hours after corneal transplantation, stromal edema is limited to the margins of the graft. (Hematoxylin-eosin, X l 2 . ) ( B ) A t higher magnification, the epithelium has already bridged the gap between donor and recipient tissues, resulting in a thinning of both donor and recipient epithelium adjacent to the wound margin. There is a relatively acellular area near the wound edges. Note depth of the graft, as indicated by the arrows marking the donor-recipient interface. (Hematoxylin-eosin, Xl2S.)
postoperative course in the eight eyes with small perforations was benign. There was edema of the graft localized in an area 1 to 2 mm around the penetration site, and lasting from nine to 13 days. When the edema had cleared, slitlamp examination revealed an intact endothelium covering the defect in Descemet's membrane at the site of perforation. In six of the eight eyes with small anterior chamber perforations, a few fine vessels extended from the corneoscleral limbus to the edge of the graft ; in the other two eyes, the vessels penetrated 1 to 2 mm into the graft. In two eyes, the size of the corneal perforation was larger than 2 mm (3mm in one and 4 mm in the other). Both grafts became partially cloudy and vascularized. The eye with the 3-mm perforation developed permanent cloudiness and vascularization in onethird of the graft area. The eye with the 4-mm perforation also had one-half of the graft bed denuded of Descemet's membrane and endothelium, and this graft became cloudy and vascularized over more than twothirds its area.
In the 100 operated eyes, not a single instance of infection was noted, nor was there any sloughing of the graft or opening of the wound. Successful grafts were obtained in 98% of the operated eyes. These grafts remained clear during the 8- to 24-week observation period. HISTOLOGIC OBSERVATIONS
An extensive histologic description of the sequence of changes in the graft and the recipient cornea will not be given, since the process of corneal wound healing has already been studied and discussed extensively. However, for completeness of the present study, histologic observations were made on the corneas of eyes enucleated 24 hours, 3 days, 10 days, 2 weeks, and 8 months after lamellar corneal transplantation, and these findings will be outlined briefly. At 24 hours following the grafting procedure, the epithelium had already bridged the gap between the donor and recipient stroma, and there was slight stromal edema near the 9-9
V O L . 66, N O . 6
LAMELLAR
CORNEAL
TRANSPLANTATION
IIIS
Fig. S (Khodadoust). Three days after transplantation, a mild infiltrate of the stroma at the graft margins signals the active repair process which is taking place. The epithelium has almost completely filled in the surface defect, although a discrete basal epithelial layer has not yet formed at the repair site. The arrows indicate the donor-recipient junction. (Hematoxylin-eosin, X l 5 0 . )
w o u n d edges as well as a relatively acellular
gained their normal compactness
area (fig. 4 - A ) . Both the donor and recipient
T h e donor-recipient interface could hardly be
stroma were covered with a normal thickness
recognized at the base of the graft, and the
of
cellular infiltration at the w o u n d margins was
epithelium except near the w o u n d m a r -
fibroblasts
(fig. 6 ) .
gins, where the epithelial thickness was re-
reduced to a f e w
duced to a layer two to three cells deep (fig.
round cells (fig. 6 - B ) . T h e epithelium over
and occasional
4 - B ) . T h i s epithelial thinning was due to the
the w o u n d n o w had a well-developed basal
sliding of those cells involved in the repair
layer. T h e leveling tendency of the epithe-
and closure of the adjacent corneal wound.
lium at the junction of the donor and recipi-
three days after transplantation, the
ent stroma was apparent, with the epithelium
relatively acellular area of the graft and re-
increasing its thickness in order to restore
cipient stroma described above was populated
the original smooth corneal surface.
At
with a sparse infiltrate of polymorphonuclear
At eight weeks, the histologic picture of
leukocytes, fibroblasts, and occasional m o n o -
the grafted cornea was the same centrally as
cytes (fig. 5 ) . T h e r e w a s less stromal edema.
that of a normal ungrafted cornea. A small,
T h e epithelium over the donor and recipient
well-healed scar lay at the junction of donor
stroma adjacent to the w o u n d was three to
and recipient tissues at the periphery.
four cells thick and mitotic activity could be COMMENT
seen in the basal layer of epithelium on either side, although a discrete basal layer had
1. T h e usefulness of lamellar keratoplasty
not yet formed at the repair site. Elsewhere,
in
the
normal
volved in allograft rejection depends upon
thickness, with n o evidence of destruction or
the ability to p e r f o r m reproducibly success-
donor
epithelium
retained
its
abnormal histologic change.
studying
the
immunologic
factors
in-
ful corneal grafts whose clarity (prior to the
At ten days after transplantation, the stro-
onset o f graft rejection)
can be depended
mal edema near the w o u n d edge had disap-
upon. W e report here a technique permitting
peared,
successful
and the
stromal lamellae
had
re-
deep-lamellar
8-mm
corneal
1116
A M E R I C A N JOURNAL OF O P H T H A L M O L O G Y
D E C E M B E R , 1968
Fig. 6 (Khodadoust). ( A ) Ten days after transplantation, stromal edema has disappeared and the donor-recipient interface can scarcely be recognized at the base of the graft. (Hematoxylin-eosin, X l 2 . ) ( B ) A t this time, the cellular infiltrate at the wound margins is reduced to a few fibroblasts and occasional round cells. The epithelium over the wound has completed the leveling process, and now has a well developed basal layer. (Hematoxylin-eosin, X12S.)
grafts in the rabbit in 98 of 100 consecutive procedures. 2. W e were impressed by the minimal reaction and relative clarity of these lamellar grafts in the early postoperative period. The technique used in separating the lamellar corneal disc from the deep stromal fibers causes minimal embarrassment of the tissue ; there is no pulling or folding of the disc and the overlying epithelium remains intact. A c cidental perforation into the anterior chamber during deep dissection does not necessarily damage the graft. If the perforation is less than 2 mm in size, the resultant localized edema at the perforation site will clear within two weeks with healing of the endothelium. A break larger than 2 mm in Descemet's membrane and in the endothelium causes cloudiness and subsequent vascularization at the site of perforation. This is probably due to curling of Descemet's membrane and slow endothelial repair, resulting
in persistent edema, vascularization, and scar tissue formation. 3. Good suturing technique is important for successful grafting. The use of a dissecting microscope is essential for correct suture placement and tension, and we customarily use a 16X magnification. Using the microscope, we have not had any difficulty with the wound notching described by Shafer. Although we routinely use 8-0 virgin silk suture, we have also used two other suture materials to secure the graft into the corneal bed. Very good results were obtained earlier with an experimental 7-0 absorbable collagen suture, but this material is no longer available. W e have had better technical results with fine black nylon thread of 30p. diameter than with 7-0 collagen suture. 2
4. The timing of suture removal also affects the outcome of the graft procedure. The longer the sutures are left in place, the greater will be the growth of vessels through
V O L . 66, N O . 6
LAMELLAR CORNEAL T R A N S P L A N T A T I O N
the recipient cornea toward the grafted corneal disc. In some experimental situations, heavy vascularization is desirable. This may be achieved by leaving the sutures in place beyond seven days. Eccentric placement of the graft has also been employed intentionally in order to have more vessels clustered at one edge of the graft. The vessels growing in from the corneoscleral limbus will first reach that portion of the graft which is nearest the limbus. But beyond this, placement of the graft near the limbus seems to incite a higher degree of vascularization of the graft than can be accounted for by the simple explanation that the vessels have a shorter distance to grow to reach the wound edge. This phenomenon is not well understood. 5. Fortunately, postoperative infection has not been a problem in this series of operations on rabbits. We attribute this lack of complications to a variety of factors. Irrigation of the conjunctival sac prior to surgery and use of the sterile plastic drape minimize contamination of the eye during surgery. Careful apposition of the corneal wound edges, the careful handling of the tissue so that a minimal amount of epithelium is destroyed, and the routine use of antibiotic ointment after surgery minimize the growth of organisms postoperatively. W e do not suture the eyelids closed at the end of the surgical procedure. It is possible that leaving the lids free permits a more physiologic distribution of the tear film over the corneal graft and that the beneficial properties of the tears are important postoperatively. 6. A combination of promazine HCl and pentobarbital sodium provides satisfactory anesthesia throughout the surgical procedure. The technique is simple to perform, and the mortality rate in more than 500 operations done during the past two years has
1117
been less than 1%. On rare occasions, a rabbit will develop respiratory arrest early in the operative procedure. Ventilation can be restarted easily by placing a rubber tube in the mouth of the rabbit, occluding its nostrils, and then blowing gently on the other end of the tube to inflate the lungs. W e sometimes use a tube under the plastic drape to circulate a small flow of oxygen to the animal in order to prevent rebreathing of expired air. SUMMARY
A technique for deep lamellar corneal transplantation in the rabbit is described. Typical gross and histologic results are illustrated. With this technique, clear grafts were obtained in 98% of the operated eyes in a consecutive series of 100 corneal allograft transplants. 601 North Broadway (21205) REFERENCES 1. von Hippel, A. : Eine neue Methode der Hornhauttransplantation. Arch. f. Ophth. 34(Abt. 1) :108, 1888. 2. Shafer, D. M. : Experimental corneal grafts of the lamellar partial type. Am. J. Ophth. 33 :26, 1950. 3. Kornblueth, W . and Nelken, E. : Partial lamellar corneal grafts in rabbits. Histologic observations. Am. J. Ophth. 45:242, 1958. 4. Davis, F. A. : The anatomy and histology of the eye and orbit of the rabbit. Tr. Am. Ophth. Soc. 27:401, 1929. 5. von Bahr, G. : Corneal thickness : Its measurement and changes. Am. J. Ophth. 42:251, 1956. 6. Bonnefon, G. and Lacoste, A . : Recherches histologiques sur la greffe cornéenne autoplastique. Arch. d'Ophtal. 33 :206, 1913. 7. Weimar, V . L. : The sources of fibroblasts in corneal wound repair: A quantitative analysis. Arch. Ophth. 60:93, 1958. 8. Robb, R. M . and Kuwabara, T. : Corneal wound healing. II. An autoradiographic study of the cellular components. Arch. Ophth. 72 :401, 1964. 9. Kitano, S. and Goldman, J. N. : Cytologic and histochemical changes in corneal wound repair. Arch. Ophth. 76:345, 1966.