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Reconstruction of the Corneal Epithelium by Limbal Allograft Transplantation for Severe Ocular Surface Disorders
Reconstruction of the Comeal Epithelium by Limbal Allograft Transplantation for Severe Ocular Surface Disorders Kazuo Tsubota, MD,l ·2 Ikuko Toda, MD,l· 2 Hiroshi Saito, MD,J.3 Naoshi Shinozaki, 1•3 ]un Shimazaki, MD 1•2 Purpose: Although penetrating keratoplasty is an established surgical procedure, it often is ineffective for severe ocular surface diseases such as alkali burns or limbal deficiency. The authors have performed limbal allograft transplantation for the recon struction of the corneal epithelium. Methods: A total of nine patients (3 with chemical injury, 3 with limbal deficiency with unknown etiology, 2 with moderate ocular pemphigoid [OCP], and 1 with traumatic limbal deficiency) were treated by limbal allograft transplantation. Penetrating kerato plasties were performed in all patients with the exception of one with OCP. Patients received cyclosporine both systemically (1 0 mgfkg) and topically (0.05%) as well as high-dose intravenous dexamethasone (8 mg). Results: The corneal epithelium was reconstructed in all patients, although two showed partial increased fluorescein permeability and two others required a second surgery. The other five epithelia remained clear at mean follow-up of 12.3 months, with two episodes of graft rejection which were controlled successfully by medication. Conclusions: Limbal allograft transplantation with intensive immunosuppression by cyclosporine and high-dose steroids appears to be a promising surgical intervention for the reconstruction of corneas affected by severe ocular surface disease. Ophthalmology 1995; 102:1486-1496
Penetrating keratoplasty (PK) is an established surgical procedure for treating corneal opacity. However, for those patients with severe ocular surface abnormalities such as persistent epithelial defect, corneal vascularization, and epithelial irregularity, simple PK often is not successful. The causes of these conditions include chemical injury, contact lens-related epithelial defects, ocular pemphigoid Originally received: March 17, 1995. Revision accepted: June 8, 1995. 1 Department of Ophthalmology, Tok yo Dental College, Chiba. 2 Department of Ophthalmology, Keio University School of Medicine, Tokyo. 3 Department of Ophthalmology, Kyorin University School of Medicine, Tokyo. Reprint requests to Kazuo Tsubota, MD, Department of Ophthalmology, Tokyo Dental College, 11-13 Sugano 5 Chome, lchikawa-shi. Chiba, Japan 272.
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(OCP), and limbal deficiency of unknown etiology. To reconstruct a stable corneal epithelium, Thoft and Kinoshita'-4 developed the technique ofkeratoepithelio plasty, with good clinical results. The procedure involves the transplantation of corneal epithelium as well as the reconstruction of the substrate by implanting the donor Bowman layer in the recipient's lim bal position. After the procedure, the transplanted corneal epithelium covers the recipient cornea, providing a healthier and more stable epithelium. The corneal stem cell theory gives new insight into severe ocular surface abnormalities which suggests that corneal and conjunctival surfaces normally are covered by their respective epithelia, which are presumably sup plied by their own stem cells. s-? Corneal stem cells appear to concentrate at the limbus, 5- 7 while the conjunctival stem cells may be at the fornix .8 If the corneal stem cells are partially or totally depleted, the corneal surface sub
T subota et al · Limbal Allograft Transplantation sequently is covered by conjunctival epithelium. Since the limbal area is exposed directly to the environment and is influenced easily by such injuries as alkali burns, corneal stem cell deficiency can occur so that the corneal surface is covered by the proliferated conjunctival epi thelium. If the conjunctival stem cells also are depleted, which is rare because of the larger conjunctival area and the protected location (e.g., fornices) of the stem cells, the ocular surface eventually is covered by totally keratinized epithelium, which sometimes may be seen at the end stage of OCP or Stevens-Johnson syndrome. For patients with limbal cell deficiency, corneal stem cell transplant has been considered. 9 - 12 In rabbit models, several studies have established the efficacy of this pro cedure. 11 Because the limbal area includes many antigen presenting cells, such as Langerhans cells, the possibility of rejection might be higher than for regular PK. 13 To prevent rejection, limbal autograft transplantation first was tried clinically, and good surgical results were ob tained.14·15 However, there are some practical disadvan tages of this procedure. When patients have bilateral ocu lar surface disease such as chemical injury or OCP, au tograft transplantation is impossible. Patients' reluctance to harvest from the good eye also limits the applicability of this procedure. As such, limbal allograft transplantation becomes necessary in some cases. 12·16 In this study, we performed limbal allograft transplantation using eye-bank corneas stored for more than 4 days in preservation media, coupled with strong immunosuppression by systemic and topical cyclosporine. Although relatively few patients were treated, we were able to reconstruct a stable corneal epi thelium in all nine patients, with concomitant improve ment in visual acuity.
Materials and Methods Viability of Limbal Cells in Donor Cornea Because ordinary eye-bank storage media were developed to optimize preservation of the endothelium, rather than the epithelium, we were concerned with the storage con ditions of the limbal epithelium. We examined the corneal and limbal epithelia in eye-bank corneas (n = 35) stored for 5 days in Optisol GS medium (Chiron Ophthalmics, Irvine, CA). The age ofdonors ranged from 18 to 86 (mean ± standard deviation, 68.9 ± 12.1 years). The average storage time was 114.5 ± 40.2 hours. The basal part of the limbal epithelium always was intact (35/35), whereas the central corneal epithelium occasionally was missing or damaged (27/35) (Fig I). This was confirmed by ob serving the section of the tissue with a light microscope. To confirm the viability of the limbal epithelium of the donor cornea, part of the eye-bank limbal tissue was cultured in rabbit corneal epithelium growth medium (Kurabou, Inc, Tokyo, Japan) which included insulin (5 !Lg/ml), epidermal growth factor ( 10 ng/ml), hydrocorti sone (0.5 mg/ml), gentamicin (50 !lgfml) , amphotericin B (0.25 mg/ml), and 0.4% vol/vol bovine pituitary extract. Limbal tissue was cut into small pieces and cultured in
rabbit corneal epithelium growth medium in 35-mm plastic dishes and placed in a 5% carbon dioxide incubator for 20 to 30 days. The medium was changed every other day. When cell growth was identified by microscopic ob servation (Fig 2A), the limbal tissue was replaced in an other dish 12 days after the primary culture, whereas the cells in the first dish were treated with 5% trypsin (2 ml) for 30 seconds, and the cells were removed and placed in a 15-ml centrifugation tube after nutrizing with 2 ml nu trizing solution. Medium (2 ml) was added to the tube and placed in 35-mm plastic dishes, which were incubated in a 5% carbon dioxide incubator at 37°C for the sec ondary culture after centrifugated with 400g. When the cells reached 80% confluency (Fig 2B), the same procedure was repeated. During the culturing, the limbal tissue (5 X 3 mm) (n = 35) was placed in rabbit corneal epithelium growth me dium for 0, 5, 15, and 25 days, we then took a thin slice from each piece of tissue which then was fixed in Dent fixative (20% dimethyl sulfoxide and 80% methanol) for 2 hours at 4 oc. The tissue then was placed in 70% ethanol for I hour at 4 oc. After being washed three times in phosphate-buffered saline for 20 minutes, the tissue was placed in 50% block ace to prevent nonspecific binding of the antibody. We stained the limbal tissue with anti proliferation cell nuclear antigen monoclonal antibody to examine the mitotic activity of the limbal basal epithe lium. 17 Antiproliferation cell nuclear antigen I-J mono clonal antibody (clone PCIO) was diluted 100 times by 10% block ace-0.5% Tween80 as a primary antibody, and the tissue was placed in the primary antibody overnight at 4°C. The tissue then was washed thoroughly. Fluores cein isothiocynate-goat anti-mouse lgG, which was di luted 50 times with I 0% block ace-0.5 % Tween80, was used for the secondary antibody. The tissue was placed in the secondary antibody for 2 hours at room temperature and then washed. After slicing the tissue with a sharp blade in widths of0.1 to 0.2 mm, we mounted the sections on glass slides containing 90% glycerol in TRIS-buffered saline and observed the specimens by fluorescent mi croscopy.
Patients Nine patients (5 men, 4 women) (average age, 67.7 ± 16.2 years; range, 34-90 years of age) with severe ocular surface conditions were treated by limbal allograft transplantation. The diagnosis included three patients with chemical injury, three with oflimbal deficiency of unknown etiology, two with moderate OCP, and one with extensive mechanical injury to the limbal area (Table 1). The corneal epithelia had frequent defects and were compromised by conjunctivization with vessel invasion and increased fluorescein permeability and rose-bengal staining. The surface irregularities were prominent in all patients. Persistent epithelial defects were seen at the time of surgery in cases I, 2, 3, 5, and 9. Impression cytology showed transdifferentiation of the conjunctival epithelium in cases I, 3, 5, 8, and 9, with goblet cells in cases 8 and 9. When the ocular sur
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Top, Figure 1. The limbal and corneal epithelium of eye-bank cornea stored in Optisol GS for 5 days (65-year-old male donor; death to enucleation time, 5 hours; encleation to preservation time, 2 hours). Notice the limbal epithelium (arrow) is intact, whereas the corneal epithelium (double arrows) is damaged. Bottom, Figure 2. A, proliferation of human limbal epithelium from the corneal button. The cells migrate from the limbus onto the culture dish (arrow). B, cultured limbal epithelium (second passage). The cells proliferated and reached confluence 14 days after the passage.
face inflammation was severe, goblet cells generally were not observed. In addition to the epithelial abnormalities, eight patients had opaque corneas, five were aphakic, three had cataracts, and three had glaucoma. All of the aphakic patients had some complications, such as ex tensive anterior synechiae, vitreous loss, or iris neo vascularization. Visual acuities were less than hand motions (5 patients), counting fingers (l patient), and 20/200 to 20/600 (3 patients). All patients had poor vision and were considered inappropriate candidates for corneal transplantation at other institutions. Of the fellow eyes, three patients had null vision (cases 6, 8, and 9) and three had visual acuity of less than 0.04
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(cases l, 4, and 5), whereas only three had functioning fellow eyes (cases 2, 3, and 7). At the time ofthe surgery, six of the patients had been considered socially blind.
Ocular Surface Evaluation of the Patients Preoperatively, three aspects of the ocular surface were evaluated: ( l) condition of the corneal epithelium, (2) condition of the conjunctival epithelium, and (3) condi tion of the tear dynamics. The corneal epithelium was abnormal in all patients. Seven patients did not have in flamed conjunctivae, but the patients with OCP had chronic inflammation and symblephara (cases 2 and 5).
Tsubota et al · Limbal Allograft Transplantation Table 1. Patient Profiles Fellow Eye (visual acuity, diagnosis)
Case No.
Age (yrs)/ Sex
Diagnosis
Visual Acuity
Complications
2 3 4 5 6 7 8 9
71/ M 90/F 54/M 34/ M 62/ F 69/ F 79/F 77/M 73/ M
Alkali burn OCP Alkali burn Injury OCP LD LD LD Acid burn
20/ 1000 HM HM HM LP HM 20/2000 20/600 HM
Aphakia, BK BK Cataract, BK Aphakia, leukoma, glaucoma Cataract, leukoma Aphakia, BK, glaucoma Aphakia, BK Aphakia, BK, glaucoma Cataract
20/ 40, alkali burn HM,OCP 20/500, alkali burn 20/ 15, normal 20/15, normal Null, phthisis bulbi 20/1000, LD +aphakia Null, phthisis bulbi Null, enucleated
BK = bullous keratopathy; OCP = ocular pemphigoid; HM = hand motions; LP = light perception; LD = limbal deficiency of unknown etiology.
For the evaluation of tear dynamics, we combined the Schirmer test with anesthesia and the tear clearance test (Table 2). The tear function index (TFI) reflects both pro duction and drainage, and is the product result of the Schirmer and tear clearance tests. When the TFI was less than 34, tear function was considered as bad as in Sjogren syndrome. When the TFI was less than 96, tear function was considered to be moderate, as in non-Sjogren dry eye. Tear function was considered to be good when the TFI was more than 97. 18 After surgery, patients were evaluated for visual acuity and ocular surface conditions, especially of the corneal epithelium. Vital staining by fluorescein and rose bengal were examined carefully by slit lamp. Specular microscopy with the specular microscopic lens 19 also was performed in cases I, 5, 7, and 9. The mean follow-up period was 12.3 months.
Surgical Procedures The vascularized conjunctiva around the patient's limbus was removed to expose the bare limbus (Fig 3A). When necessary, PK, extracapsular cataract extraction, anterior segment reconstruction, and IOL implantation were per formed (Table 3). After anterior segment surgeries, limbal transplantation was performed. The central donor cornea was trephined with the ap propriate diameter for PK. The remaining peripheral cor nea with scleral rim then was cut into two pieces (Fig 3B). First, the scleral portion was removed from the rim with micro-scissors while the conjunctival portion was pre served as much as possible (Fig 3C). The stroma of the partially cut rim was removed from the epithelial portion with scissors {Fig 3D), so that the limbal tissue consisted of only Bowman layer, a small amount of stromal tissue, and corneal and limbal epithelium. These two pieces were placed on the recipient limbus with 10-0 nylon sutures (Figs 3E and 3F). Subconjunctival injections of genta micin (I 0 mg) and dexamethasone (2 mg) were given at the end of surgery. The ocular surface then was covered
with medical-use soft contact lenses (Breath-0, Toray, Tokyo, Japan) until the slit-lamp examination the follow ing day.
Preoperative and Postoperative Management The patients started oral cyclosporine ( lO mg/kg) 2 days before the surgery and maintained a serum level of 100 to 150 ng/ml for I month after surgery; the dosage then tapered. Intravenous dexamethasone (8 mg) was given for the first 3 days and tapered within I2 days. Eye drops included ofloxacin (Tarivit) (5 times daily), 0.1 % dexamethasone (5 times daily), betamethasone (5 times daily), and 0.05% cyclosporine diluted in 4% alpha-cyclod extran (Nacalai Tesque) (5 times daily). The cyclosporine drops were made in our laboratory using cyclosporine pow der.20 The topical antibiotics and dexamethasone eyedrops were tapered because ofthe possibility ofelevated intraocular pressure, but otherwise were continued. In contrast, the cy closporine drops always were used continuously. Table
2. The Tear Function in Each Patient
Case No.
Schirmer 1* (mm)
Clearance Test
TFI
Interpretation of Tear Function
1 2 3 4 5 6
8 8 5 34 7 35 5
1X 1X 4X 16X 8X 4X 8X 8X 8x
8 8 20 544 56 140 40 104 152
Poor Poor Poor Good Moderate Good Moderate Good Good
7
8 9
TFI
13
19
=
tear function index.
* Schirmer test with topical anesthesia.
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Figure 3. The surgical proce dure of limbal allograft trans plantation. A, the recipient vascularized limbal tissue was removed by microscissors. The separation of the limbal tissue was from the sclera. B, the cor neal rim was cut into two pieces after removal of the central clear corneal button for pene trating keratoplasty. C, the limbal epithelium was removed carefully ftom the sclera. D, the stromal portion of the corneal rim was removed from the complex of Bowman layer and limbal epithelium. E and F, the limbal allografts were placed at the 12- and 6-o'clock positions by 10-0 nylon interrupted su tures.
Results Confirmation of the Viability of Limbal Cells in Donor Cornea Results of the preliminary histologic examination showed limbal epithelium in all 35 patients, whereas the corneal epithelium was damaged in 27 (Fig 1). In all cultures (n = 35), epithelial proliferation was confirmed (Figs 2A and
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2B) by the epithelial cell growth from the limbal tissue during the culturing and isolated to culture the cell for further generations. The limbal tissue which was cultured in the medium for 5, 15, and 25 days had the cell proliferation in the basement of limbal epithelium, as identified by prolifer ation cell nuclear antigen staining (Figs 4A and 4B). At day 0 ofculturing, the basement of the some limbal tissue had no proliferation (n = 2) or a few proliferating cells (n
T subota et al · Limbal Allograft Transplantation Table 3. Initial Surgical Procedures Surgical Procedures
Case No.
1 2 3
4 5 6
7
8 9
LT LT LT LT LT LT LT LT LT
+ PK + ECCE + IOL + + + + + + +
(PK + ECCE + IOL)* PK + IOL + vitrectomy PK +ECCE+ IOL PK + vitrectomy PK + IOL + vitrectomy PK + ECCE + vitrectomy PK +ECCE+ IOL
LT = limbal transplantation; PK = penetrating keratoplasty; ECCE = extracapsular cataract extraction; IOL = intraocular lens, insertion of posterior chamber IOL or anterior chamber IOL. • In case 3, PK
+ ECCE + IOL was performed in the later stage.
= 28) (Table 4), whereas over 15 days of culturing, the number of proliferating cells increased in all of the limbal epithelium (n = 35) and the site of proliferation extended (n = 29) or moved (n = 6) toward the center of cornea.
Surgical Results of Limbal Transplantation Results of slit-lamp examination showed corneal epithe lium without vessel invasion in all patients for the average observation period of 12.3 months. Rose-bengal and flu orescein staining showed no epithelial defects or significant hyperpermeability, although keratitis occasionally was observed, which could be treated successfully by frequent use of artificial tears. When subtle staining of the corneal epithelium was detected, we took particular care of the ocular surface, including the frequent use of artificial tears and wearing of protective glasses. 21 •22 Despite these mea sures, partial conjunctival invasion into cornea occurred in cases 1 and 5, requiring a second limbal transplantation to reconstruct the corneal epithelia. The other two patients (2 and 3) had fair corneal epithelia postoperatively, which were much better than preoperatively, although somewhat irregular profiles still were seen when observed by pho tokeratoscope. In two patients (7 and 9), endothelial-type rejection developed that required systemic steroids and cyclosporine; both were controlled successfully, with stable corneal epithelia. The other three patients had completely clear and stable corneal epithelia (Table 5). Corneal epi thelial evaluation by specular microscopy showed large epithelial cells with occasional elongated or extra-large cells, showing cellular-level abnormalities after limbal al lograft transplantation. The successful five patients had good tear function, with a TFI of more than 97, except case 7 (TFI of 40), whereas the other compromised patients had worse tear function with a TFI of 8 in two, 16 in one, and 56 in one (Tables 3 and 4). Visual acuity improved in all nine patients. Even though the limbal transplantation provided a stable ocular
surface, the final vision did not return to normal in many patients. The reasons include high and irregular astig matism, whereas some patients had apparent reasons such as glaucoma (cases 4, 6, and 8) and macular degeneration (case 7). In other patients, the apparent reason was not found. However, the initial visual acuity was impaired that all patients were satisfied with the results, including the five blind patients who realized functional vision and were able to manage their activities of daily living after surgery.
Case Reports Case 3. A 54-year-old man had an alkali burn 2 years before
presenting to our hospital. The right cornea was totally conjunctivalized (Figs SA and 5B) with vessel invasion and recurrent epithelial breakdown causing recurrent pain. Visual acuity was hand motions. The Schirmer test with anesthesia was 5 mm, and the tear clearance was X4, with a TFI of 20. After a limbal transplantation on March 18, 1992, the corneal epithelium improved, but the corneal thickness did not return to normal and there was stromal opacity and cataract formation. A PK combined with extracapsular cataract extraction and posterior chamber intraocular lens insertion was performed on April 15, 1993. The corneal epithelium recovered, and visual acuity improved to 20/600. No goblet cells were detected on the cornea by impression cytology, although slight vessel invasion was observed at the 1-o'clock position. The epithelium was stable for 20 months (Figs 5C and 5D) and considered fair due to the slight irregularity and vessel invasion. Specular microscopy showed the elongated epithelial cells at 1 and 6 months after the surgery (Figs 5E and SF), suggesting a lack of epithelial proliferation on the central cornea and accelerated supply from the periphery. The intraocular pressure was well controlled, although visual acuity remained at 20/600 for unknown reasons. Case 5. A 62-year-old woman had had unilateral corneal vascularization and symblepharon in the left eye for more than 10 years. The patient had been using idoxuridine for presumed herpes keratitis for more than 2 years before the onset of the above complications. We diagnosed unilateral OCP caused by topical use of idoxuridine. The cornea totally was keratinized, with an inflamed conjunctiva. Visual acuity was light perception. Although the cornea and conjunctiva were severely dry and damaged (Fig 6A), the Schirmer test with anesthesia yielded 7 mm, with a tear clearance of X8 and a TFI of 56. A limbal transplant was performed using a triple procedure (Fig 6B). Visual acuity recovered to 20/100 to 20/50, and the ocular surface was stable for 6 months (Fig 6C), after which an inferior symblepharon developed with persistent epithelial break (Fig 6D). The symblepharon then was removed and a new limbal graft was transplanted only in the inferior portion. After the second surgery, the corneal epithelium was stable through 3 months of follow-up (Fig 6E). Specular microcopy showed extra large epithelial cells, suggesting cellular level abnormalities. Case 9. A 73-year-old man had had very poor vision due to a severe acid burn more than I 0 years earlier. The fellow eye was lost due to perforating trauma. The cornea was vascularized and opaque (Fig 7A), with visual acuity of hand motions. Impression cytology showed goblet cells on the corneal epithe lium (Fig 7B). The conjunctiva looked normal, and tear function was good, with a TFI of 152. Limbal transplantation was per formed using a triple procedure. Visual acuity recovered to 20/ 100 the day after surgery and improved to 20/60 1 month after
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Top, Figure 4. Proliferation cell nuclear antigen staining of the cultured limbal epithelium. A, white arrow shows limbal portion of cornea. A few basal cells of limbal epithelium (double arrow) were stained with proliferation cell nuclear antigen after being cultured for 5 days. B, the basal portion of the limbal epi thelium which was cultured for 15 days. The region of the proliferating cells spread toward the center part of cornea. Second row through bottom row, Fig ure 5. Case 3. A, the preoperative cor· neal condition (alkali burn). The corneal epithelium was totally covered by the conjunctival epithelium with extensive vessel invasion. B, the preoperative photokeratoscopic view of the cornea. The corneal surface was very irregular. C, the postoperative corneal condition 20 months after the limbal transplant. The corneal epithelium was stable with slight vessel invasion at the 1-o'clock position. D, the postoperative photo keratoscopic view of the cornea 20 months after the limbal transplant. Al though there was some residual astig matism, the corneal epithelial irregularity was improved. E, specular microscopic view of corneal epithelium 1 month after surgery. There are elongated cells. F, specular microscopic view of corneal epithelium 6 months after surgery. The epithelial cells were irregularly confi gured, with elongated cells.
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T subota et al · Limbal Allograft Transplantation Table
No. of Limbal Tissues Examined
=
Results of Cultured Limbal Tissue Viability
Intact Central Cornea Epithelium
Intact Limbal Cornea Epithelium
No.(%)
No.(%)
8 (22.9)
35 (100)
35 PCNA
4.
PCNA Staining* of Limbal Epithelium at 0 Day
2
5 Days
+
++
28
5
0
15 Days
+
++
21
14
0
+
++
0
35
25 Days
0
+
++
0
35
proliferating cell nucleus antigen.
•- = no stained cells; + = small portion of stained cells; ++ = large portion of stained cells.
surgery (Fig 7C). The cornea has been stable for more than 3 months, without any vital staining or goblet cells. Specular mi croscopy showed regular configuration with no elongated or ab normally large cells (Fig 70). 23 ·24 Although the patient had the typical endothelial-type rejection 3 months after surgery, it was successfully controlled by medication.
Discussion We performed nine limbal allograft transplants to there constructed corneal epithelia affected by severe ocular surface disorders. The epithelia have been clear and stable for more than 1 year postoperatively in five of nine pa tients. These patients had good tear function, which may be critical for success in such cases. In two patients, partial improvement was obtained and in the other two patients, a second surgery was necessary but the patients still main tained a clinically healthy corneal epithelium. Although the specular microscope did not show an entirely normal configuration and there may still be need for extensive care for the epithelium, the clinical improvement was dramatic. In no patients were massive vessel invasion or persistent epithelial defects observed. Although ordinary PK can provide clear corneal graft for 4 or more months after surgery, the clarity is deteriorating slowly. Because we have observed these patients for more than 1 year,
Table
limbal allograft transplantation maybe a good surgical method for reconstruction. The overall success rate of corneal transplant has im proved over the last decade, which is due mostly to the introduction of viscous material, the development of modern surgical techniques, the quality control of donor corneas by modern eye banks, and the appropriate use of immunosuppression. However, patients with conditions such as corneal neovascularization, persistent epithelial defect, persistent conjunctival inflammation, and con junctival transdifferentiation of the corneal epithelium tend to have poor transplant outcomes. The patients on whom we performed limbal transplantation had very se vere ocular surface abnormalities, which were not amen able to simple PK. Our patients had not undergone eye surgery for many years, and six of the nine patients were considered legally blind or handicapped because they also had poor vision in their fellow eyes. Because the trans planted corneal epithelium cannot survive very long, 25 •26 PK alone cannot provide a long-lasting epithelium. For alkali burn and other diseases, keratoepithelioplasty, in which the corneal epithelium and Bowman layer are transplanted at the periphery of the recipient cornea, has been performed successfully by Kaufman, 1 Thoft, 3 and Kinoshita et al. 5 This procedure avoids the limbal area of the donor cornea, which includes various types ofantigen presenting cells such as Langerhans cells, 13 thereby ob-
5. Surgical Results Visual Acuity
Case No.
2 3 4 5 6 7
8 9
Corneal Epithelium
Results
Final
Best
Stable after second surgery Partial irregularity Partial irregularity Clear and stable Stable after second surgery Clear and stable Clear and stable Clear and stable Clear and stable
Re-LT Fair Fair Good Re-LT Good Good/Re Good Good/Re
20/200 20/300 20/600 20/50 20/100 20/1000 20/200 20/200 20/60
20/50 20/100 20/100 20/50 20/50 20/1000 20/200 20/200 20/60
Follow-up (mos)
7 7
11 13
15 16 17 5 2
Re = endothelial rejection episode; LT = limbal transplantation.
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Top row through third row, Figure 6. Case 5. A, preoperative corneal condition. The cor nea was very irregular with vessel invasion. The central cornea was keratinized and skin-like. B, lptraoperative condition of the cornea. The thick epithelium was removed, and penetrating keratoplasty was performed with limbal trans plantation. C, the cornea 5 months after surgery. The cornea was clear and stable, and the epi thelium was regular. D, the symblepharon started inferiorly with persistent epithelial break 6 months after surgery. E, the cornea was stable for 5 months after the second surgery. F, the specular microscopic view of the cornea 5 months after the second surgery. The cells are large, although no elongated ones are observed. Fourth row and bottom row, Figure 7. Case 9. A, preoperative condition of cornea. The cornea was covered completely by conjuncti valized epithelium with vessel invasion. B, impression cytology of the preoperative cornea. By periodic acid-Schiff staining, the goblet cells (red-stained) were observed clearly. C, postop erative cornea (1 month after the surgery). The corneal epithelium was regular and stable. D, the specular microscopic view of the corneal epithelium 3 months after surgery. The pattern of the epithelium was regular, and no elongated cells or extra-large cells were observed.
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T subota et al · Limbal Allograft Transplantation v1atmg strong immunosuppression and achieving the same level of potential rejection as simple PK. A disad vantage of keratoepithelioplasty is that it requires a very fresh cornea with a good epithelium, which may be dif ficult to obtain in the routine eye-bank system. In addition, the procedure requires two donor corneas when it is com bined with PK because it requires the central corneal ep ithelium. Last, stem cells may not be present in the central corneal epithelium/· 27 which eventually may reduce the number of phenotypic corneal cells. There has been considerable discussion about the pres ence and location of corneal epithelial stem cells. Most recent studies have shown that the stem cells are located primarily in the limbal area, 5•7•9•27 •28 although some reports suggest a wider distribution in the peripheral cornea. 29 According to the cornea stem cell theory, destruction of the limbal epithelium results in corneal epithelial break down followed by conjunctival-epithelial invasion of the cornea, which has been confirmed in a rabbit model. 9 This suggests that if an alkali burn destroyed the whole limbal area, the cornea would break down severely, whereas a partial loss of the limbal area would result in only slight epithelial abnormalities. Mild cases of OCP and Stevens-Johnson syndrome also result in the con junctival epithelialization of the cornea. In our patients with bullous keratopathy, limbal deficiency with unknown etiology also occurred, and the corneal epithelium had severe breakdown and vessel invasion. The limbal dys function may be related to the recurrent epithelial erosion, which requires stem-cell proliferation for proper wound healing. Accelerated mitosis in the corneal limbal stem cells may result in dysfunction of the corneal stem cells in the long term, although this requires further study. Conjunctivization was confirmed in some patients by the presence of goblet cells on impression cytology. 30 In those patients, the need for stem cell transplantation is comparable to that of bone marrow transplantation for patients with aplastic anemia. The first trials of human corneal stem cell transplant were performed by Kenyon et al 14• 15 through limbal autograft transplantation. They transplanted the limbal area, presumably including stem cells, from the good fellow eye to the recipient eye. The results were promising, with the re-establishment of the corneal epithelium after limbal autograft transplantation. We have applied this concept for the reconstruction of severe corneal surface disorders as first reported by Pfister 12 and Tsai and Tseng. 16 Although partial removal of the limbus from the fellow eye was shown by Kenyon et a! to be safe, many patients are reluctant to interfere with the good eye. Moreover, some of the patients who have had a chemical injury were affected bilaterally, so that limbal autograft transplantation was impossible. Re sults of histopathologic examination showed that the lim hal cells were viable deep in the epithelium, whereas the more superficial cells usually were damaged during pres ervation in the medium. The corneas were stored in chondroitin-supplemented preservation medium for an average of 5 days. Although it has been noted that this medium is not ideal for epithelial preservation, 31 •32 limbal cells were viable. The cell culture using the limbal area
showed strong epithelial cell proliferation, which sup ported the histologic observation that preservation of the limbal cells was possible. The greatest disadvantage of limbal allograft trans plantation is the increased risk of rejection because the limbal area contains antigen-presenting cells. We observed typical endothelial-type rejection in two of nine patients, despite the strong immunosuppression. In our series, we used high doses of intravenous steroids for the first 8 days after surgery, oral cyclosporine 2 days before and 1 month after surgery, and long-term cyclosporine and dexameth asone eye drops. 20 This immunosuppression was much stronger than for the usual PK. Although the mechanism of cyclosporine is not totally understood, it suppresses interleukin-2 and various types of cytotoxic compounds such as granzyme A and perforin from activated T cells and has been indispensable for kidney, liver, bone marrow, and other transplants. 33- 36 Although rejection is not sup pressed completely, we believe that the combination of oral and topical cyclosporine is critical to our successful limbal transplants. Although limbal transplantation is probably successful because of the transplantation of stem cells, other factors may play roles. The procedure provides a healthy Bowman layer at the periphery of the recipient cornea which may serve as a good substrate. It has been reported that different substrates can modify cellular expression, 37 and the pres ence of a healthy Bowman layer may transdifferentiate the recipient conjunctival epithelium to a corneal phe notype. Another factor may be the suppression of periph eral neovascularization by the corneal stroma. The cornea itself is a known suppressor of new vessels. These three mechanisms may work together for the re-establishment of a normal corneal epithelium. In our surgical technique, we remove most of the stromal tissue from the limbal tissue, so that only the corneal epithelium and Bowman layer with a small por tion of stroma are transplanted. This procedure reduces the amount of transplanted antigen-presenting cells be cause the peripheral stroma includes many active cells. The duration of storage also may have some effect on the antigen-presenting cells. The use oflimbal tissue from the same donor as the PK tissue can save a donor cornea using only one donor eye for the combined procedure of PK and limbal transplantation. Limbal transplantation, including cutting the limbal tissue into two parts, remov ing the stromal tissue, and suturing with two or three 10 0 nylon interrupted sutures takes only 5 to 10 minutes. We currently are using limbal transplantation for severe corneal epithelial abnormalities without limbal cells. Be cause the technique is easy and rejection can be controlled using immunosuppressants, the procedure may have a wider applicability. Other ocular surface disorders such as partial limbal deficiency, gelatinous drop-like dystro phy, and persistent epithelial defect can be good indica tions for limbal transplantation. Acknowledgments. The authors thank Scheffer Tseng, MD, PhD, Kenneth R. Kenyon, MD, Ray Tsai, MD, and An drew Huang, MD, for their initiation of this procedure and their discussions concerning limbal transplantation. They also thank
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Hsu Ke-Ping, MD, Yukiko Yagi, ORT, Tokuaki Sugawara, OMA, and Saori Nishijima, ORT, for the ocular surface ex aminations.
References I. Kaufman HE. Keratoepithelioplasty for the replacement of damaged corneal epithelium [editorial]. Am J Ophthalmol 1984;97: 100-1. 2. Kinoshita S, Ohashi Y, Ohji M, Manabe R. Long-term re sults of keratoepithelioplasty in Mooren's ulcer. Ophthal mology 1991 ;98:438-45. 3. Thoft RA. Keratoepithelioplasty. Am J Ophthalmol 1984;97: 1-6. 4. Turgeon PW, Nauheim RC, Roat MI, et al. Indications for keratoepithelioplasty. Arch Ophthalmol 1990; 108:233-6. 5. Kinoshita S, Kiorpes T, Friend J, Thoft RA. Limbal epi thelium in ocular surface wound healing. Invest Ophthalmol Vis Sci 1982;23:73-80. 6. Davanger M, Evensen A. Role of the peripapillary structure in renewal of corneal epithelium. Nature 1970;229:560-1. 7. Tseng SCG. Concept and application of limbal stem cells. Eye 1989;3:141-57. 8. Wei ZG, Wu RM, Lavker R, Sun IT. In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral con junctival epithelia. Implications on conjunctival epithelial transdifferentiation and stem cells. Invest Ophthalmol Vis Sci 1993;34:1814-28. 9. Huang A, Tseng SC. Corneal epithelial wound healing in the absence of limbal epithelium . Invest Ophthalmol Vis Sci 1991 ;32:96-1 05. 10. Tseng SC, Tsai RJ. Limbal transplantation for ocular surface reconstruction: a review. Fortschr Ophthalmol 1991 ;88:236 42. I I . Tsai RJ, Sun TT, Tseng SC. Comparison of limbal and conjunctival autograft transplantation in corneal surface re construction in rabbits. Ophthalmology 1990;97:446-55. 12. Pfister R. Corneal stem cell disease: concepts, categorization, and treatment by auto- and homotransplantation oflimbal stem cells. CLAO J 1994;20:64-72. 13. Williams KA, Coster DJ. The role of the limbus in corneal allograft rejection. Eye 1989;3: 158-66. 14. Kenyon KR, Tseng SC. Limbal autograft transplantation for ocular surface disorders. Ophthalmology 1989;96:709 22. 15. Kenyon KR. Limbal autograft transplantation for chemical and thermal burns. Dev Ophthalmol 1989; 18:53-8. 16. Tsai R, Tseng SC. Human allograft limbal transplantation for corneal surface reconstruction. Cornea. 1994; 13:389 400. 17. Bravo R, MacDonald-Bravo H. Existence oftwo populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Bioi I 987; I 05:1549-54. 18. Hsu K, Yagi Y, Toda I, Tsubota K. Tear function index: a new measure of dry eye. Arch Ophthalmol 1995;113:84 88.
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I 9. Tsubota K. A contact lens for specular microscopic obser vation. Am J Ophthalmol 1988;106:627-8. 20. Kanai A, Alba R, Takano T, eta!. The effect on the cornea of alpha cyclodextrin vehicle for cyclosporine eye drops. Transplantation Proceedings 1989;21 :3 I 50-2. 21. Tsubota K. The effect ofwearing spectacles on the humidity ofthe eye. Am J Ophthalmol 1989;108:92-3. 22. Tsubota K, Yamada M, Urayama K. Spectacle side panels and moist inserts for the treatment of dry eye patients. Cor nea 1994; 13:202-9. 23. Tsubota K, Yamada M, Naoi S. Specular microscopic ob servation of normal human corneal epithelium. Ophthal mology 1992;99:89-94. 24. Tsubota K, Yamada M, Naoi S. Specular microscopic ob servation of human corneal epithelial abnormalities. Oph thalmology 1991 ;98: 184-91. 25. Kinoshita S, Friend J, Thoft RA. Sex chromatin of donor corneal epithelium in rabbits. Invest Ophthalmol Vis Sci 1981;21:434-41. 26. Dahlman C. On the fate of the corneal graft. Acta Ophthal mol 1957;35:286-302. 27. Costarelis G, Cheng S, Dong G, Sun IT, Lavker R. Existence of slow-cycling limbal epithelial basal cells that can be pref erentially stimulated to proliferate: implications on epithelial stem cells. Cell 1989;57:201-9. 28. Greiner J, Covington H, Allansmith M. The human limbus. A scanning electron microscopic study. Arch Ophthalmo1 1979;97: 1159-65. 29. Lauweryns B, van den Oord J, de Vos R, Missotten L. A new epithelial cell type in the human cornea. Invest Ophthalmol Vis Sci 1993;4: 1983-90. 30. Nelson J, Havener V, Cameron J. Cellulose acetate impres sions of the ocular surface: dry eye state. Arch Ophthalmol 1982; 10 I: 1869-72. 31. Farge E, Fort R, Wilhe1mus K, et a!. Morphologic changes ofK-sol preserved human corneas. Cornea 1989;8:159-69. 32. Lindstrom R, Kaufman H, Skelnik D, Laing RA, Lass J, Musch D, et al. Optisol corneal storage medium. Am J Ophthalmol 1992; 114:345-56. 33. Liu C, Rafil S, Granelli-Piperno A, Trapani J, Young J. Perforin and serine esterase gene expression in stimulated human T cells. J Exp Med 1989;170:2105-18. 34. Lancki D, Kaper B, Fitch F. The requirements for triggering of lysis by cytolytic T lymphocyte clones II. Cyclosporine A inhibits TCR-mediated exocytosis but only selectively in hibits TCR-mediated lytic activity by cloned CTL. J lm munol 1989;142:416-24. 35. Lu P, Garcia-Sanz J, Lichtenheld M, Podack E. Perforin expression in human peripheral blood mononuclear cells. J Immunol 1992;148:3354-60. 36. Griffiths G, Alpert S, Lambert E, McGuire J, Weissman I. Perforin and granzyme A expression identifying cytolytic lymphocytes in rheumatoid arthritis. Proc Nat! Acad Sci USA 1992;89:549-53. 37. Tsai R, Tseng SC. Substrate modulation of cultured rabbit conjunctival epithelial cell differentiation and morphology. Invest Ophthalmol Vis Sci 1988;29: 1565-76.
Penetrating keratoplasty (PK) is an established surgical procedure for treating corneal opacity. However, for those patients with severe ocular surface abnormalities such as persistent epithelial defect, corneal vascularization, and epithelial irregularity, simple PK often is not successful. The causes of these conditions include chemical injury, contact lens-related epithelial defects, ocular pemphigoid Originally received: March 17, 1995. Revision accepted: June 8, 1995. 1 Department of Ophthalmology, Tok yo Dental College, Chiba. 2 Department of Ophthalmology, Keio University School of Medicine, Tokyo. 3 Department of Ophthalmology, Kyorin University School of Medicine, Tokyo. Reprint requests to Kazuo Tsubota, MD, Department of Ophthalmology, Tokyo Dental College, 11-13 Sugano 5 Chome, lchikawa-shi. Chiba, Japan 272.
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(OCP), and limbal deficiency of unknown etiology. To reconstruct a stable corneal epithelium, Thoft and Kinoshita'-4 developed the technique ofkeratoepithelio plasty, with good clinical results. The procedure involves the transplantation of corneal epithelium as well as the reconstruction of the substrate by implanting the donor Bowman layer in the recipient's lim bal position. After the procedure, the transplanted corneal epithelium covers the recipient cornea, providing a healthier and more stable epithelium. The corneal stem cell theory gives new insight into severe ocular surface abnormalities which suggests that corneal and conjunctival surfaces normally are covered by their respective epithelia, which are presumably sup plied by their own stem cells. s-? Corneal stem cells appear to concentrate at the limbus, 5- 7 while the conjunctival stem cells may be at the fornix .8 If the corneal stem cells are partially or totally depleted, the corneal surface sub
T subota et al · Limbal Allograft Transplantation sequently is covered by conjunctival epithelium. Since the limbal area is exposed directly to the environment and is influenced easily by such injuries as alkali burns, corneal stem cell deficiency can occur so that the corneal surface is covered by the proliferated conjunctival epi thelium. If the conjunctival stem cells also are depleted, which is rare because of the larger conjunctival area and the protected location (e.g., fornices) of the stem cells, the ocular surface eventually is covered by totally keratinized epithelium, which sometimes may be seen at the end stage of OCP or Stevens-Johnson syndrome. For patients with limbal cell deficiency, corneal stem cell transplant has been considered. 9 - 12 In rabbit models, several studies have established the efficacy of this pro cedure. 11 Because the limbal area includes many antigen presenting cells, such as Langerhans cells, the possibility of rejection might be higher than for regular PK. 13 To prevent rejection, limbal autograft transplantation first was tried clinically, and good surgical results were ob tained.14·15 However, there are some practical disadvan tages of this procedure. When patients have bilateral ocu lar surface disease such as chemical injury or OCP, au tograft transplantation is impossible. Patients' reluctance to harvest from the good eye also limits the applicability of this procedure. As such, limbal allograft transplantation becomes necessary in some cases. 12·16 In this study, we performed limbal allograft transplantation using eye-bank corneas stored for more than 4 days in preservation media, coupled with strong immunosuppression by systemic and topical cyclosporine. Although relatively few patients were treated, we were able to reconstruct a stable corneal epi thelium in all nine patients, with concomitant improve ment in visual acuity.
Materials and Methods Viability of Limbal Cells in Donor Cornea Because ordinary eye-bank storage media were developed to optimize preservation of the endothelium, rather than the epithelium, we were concerned with the storage con ditions of the limbal epithelium. We examined the corneal and limbal epithelia in eye-bank corneas (n = 35) stored for 5 days in Optisol GS medium (Chiron Ophthalmics, Irvine, CA). The age ofdonors ranged from 18 to 86 (mean ± standard deviation, 68.9 ± 12.1 years). The average storage time was 114.5 ± 40.2 hours. The basal part of the limbal epithelium always was intact (35/35), whereas the central corneal epithelium occasionally was missing or damaged (27/35) (Fig I). This was confirmed by ob serving the section of the tissue with a light microscope. To confirm the viability of the limbal epithelium of the donor cornea, part of the eye-bank limbal tissue was cultured in rabbit corneal epithelium growth medium (Kurabou, Inc, Tokyo, Japan) which included insulin (5 !Lg/ml), epidermal growth factor ( 10 ng/ml), hydrocorti sone (0.5 mg/ml), gentamicin (50 !lgfml) , amphotericin B (0.25 mg/ml), and 0.4% vol/vol bovine pituitary extract. Limbal tissue was cut into small pieces and cultured in
rabbit corneal epithelium growth medium in 35-mm plastic dishes and placed in a 5% carbon dioxide incubator for 20 to 30 days. The medium was changed every other day. When cell growth was identified by microscopic ob servation (Fig 2A), the limbal tissue was replaced in an other dish 12 days after the primary culture, whereas the cells in the first dish were treated with 5% trypsin (2 ml) for 30 seconds, and the cells were removed and placed in a 15-ml centrifugation tube after nutrizing with 2 ml nu trizing solution. Medium (2 ml) was added to the tube and placed in 35-mm plastic dishes, which were incubated in a 5% carbon dioxide incubator at 37°C for the sec ondary culture after centrifugated with 400g. When the cells reached 80% confluency (Fig 2B), the same procedure was repeated. During the culturing, the limbal tissue (5 X 3 mm) (n = 35) was placed in rabbit corneal epithelium growth me dium for 0, 5, 15, and 25 days, we then took a thin slice from each piece of tissue which then was fixed in Dent fixative (20% dimethyl sulfoxide and 80% methanol) for 2 hours at 4 oc. The tissue then was placed in 70% ethanol for I hour at 4 oc. After being washed three times in phosphate-buffered saline for 20 minutes, the tissue was placed in 50% block ace to prevent nonspecific binding of the antibody. We stained the limbal tissue with anti proliferation cell nuclear antigen monoclonal antibody to examine the mitotic activity of the limbal basal epithe lium. 17 Antiproliferation cell nuclear antigen I-J mono clonal antibody (clone PCIO) was diluted 100 times by 10% block ace-0.5% Tween80 as a primary antibody, and the tissue was placed in the primary antibody overnight at 4°C. The tissue then was washed thoroughly. Fluores cein isothiocynate-goat anti-mouse lgG, which was di luted 50 times with I 0% block ace-0.5 % Tween80, was used for the secondary antibody. The tissue was placed in the secondary antibody for 2 hours at room temperature and then washed. After slicing the tissue with a sharp blade in widths of0.1 to 0.2 mm, we mounted the sections on glass slides containing 90% glycerol in TRIS-buffered saline and observed the specimens by fluorescent mi croscopy.
Patients Nine patients (5 men, 4 women) (average age, 67.7 ± 16.2 years; range, 34-90 years of age) with severe ocular surface conditions were treated by limbal allograft transplantation. The diagnosis included three patients with chemical injury, three with oflimbal deficiency of unknown etiology, two with moderate OCP, and one with extensive mechanical injury to the limbal area (Table 1). The corneal epithelia had frequent defects and were compromised by conjunctivization with vessel invasion and increased fluorescein permeability and rose-bengal staining. The surface irregularities were prominent in all patients. Persistent epithelial defects were seen at the time of surgery in cases I, 2, 3, 5, and 9. Impression cytology showed transdifferentiation of the conjunctival epithelium in cases I, 3, 5, 8, and 9, with goblet cells in cases 8 and 9. When the ocular sur
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Top, Figure 1. The limbal and corneal epithelium of eye-bank cornea stored in Optisol GS for 5 days (65-year-old male donor; death to enucleation time, 5 hours; encleation to preservation time, 2 hours). Notice the limbal epithelium (arrow) is intact, whereas the corneal epithelium (double arrows) is damaged. Bottom, Figure 2. A, proliferation of human limbal epithelium from the corneal button. The cells migrate from the limbus onto the culture dish (arrow). B, cultured limbal epithelium (second passage). The cells proliferated and reached confluence 14 days after the passage.
face inflammation was severe, goblet cells generally were not observed. In addition to the epithelial abnormalities, eight patients had opaque corneas, five were aphakic, three had cataracts, and three had glaucoma. All of the aphakic patients had some complications, such as ex tensive anterior synechiae, vitreous loss, or iris neo vascularization. Visual acuities were less than hand motions (5 patients), counting fingers (l patient), and 20/200 to 20/600 (3 patients). All patients had poor vision and were considered inappropriate candidates for corneal transplantation at other institutions. Of the fellow eyes, three patients had null vision (cases 6, 8, and 9) and three had visual acuity of less than 0.04
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(cases l, 4, and 5), whereas only three had functioning fellow eyes (cases 2, 3, and 7). At the time ofthe surgery, six of the patients had been considered socially blind.
Ocular Surface Evaluation of the Patients Preoperatively, three aspects of the ocular surface were evaluated: ( l) condition of the corneal epithelium, (2) condition of the conjunctival epithelium, and (3) condi tion of the tear dynamics. The corneal epithelium was abnormal in all patients. Seven patients did not have in flamed conjunctivae, but the patients with OCP had chronic inflammation and symblephara (cases 2 and 5).
Tsubota et al · Limbal Allograft Transplantation Table 1. Patient Profiles Fellow Eye (visual acuity, diagnosis)
Case No.
Age (yrs)/ Sex
Diagnosis
Visual Acuity
Complications
2 3 4 5 6 7 8 9
71/ M 90/F 54/M 34/ M 62/ F 69/ F 79/F 77/M 73/ M
Alkali burn OCP Alkali burn Injury OCP LD LD LD Acid burn
20/ 1000 HM HM HM LP HM 20/2000 20/600 HM
Aphakia, BK BK Cataract, BK Aphakia, leukoma, glaucoma Cataract, leukoma Aphakia, BK, glaucoma Aphakia, BK Aphakia, BK, glaucoma Cataract
20/ 40, alkali burn HM,OCP 20/500, alkali burn 20/ 15, normal 20/15, normal Null, phthisis bulbi 20/1000, LD +aphakia Null, phthisis bulbi Null, enucleated
BK = bullous keratopathy; OCP = ocular pemphigoid; HM = hand motions; LP = light perception; LD = limbal deficiency of unknown etiology.
For the evaluation of tear dynamics, we combined the Schirmer test with anesthesia and the tear clearance test (Table 2). The tear function index (TFI) reflects both pro duction and drainage, and is the product result of the Schirmer and tear clearance tests. When the TFI was less than 34, tear function was considered as bad as in Sjogren syndrome. When the TFI was less than 96, tear function was considered to be moderate, as in non-Sjogren dry eye. Tear function was considered to be good when the TFI was more than 97. 18 After surgery, patients were evaluated for visual acuity and ocular surface conditions, especially of the corneal epithelium. Vital staining by fluorescein and rose bengal were examined carefully by slit lamp. Specular microscopy with the specular microscopic lens 19 also was performed in cases I, 5, 7, and 9. The mean follow-up period was 12.3 months.
Surgical Procedures The vascularized conjunctiva around the patient's limbus was removed to expose the bare limbus (Fig 3A). When necessary, PK, extracapsular cataract extraction, anterior segment reconstruction, and IOL implantation were per formed (Table 3). After anterior segment surgeries, limbal transplantation was performed. The central donor cornea was trephined with the ap propriate diameter for PK. The remaining peripheral cor nea with scleral rim then was cut into two pieces (Fig 3B). First, the scleral portion was removed from the rim with micro-scissors while the conjunctival portion was pre served as much as possible (Fig 3C). The stroma of the partially cut rim was removed from the epithelial portion with scissors {Fig 3D), so that the limbal tissue consisted of only Bowman layer, a small amount of stromal tissue, and corneal and limbal epithelium. These two pieces were placed on the recipient limbus with 10-0 nylon sutures (Figs 3E and 3F). Subconjunctival injections of genta micin (I 0 mg) and dexamethasone (2 mg) were given at the end of surgery. The ocular surface then was covered
with medical-use soft contact lenses (Breath-0, Toray, Tokyo, Japan) until the slit-lamp examination the follow ing day.
Preoperative and Postoperative Management The patients started oral cyclosporine ( lO mg/kg) 2 days before the surgery and maintained a serum level of 100 to 150 ng/ml for I month after surgery; the dosage then tapered. Intravenous dexamethasone (8 mg) was given for the first 3 days and tapered within I2 days. Eye drops included ofloxacin (Tarivit) (5 times daily), 0.1 % dexamethasone (5 times daily), betamethasone (5 times daily), and 0.05% cyclosporine diluted in 4% alpha-cyclod extran (Nacalai Tesque) (5 times daily). The cyclosporine drops were made in our laboratory using cyclosporine pow der.20 The topical antibiotics and dexamethasone eyedrops were tapered because ofthe possibility ofelevated intraocular pressure, but otherwise were continued. In contrast, the cy closporine drops always were used continuously. Table
2. The Tear Function in Each Patient
Case No.
Schirmer 1* (mm)
Clearance Test
TFI
Interpretation of Tear Function
1 2 3 4 5 6
8 8 5 34 7 35 5
1X 1X 4X 16X 8X 4X 8X 8X 8x
8 8 20 544 56 140 40 104 152
Poor Poor Poor Good Moderate Good Moderate Good Good
7
8 9
TFI
13
19
=
tear function index.
* Schirmer test with topical anesthesia.
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Figure 3. The surgical proce dure of limbal allograft trans plantation. A, the recipient vascularized limbal tissue was removed by microscissors. The separation of the limbal tissue was from the sclera. B, the cor neal rim was cut into two pieces after removal of the central clear corneal button for pene trating keratoplasty. C, the limbal epithelium was removed carefully ftom the sclera. D, the stromal portion of the corneal rim was removed from the complex of Bowman layer and limbal epithelium. E and F, the limbal allografts were placed at the 12- and 6-o'clock positions by 10-0 nylon interrupted su tures.
Results Confirmation of the Viability of Limbal Cells in Donor Cornea Results of the preliminary histologic examination showed limbal epithelium in all 35 patients, whereas the corneal epithelium was damaged in 27 (Fig 1). In all cultures (n = 35), epithelial proliferation was confirmed (Figs 2A and
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2B) by the epithelial cell growth from the limbal tissue during the culturing and isolated to culture the cell for further generations. The limbal tissue which was cultured in the medium for 5, 15, and 25 days had the cell proliferation in the basement of limbal epithelium, as identified by prolifer ation cell nuclear antigen staining (Figs 4A and 4B). At day 0 ofculturing, the basement of the some limbal tissue had no proliferation (n = 2) or a few proliferating cells (n
T subota et al · Limbal Allograft Transplantation Table 3. Initial Surgical Procedures Surgical Procedures
Case No.
1 2 3
4 5 6
7
8 9
LT LT LT LT LT LT LT LT LT
+ PK + ECCE + IOL + + + + + + +
(PK + ECCE + IOL)* PK + IOL + vitrectomy PK +ECCE+ IOL PK + vitrectomy PK + IOL + vitrectomy PK + ECCE + vitrectomy PK +ECCE+ IOL
LT = limbal transplantation; PK = penetrating keratoplasty; ECCE = extracapsular cataract extraction; IOL = intraocular lens, insertion of posterior chamber IOL or anterior chamber IOL. • In case 3, PK
+ ECCE + IOL was performed in the later stage.
= 28) (Table 4), whereas over 15 days of culturing, the number of proliferating cells increased in all of the limbal epithelium (n = 35) and the site of proliferation extended (n = 29) or moved (n = 6) toward the center of cornea.
Surgical Results of Limbal Transplantation Results of slit-lamp examination showed corneal epithe lium without vessel invasion in all patients for the average observation period of 12.3 months. Rose-bengal and flu orescein staining showed no epithelial defects or significant hyperpermeability, although keratitis occasionally was observed, which could be treated successfully by frequent use of artificial tears. When subtle staining of the corneal epithelium was detected, we took particular care of the ocular surface, including the frequent use of artificial tears and wearing of protective glasses. 21 •22 Despite these mea sures, partial conjunctival invasion into cornea occurred in cases 1 and 5, requiring a second limbal transplantation to reconstruct the corneal epithelia. The other two patients (2 and 3) had fair corneal epithelia postoperatively, which were much better than preoperatively, although somewhat irregular profiles still were seen when observed by pho tokeratoscope. In two patients (7 and 9), endothelial-type rejection developed that required systemic steroids and cyclosporine; both were controlled successfully, with stable corneal epithelia. The other three patients had completely clear and stable corneal epithelia (Table 5). Corneal epi thelial evaluation by specular microscopy showed large epithelial cells with occasional elongated or extra-large cells, showing cellular-level abnormalities after limbal al lograft transplantation. The successful five patients had good tear function, with a TFI of more than 97, except case 7 (TFI of 40), whereas the other compromised patients had worse tear function with a TFI of 8 in two, 16 in one, and 56 in one (Tables 3 and 4). Visual acuity improved in all nine patients. Even though the limbal transplantation provided a stable ocular
surface, the final vision did not return to normal in many patients. The reasons include high and irregular astig matism, whereas some patients had apparent reasons such as glaucoma (cases 4, 6, and 8) and macular degeneration (case 7). In other patients, the apparent reason was not found. However, the initial visual acuity was impaired that all patients were satisfied with the results, including the five blind patients who realized functional vision and were able to manage their activities of daily living after surgery.
Case Reports Case 3. A 54-year-old man had an alkali burn 2 years before
presenting to our hospital. The right cornea was totally conjunctivalized (Figs SA and 5B) with vessel invasion and recurrent epithelial breakdown causing recurrent pain. Visual acuity was hand motions. The Schirmer test with anesthesia was 5 mm, and the tear clearance was X4, with a TFI of 20. After a limbal transplantation on March 18, 1992, the corneal epithelium improved, but the corneal thickness did not return to normal and there was stromal opacity and cataract formation. A PK combined with extracapsular cataract extraction and posterior chamber intraocular lens insertion was performed on April 15, 1993. The corneal epithelium recovered, and visual acuity improved to 20/600. No goblet cells were detected on the cornea by impression cytology, although slight vessel invasion was observed at the 1-o'clock position. The epithelium was stable for 20 months (Figs 5C and 5D) and considered fair due to the slight irregularity and vessel invasion. Specular microscopy showed the elongated epithelial cells at 1 and 6 months after the surgery (Figs 5E and SF), suggesting a lack of epithelial proliferation on the central cornea and accelerated supply from the periphery. The intraocular pressure was well controlled, although visual acuity remained at 20/600 for unknown reasons. Case 5. A 62-year-old woman had had unilateral corneal vascularization and symblepharon in the left eye for more than 10 years. The patient had been using idoxuridine for presumed herpes keratitis for more than 2 years before the onset of the above complications. We diagnosed unilateral OCP caused by topical use of idoxuridine. The cornea totally was keratinized, with an inflamed conjunctiva. Visual acuity was light perception. Although the cornea and conjunctiva were severely dry and damaged (Fig 6A), the Schirmer test with anesthesia yielded 7 mm, with a tear clearance of X8 and a TFI of 56. A limbal transplant was performed using a triple procedure (Fig 6B). Visual acuity recovered to 20/100 to 20/50, and the ocular surface was stable for 6 months (Fig 6C), after which an inferior symblepharon developed with persistent epithelial break (Fig 6D). The symblepharon then was removed and a new limbal graft was transplanted only in the inferior portion. After the second surgery, the corneal epithelium was stable through 3 months of follow-up (Fig 6E). Specular microcopy showed extra large epithelial cells, suggesting cellular level abnormalities. Case 9. A 73-year-old man had had very poor vision due to a severe acid burn more than I 0 years earlier. The fellow eye was lost due to perforating trauma. The cornea was vascularized and opaque (Fig 7A), with visual acuity of hand motions. Impression cytology showed goblet cells on the corneal epithe lium (Fig 7B). The conjunctiva looked normal, and tear function was good, with a TFI of 152. Limbal transplantation was per formed using a triple procedure. Visual acuity recovered to 20/ 100 the day after surgery and improved to 20/60 1 month after
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Top, Figure 4. Proliferation cell nuclear antigen staining of the cultured limbal epithelium. A, white arrow shows limbal portion of cornea. A few basal cells of limbal epithelium (double arrow) were stained with proliferation cell nuclear antigen after being cultured for 5 days. B, the basal portion of the limbal epi thelium which was cultured for 15 days. The region of the proliferating cells spread toward the center part of cornea. Second row through bottom row, Fig ure 5. Case 3. A, the preoperative cor· neal condition (alkali burn). The corneal epithelium was totally covered by the conjunctival epithelium with extensive vessel invasion. B, the preoperative photokeratoscopic view of the cornea. The corneal surface was very irregular. C, the postoperative corneal condition 20 months after the limbal transplant. The corneal epithelium was stable with slight vessel invasion at the 1-o'clock position. D, the postoperative photo keratoscopic view of the cornea 20 months after the limbal transplant. Al though there was some residual astig matism, the corneal epithelial irregularity was improved. E, specular microscopic view of corneal epithelium 1 month after surgery. There are elongated cells. F, specular microscopic view of corneal epithelium 6 months after surgery. The epithelial cells were irregularly confi gured, with elongated cells.
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T subota et al · Limbal Allograft Transplantation Table
No. of Limbal Tissues Examined
=
Results of Cultured Limbal Tissue Viability
Intact Central Cornea Epithelium
Intact Limbal Cornea Epithelium
No.(%)
No.(%)
8 (22.9)
35 (100)
35 PCNA
4.
PCNA Staining* of Limbal Epithelium at 0 Day
2
5 Days
+
++
28
5
0
15 Days
+
++
21
14
0
+
++
0
35
25 Days
0
+
++
0
35
proliferating cell nucleus antigen.
•- = no stained cells; + = small portion of stained cells; ++ = large portion of stained cells.
surgery (Fig 7C). The cornea has been stable for more than 3 months, without any vital staining or goblet cells. Specular mi croscopy showed regular configuration with no elongated or ab normally large cells (Fig 70). 23 ·24 Although the patient had the typical endothelial-type rejection 3 months after surgery, it was successfully controlled by medication.
Discussion We performed nine limbal allograft transplants to there constructed corneal epithelia affected by severe ocular surface disorders. The epithelia have been clear and stable for more than 1 year postoperatively in five of nine pa tients. These patients had good tear function, which may be critical for success in such cases. In two patients, partial improvement was obtained and in the other two patients, a second surgery was necessary but the patients still main tained a clinically healthy corneal epithelium. Although the specular microscope did not show an entirely normal configuration and there may still be need for extensive care for the epithelium, the clinical improvement was dramatic. In no patients were massive vessel invasion or persistent epithelial defects observed. Although ordinary PK can provide clear corneal graft for 4 or more months after surgery, the clarity is deteriorating slowly. Because we have observed these patients for more than 1 year,
Table
limbal allograft transplantation maybe a good surgical method for reconstruction. The overall success rate of corneal transplant has im proved over the last decade, which is due mostly to the introduction of viscous material, the development of modern surgical techniques, the quality control of donor corneas by modern eye banks, and the appropriate use of immunosuppression. However, patients with conditions such as corneal neovascularization, persistent epithelial defect, persistent conjunctival inflammation, and con junctival transdifferentiation of the corneal epithelium tend to have poor transplant outcomes. The patients on whom we performed limbal transplantation had very se vere ocular surface abnormalities, which were not amen able to simple PK. Our patients had not undergone eye surgery for many years, and six of the nine patients were considered legally blind or handicapped because they also had poor vision in their fellow eyes. Because the trans planted corneal epithelium cannot survive very long, 25 •26 PK alone cannot provide a long-lasting epithelium. For alkali burn and other diseases, keratoepithelioplasty, in which the corneal epithelium and Bowman layer are transplanted at the periphery of the recipient cornea, has been performed successfully by Kaufman, 1 Thoft, 3 and Kinoshita et al. 5 This procedure avoids the limbal area of the donor cornea, which includes various types ofantigen presenting cells such as Langerhans cells, 13 thereby ob-
5. Surgical Results Visual Acuity
Case No.
2 3 4 5 6 7
8 9
Corneal Epithelium
Results
Final
Best
Stable after second surgery Partial irregularity Partial irregularity Clear and stable Stable after second surgery Clear and stable Clear and stable Clear and stable Clear and stable
Re-LT Fair Fair Good Re-LT Good Good/Re Good Good/Re
20/200 20/300 20/600 20/50 20/100 20/1000 20/200 20/200 20/60
20/50 20/100 20/100 20/50 20/50 20/1000 20/200 20/200 20/60
Follow-up (mos)
7 7
11 13
15 16 17 5 2
Re = endothelial rejection episode; LT = limbal transplantation.
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Top row through third row, Figure 6. Case 5. A, preoperative corneal condition. The cor nea was very irregular with vessel invasion. The central cornea was keratinized and skin-like. B, lptraoperative condition of the cornea. The thick epithelium was removed, and penetrating keratoplasty was performed with limbal trans plantation. C, the cornea 5 months after surgery. The cornea was clear and stable, and the epi thelium was regular. D, the symblepharon started inferiorly with persistent epithelial break 6 months after surgery. E, the cornea was stable for 5 months after the second surgery. F, the specular microscopic view of the cornea 5 months after the second surgery. The cells are large, although no elongated ones are observed. Fourth row and bottom row, Figure 7. Case 9. A, preoperative condition of cornea. The cornea was covered completely by conjuncti valized epithelium with vessel invasion. B, impression cytology of the preoperative cornea. By periodic acid-Schiff staining, the goblet cells (red-stained) were observed clearly. C, postop erative cornea (1 month after the surgery). The corneal epithelium was regular and stable. D, the specular microscopic view of the corneal epithelium 3 months after surgery. The pattern of the epithelium was regular, and no elongated cells or extra-large cells were observed.
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T subota et al · Limbal Allograft Transplantation v1atmg strong immunosuppression and achieving the same level of potential rejection as simple PK. A disad vantage of keratoepithelioplasty is that it requires a very fresh cornea with a good epithelium, which may be dif ficult to obtain in the routine eye-bank system. In addition, the procedure requires two donor corneas when it is com bined with PK because it requires the central corneal ep ithelium. Last, stem cells may not be present in the central corneal epithelium/· 27 which eventually may reduce the number of phenotypic corneal cells. There has been considerable discussion about the pres ence and location of corneal epithelial stem cells. Most recent studies have shown that the stem cells are located primarily in the limbal area, 5•7•9•27 •28 although some reports suggest a wider distribution in the peripheral cornea. 29 According to the cornea stem cell theory, destruction of the limbal epithelium results in corneal epithelial break down followed by conjunctival-epithelial invasion of the cornea, which has been confirmed in a rabbit model. 9 This suggests that if an alkali burn destroyed the whole limbal area, the cornea would break down severely, whereas a partial loss of the limbal area would result in only slight epithelial abnormalities. Mild cases of OCP and Stevens-Johnson syndrome also result in the con junctival epithelialization of the cornea. In our patients with bullous keratopathy, limbal deficiency with unknown etiology also occurred, and the corneal epithelium had severe breakdown and vessel invasion. The limbal dys function may be related to the recurrent epithelial erosion, which requires stem-cell proliferation for proper wound healing. Accelerated mitosis in the corneal limbal stem cells may result in dysfunction of the corneal stem cells in the long term, although this requires further study. Conjunctivization was confirmed in some patients by the presence of goblet cells on impression cytology. 30 In those patients, the need for stem cell transplantation is comparable to that of bone marrow transplantation for patients with aplastic anemia. The first trials of human corneal stem cell transplant were performed by Kenyon et al 14• 15 through limbal autograft transplantation. They transplanted the limbal area, presumably including stem cells, from the good fellow eye to the recipient eye. The results were promising, with the re-establishment of the corneal epithelium after limbal autograft transplantation. We have applied this concept for the reconstruction of severe corneal surface disorders as first reported by Pfister 12 and Tsai and Tseng. 16 Although partial removal of the limbus from the fellow eye was shown by Kenyon et a! to be safe, many patients are reluctant to interfere with the good eye. Moreover, some of the patients who have had a chemical injury were affected bilaterally, so that limbal autograft transplantation was impossible. Re sults of histopathologic examination showed that the lim hal cells were viable deep in the epithelium, whereas the more superficial cells usually were damaged during pres ervation in the medium. The corneas were stored in chondroitin-supplemented preservation medium for an average of 5 days. Although it has been noted that this medium is not ideal for epithelial preservation, 31 •32 limbal cells were viable. The cell culture using the limbal area
showed strong epithelial cell proliferation, which sup ported the histologic observation that preservation of the limbal cells was possible. The greatest disadvantage of limbal allograft trans plantation is the increased risk of rejection because the limbal area contains antigen-presenting cells. We observed typical endothelial-type rejection in two of nine patients, despite the strong immunosuppression. In our series, we used high doses of intravenous steroids for the first 8 days after surgery, oral cyclosporine 2 days before and 1 month after surgery, and long-term cyclosporine and dexameth asone eye drops. 20 This immunosuppression was much stronger than for the usual PK. Although the mechanism of cyclosporine is not totally understood, it suppresses interleukin-2 and various types of cytotoxic compounds such as granzyme A and perforin from activated T cells and has been indispensable for kidney, liver, bone marrow, and other transplants. 33- 36 Although rejection is not sup pressed completely, we believe that the combination of oral and topical cyclosporine is critical to our successful limbal transplants. Although limbal transplantation is probably successful because of the transplantation of stem cells, other factors may play roles. The procedure provides a healthy Bowman layer at the periphery of the recipient cornea which may serve as a good substrate. It has been reported that different substrates can modify cellular expression, 37 and the pres ence of a healthy Bowman layer may transdifferentiate the recipient conjunctival epithelium to a corneal phe notype. Another factor may be the suppression of periph eral neovascularization by the corneal stroma. The cornea itself is a known suppressor of new vessels. These three mechanisms may work together for the re-establishment of a normal corneal epithelium. In our surgical technique, we remove most of the stromal tissue from the limbal tissue, so that only the corneal epithelium and Bowman layer with a small por tion of stroma are transplanted. This procedure reduces the amount of transplanted antigen-presenting cells be cause the peripheral stroma includes many active cells. The duration of storage also may have some effect on the antigen-presenting cells. The use oflimbal tissue from the same donor as the PK tissue can save a donor cornea using only one donor eye for the combined procedure of PK and limbal transplantation. Limbal transplantation, including cutting the limbal tissue into two parts, remov ing the stromal tissue, and suturing with two or three 10 0 nylon interrupted sutures takes only 5 to 10 minutes. We currently are using limbal transplantation for severe corneal epithelial abnormalities without limbal cells. Be cause the technique is easy and rejection can be controlled using immunosuppressants, the procedure may have a wider applicability. Other ocular surface disorders such as partial limbal deficiency, gelatinous drop-like dystro phy, and persistent epithelial defect can be good indica tions for limbal transplantation. Acknowledgments. The authors thank Scheffer Tseng, MD, PhD, Kenneth R. Kenyon, MD, Ray Tsai, MD, and An drew Huang, MD, for their initiation of this procedure and their discussions concerning limbal transplantation. They also thank
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Volume 102, Number 10, October 1995
Hsu Ke-Ping, MD, Yukiko Yagi, ORT, Tokuaki Sugawara, OMA, and Saori Nishijima, ORT, for the ocular surface ex aminations.
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I 9. Tsubota K. A contact lens for specular microscopic obser vation. Am J Ophthalmol 1988;106:627-8. 20. Kanai A, Alba R, Takano T, eta!. The effect on the cornea of alpha cyclodextrin vehicle for cyclosporine eye drops. Transplantation Proceedings 1989;21 :3 I 50-2. 21. Tsubota K. The effect ofwearing spectacles on the humidity ofthe eye. Am J Ophthalmol 1989;108:92-3. 22. Tsubota K, Yamada M, Urayama K. Spectacle side panels and moist inserts for the treatment of dry eye patients. Cor nea 1994; 13:202-9. 23. Tsubota K, Yamada M, Naoi S. Specular microscopic ob servation of normal human corneal epithelium. Ophthal mology 1992;99:89-94. 24. Tsubota K, Yamada M, Naoi S. Specular microscopic ob servation of human corneal epithelial abnormalities. Oph thalmology 1991 ;98: 184-91. 25. Kinoshita S, Friend J, Thoft RA. Sex chromatin of donor corneal epithelium in rabbits. Invest Ophthalmol Vis Sci 1981;21:434-41. 26. Dahlman C. On the fate of the corneal graft. Acta Ophthal mol 1957;35:286-302. 27. Costarelis G, Cheng S, Dong G, Sun IT, Lavker R. Existence of slow-cycling limbal epithelial basal cells that can be pref erentially stimulated to proliferate: implications on epithelial stem cells. Cell 1989;57:201-9. 28. Greiner J, Covington H, Allansmith M. The human limbus. A scanning electron microscopic study. Arch Ophthalmo1 1979;97: 1159-65. 29. Lauweryns B, van den Oord J, de Vos R, Missotten L. A new epithelial cell type in the human cornea. Invest Ophthalmol Vis Sci 1993;4: 1983-90. 30. Nelson J, Havener V, Cameron J. Cellulose acetate impres sions of the ocular surface: dry eye state. Arch Ophthalmol 1982; 10 I: 1869-72. 31. Farge E, Fort R, Wilhe1mus K, et a!. Morphologic changes ofK-sol preserved human corneas. Cornea 1989;8:159-69. 32. Lindstrom R, Kaufman H, Skelnik D, Laing RA, Lass J, Musch D, et al. Optisol corneal storage medium. Am J Ophthalmol 1992; 114:345-56. 33. Liu C, Rafil S, Granelli-Piperno A, Trapani J, Young J. Perforin and serine esterase gene expression in stimulated human T cells. J Exp Med 1989;170:2105-18. 34. Lancki D, Kaper B, Fitch F. The requirements for triggering of lysis by cytolytic T lymphocyte clones II. Cyclosporine A inhibits TCR-mediated exocytosis but only selectively in hibits TCR-mediated lytic activity by cloned CTL. J lm munol 1989;142:416-24. 35. Lu P, Garcia-Sanz J, Lichtenheld M, Podack E. Perforin expression in human peripheral blood mononuclear cells. J Immunol 1992;148:3354-60. 36. Griffiths G, Alpert S, Lambert E, McGuire J, Weissman I. Perforin and granzyme A expression identifying cytolytic lymphocytes in rheumatoid arthritis. Proc Nat! Acad Sci USA 1992;89:549-53. 37. Tsai R, Tseng SC. Substrate modulation of cultured rabbit conjunctival epithelial cell differentiation and morphology. Invest Ophthalmol Vis Sci 1988;29: 1565-76.