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| ORIGINAL ARTICLE |
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| Year : 1996 | Volume
: 44
| Issue : 1 | Page : 33-38 |
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Conjunctival transplantation for corneal surface reconstruction - case reports and review of literature
Alkesh Sanghvi1, Surendra Basti2
1 Sight Savers Cornea Training, Smt.Uttaradevi Rao Training Centre for Advancement of Eye Care, L.V. Prasad Eye Institute, Hyderabad - 500034, India
2 Cornea Service, L. V Prasad Eye Institute, Hyderabad - 500034, India
Correspondence Address:
Alkesh Sanghvi
Sight Savers Cornea Training, Smt.Uttaradevi Rao Training Centre for Advancement of Eye Care, L.V. Prasad Eye Institute, Hyderabad - 500034
India
 Source of Support: None, Conflict of Interest: None  | Check |
PMID: 8828304 
Corneal persistent epithelial defects (PED) can occur due to various causes. In diffuse ocular surface disease, they often occur secondary to depletion of limbal stem cells. A number of complications occur secondary to PED and successful treatment usually requires conjunctival surgeries for corneal surface reconstruction. We report two cases of chemical injury successfully treated by two such procedures. This report highlights the encouraging results of limbal transplantation and reviews the literature in the management of PED with limbal stem cell loss. Keywords: Chemical Injury - Corneal Opacity - Limbal Transplantation -Conjunctival Transplantation - Persistent Epithelial Defect - Stem cells.
How to cite this article:
Sanghvi A, Basti S. Conjunctival transplantation for corneal surface reconstruction - case reports and review of literature. Indian J Ophthalmol 1996;44:33-8 |
The importance of having a regular ocular surface with well differentiated phenotypes of corneal and conjunctival epithelia for a physiologically functioning eye has been well recognised.[1] The conjunctiva plays several important roles - it serves as a reservoir for tears and contributes to the secretions that make-up tears by production of mucus. Conjunctival epithelium and its secretions form an important barrier to the external environment and infection, and are also important in ocular wound healing. The primary function of the corneal epithelium is to provide a smooth refracting surface at the front of the eye, in conjunction with the tear film. It also serves as a relatively impermeable barrier to water soluble materials from the tear film.
Extensive external ocular inflammation or tissue damage by injury or disease results in failure of surface epithelial repair. Especially in conditions associated with limbal stem cell loss, poor corneal epithelial healing results and can lead to persistent epithelial defects.[2] These are harbingers of complications such as stromal ulceration and can thus cause severe ocular morbidity.
It is to avert the above mentioned perils of epithelial nonhealing that treatment after a chemical burn is largely focused on reinstating an intact ocular surface. Noninvasive measures such as lubricants, patching and contact lenses have been the mainstay of treatment to promote resurfacing by proliferation of the nontraumatized epithelial cells.[3] In situations associated with widespread ocular surface damage however, the residual surface epithelium may be inadequate, leading to epithelial nonhealing. In such situations, augmenting the residual stem cell pool with fresh cells has been a surgical modality that has evinced considerable interest among clinicians recently. We managed two patients with this approach and present our experience.
| Case reports | |  |
| Case l | | |
A 30 year old male sustained chemical injury in the right eye (RE) due to accidental fall of chemical powders during 'holi' festival in March 1994. He was seen by his ophthalmologist, who had prescribed him topical betamethasone (0.1%) six hourly, neosporin eye ointment at bed time and tablet acetazolamide 250mg twice daily. Since he did not improve with the medications, he came to us on May 23, 1994. On examination, he was noted to have a visual acuity of counting fingers at 2 meters, N36 in RE and 6/6, N6 in left eye (LE). The LE was found to be clinically unremarkable. The right eye showed conjunctival injection. The cornea showed an epithelial defect measuring 7mm X 7mm[Figure - 1]. There was scarring underlying the edges of the defect and superficial vascularization at the limbus. Intraocular pressure (IOP) was within normal limits. Based on the clinical findings a diagnosis of chemical injury of the RE with persistent epithelial defect was made and treatment instituted. An ultra thin soft bandage contact lens (U4 series, Bausch and Lomb Inc., Rochester, NY) was inserted in RE and the patient was advised use of topical chloramphenicol (0.4%) six hourly and betamethasone (0.1%) twelve hourly and was regularly followed-up. On June 15, 1994, the condition had not significantly improved and the epithelial defect was seen to persist. The underlying stroma was relatively clear and conjunctival epithelium was seen growing in 360 degrees. Since the epithelial defect was unresponsive to medical therapy, limbal conjunctival autografting was done on June 20, 1994, under general anaesthesia.
| Surgical Procedure | | |
Grafting was done as a 3 step procedure:
Step 1 - RE
Barraquer wire speculum was placed to separate the lids and superior and inferior rectus bridle sutures were applied using 6.0 silk. Conjunctival overgrowth at limbus was separated with Weckcel sponges and iris repositor. Peritomy with excision of 5mm frill of conjunctiva was done. Superficial Keratectomy was performed with a Tookes' Knife, leaving a smooth corneal bed.
Step 2 - LE
Lid speculum was placed and 6.0 silk superior and inferior rectus bridle sutures applied. Four limbal conjunctival grafts dissected as follows: A perpendicular, l/3rd stromal thickness deep incision was made in the cornea parallel to the limbus and 1mm inside it, using #15 blade on a Bard-Parker handle. The cut edge was gently lifted with a nontoothed Pierse Hoskin's forceps and dissection was continued peripherally using a crescent knife, to obtain a limbal keratectomy. Grafts from 3-O and 9-0 clock measured 2.5mm x 2.5mm and were of 1/3rd stromal thickness, while 12-O and 6-O clock grafts were of similar thickness but measured 2.5mm vertically x 3.5mm horizontally. The grafts were placed in Ringer lactate solution.
Step 3 - RE
Autografts were sutured at the corresponding limbus. Grafts from a given location were sutured to the corresponding site in the recipient bed. Care was taken to ensure that limbal cornea was towards cornea and conjunctiva towards conjunctival side. Four 10-0 nylon sutures were used per graft and the suture knots were buried. A bandage Lens was placed in RE and soframycin eye ointment instilled in LE. Both eyes were patched.
Postoperatively the patient was advised to instill in RE, topical betamethasone (0.1%) three hourly, chloramphenicol (0.4%) six hourly and cyclopentolate (1%) twelve hourly and in LE, chloramphenicol (0.4%) six hourly and hydroxypropylmethylcellulose (0.7%) two hourly and was regularly followed-up. The clinical picture on the first postoperative day in the RE and LE is shown in [Figure - 2] and [Figure - 3] respectively. The donor sites showed complete reepithelialization when examined one week after surgery. The chloramphenicol was discontinued after a week and the lubricant after two weeks in the LE. Re-epithelialization of the right eye was complete two weeks after surgery. When examined two months after surgery [Figure - 4] [Figure - 5], vision in RE improved to 6/18 with a totally re-epithelialized cornea. The LE was unremarkable except for the presence of subepithelial scarring at the donor sites. The bandage contact lens was removed from the RE and the medications were tapered off. Topical hydroxypropylmethylcellulose (0.7%) six hourly was prescribed for the right eye. He was advised to return in February 1995, but did not report for review.
| Case 2 | | |
A 38-year-old male had an alkali injury due to fall of liquor ammonia in his right eye in February 1993. He had consulted his ophthalmologist who had prescribed him topical betamethasone (0.1%) eight hourly, hydroxypropylmethylcellulose (0.7%) eight hourly and tablet prednisolone l0 mg daily. Since he did not notice much improvement over five months, he came to us. He was examined by us on July 1, 1993, and was noted to have a visual acuity of finger counting at one meter in RE and 6/6 in LE. The left eye was found to be clinically unremarkable. The right cornea [Figure:6] showed an epithelial defect (measuring 6.4 x 6.4mm), with an elevated, undermined edge. There was dense superficial and deep vascularization and the surrounding cornea was oedematous. The IOP was within normal limits. Based on the above clinical picture, a diagnosis of persistent epithelial defect in right eye secondary to alkali burn was made and treatment instituted. A bandage contact lens was inserted in RE and topical chloramphenicol (0.4%) six hourly and hourly hydroxypropylmethylcellulose (0.7%) prescribed. The patient was periodically reviewed but the clinical progress was unsatisfactory and the epithelial defect was unresponsive to the initiated treatment. In March 1994, the defect measured 4.0mm x 4.0mm with 360 degree superficial and deep vascularization and stromal scarring. Just above the visual axis and towards 2-O clock position, the cornea was found to be very thin (80 to 90% thinning). In view of the persistent epithelial defect, the irregular corneal surface, stromal scarring with dense vascularization, conjunctival autograft transplantation was preferred for corneal surface reconstruction.
| Surgical Procedure | | |
Surgery was performed on March 25, 1994, under general anaesthesia. Brief details of the surgical procedure are as follows:
Step 1 - LE
The lids were separated using a Barraquer wire speculum. The globe was retracted inferiorly. In the superior quadrant, a zone 3mm x 3mm and 3mm away from the limbus was measured with callipers and marked using methylene blue dye. A conjunctival flap was dissected out by tenting-up the conjunctiva with a Pierce-Hoskins forceps and cutting with spring scissors. In a similar fashion three more flaps were dissected out from the other three quadrants. All the grafts were placed in Ringer lactate solution. The beds of the grafts were closed by suturing with 6-0 plain catgut.
Step 2 - RE
The lids were separated using Barraquer wire speculum. The conjunctiva 3mm away from the limbus was marked all around using methylene blue and a peritomy performed with spring scissors. Epithelium from the edges of the persistent epithelial defect was debrided and removed along with the contiguous conjunctiva by holding the cut edge of conjunctiva and dissecting with a crescent knife. A No.15 blade on a Bard-Parker handle was used to render the surface smooth. The donor conjunctival flaps were straddled on the limbus. Three 10-0 nylon sutures were used to secure the peripheral part of the graft to episclera. The central ends of the four grafts were included in a loose 6-0 plain catgut suture passed in a purse-string fashion. An ultra thin bandage contact lens was placed and a drop of gentamicin(0.3%) instilled. RE was patched. In the LE, soframycin ointment was instilled and the eye was left open.
Postoperatively the patient was advised to instill topical betamethasone (0.1%) three hourly, chloramphenicol (0.4%) six hourly and cyclopentolate (1%) twelve hourly in the RE and chloramphenicol (0.4%) six hourly in the LE and was regularly followed-up. The donor sites were unremarkable and the eye quiet a week after surgery at which time the topical antibiotic was discontinued. Re-epithelialization of the right eye was complete three weeks after surgery. The bandage contact lens was removed six weeks postoperatively and the eye drops tapered off. The patient was thereafter regularly followed-up and when seen on July 21, 1994, visual acuity in RE was counting fingers at one meter. The epithelial defect had resolved completely, leaving a vascularized and scarred cornea with a smooth regular surface. LE was unremarkable. He was advised to continue instillation of hydroxy-propylmethylcellulose (0.7%) four hourly in RE. The clinical picture has remained the same on subsequent follow-up visits.
| Discussion | | |
For decades, clinicians have found a direct correlation between limbal integrity and ultimate anatomic and visual outcome in patients sustaining chemical burns. Status of the limbus has hence been an important consideration in classification systems that help prognosticate cases of chemical burns.[4]
The clinical anatomy of the limbus was observed in 1921 by Vogt[5] who described ridges present in the perilimbal conjunctiva perpendicular to the cornea. In 1971, Davanger and Evanson[6] suggested that these perilimbal arrays were the source of stem cells and were cardinal for renewing corneal epithelium.
Evidence on the existence of corneal epithelial stem cells at the limbus was proved by a study performed by Schermer et al.[7] Their findings of a major 64K corneal keratin using monoclonal antibodies (AE-5) typical for the corneal epithelial phenotype, suggested a limbal location of corneal epithelial stem cells. Subsequently, Cotsarelis et al[8] and Lavker et al[9] arrived at the same conclusion, based on incorporation of tritiated thymidine into the corneal/limbal basal cells under stimulation by a tumour promoter.
There is no direct histologic proof that stem cells exist. However Zieske[10] was able to produce a monoclonal antibody to the 50K keratin believed to be specific for stem cells. Sharma and Coles[11] used a mathematically-based population balance model and made their observations on the average mitotic rate of limbal epithelium and rate of epithelial movement in rabbit corneas. They postulated that if the centripetal supply from the limbus ceases without any concurrent elevation in the rate of corneal mitosis, the corneal epithelium would lose about one-third of its volume in a one month period. They also showed that the half-time for the corneal epithelium (time needed for replacing half of all preexisting cells) is about 9 weeks. An almost complete renewal of the corneal epithelium by the progeny of the limbal stem cells occurs in about a 9 - 12 month period. Today, most clinicians believe that the corneal epithelial stem cells are confined to the limbus whereas conjunctival epithelial stem cells are diffusely distributed in the conjunctival cul-de-sac.[1]
The stem cell theory proposed by Tseng et al[1] envisages presence of two compartments: (1) Proliferative compartment: consisting of stem cells(SC) situated in the limbal basal layer, and, transient amplifying cells (TAC) situated in the corneal basal layer. (2) Differentiative compartment: comprising of postmitotic cells (PMC) and terminal differentiated cells (TDC) both situated in the cornea. The stem cells have a long life, are slow-cycling and quiescent with a low mitotic activity under the normal steady state and are poorly differentiated. On demand, mitosis occurs and one SC gives rise to one SC (SC renewal) and one TAC (TAC generation). TAC, in contrast to stem cells, are short-lived with a short life-cycle and are well differentiated. At a critical point after limited rounds of mitosis, TAC will cease to mitose and march into the differentiative compartment and become PMC. These cells are committed to terminal cellular differentiation to generate TDC.[1] Because of the junctional position of limbal epithelium between corneal and conjunctival epithelia and a unique SC content, the regional growth pressure generated by SC and TAC from the limbus can conceivably prevent conjunctival epithelial ingrowth during normal healing of a central corneal epithelial defect. Taken together, limbal SC can thus be considered as the ultimate source of corneal epithelial regeneration and serve as the junctional barrier between corneal and conjunctival epithelia. Critical limbal stem cell loss is therefore associated with PED and conjunctivalization of the cornea.
Based on the type of stem cells transplanted, Tseng et al[1] have classified conjunctival surgeries for corneal surface reconstruction as follows:
- 1. Transplantation of limbal SC
- A. Limbal transplantation: autograft or allograft
- B. Keratoepithelioplasty: allograft
- 2. Transplantation of conjunctival SC
- A. Conjunctival transplantation: autograft
- B. Conjunctival flap: autograft
Conjunctival transplantation was suggested in 1977 by Thoft[12] based on the concept of conjunctival transdifferentiation. He envisaged that conjunctival epithelium when transplanted onto the cornea would transdifferentiate into the regular, clear corneal epithelium. However while epithelial migration from the donor sites covered the entire bare corneal stroma within 7 to 10 days in most cases, the resultant phenotype was not entirely corneal. Also, these corneas exhibited progressive vascularization and lipid keratopathy
To circumvent some of these problems, Thoft[13] introduced keratoepithelioplasty as a surgical procedure to restore the integrity of the ocular surface in patients with bilateral diffuse ocular surface diseases. From donor corneas, 3mm x 3mm lenticules were fashioned from the peripheral cornea without including the limbal epithelium. These were transferred to the recipient bed with the peripheral aspect of the lenticule directed centrally, and sutured. Turgeon et al[14] highlighted the importance of using keratoepithelioplasty lenticules that necessarily included the limbal epithelium. It was found that following keratoepithelioplasty, the corneal epithelium was typically intact within one to two weeks, smooth and regular by four weeks and stable thereafter.
Conjunctival flap as a surgical procedure was first described by Scholer in 1887. Gundersen[15] in 1958 described a new technique and a number of surgical indications for conjunctival flaps, especially for recalcitrant corneal ulceration and poor epithelialization. However this procedure has its own limitations. The flap will become thinner after resolution of the PED and will eventually lift up and get detached. An eventual corneal phenotype can never be obtained since the flap has an intact vasculature which will not permit any degree of transdifferentiation to the corneal phenotype.
Limbal stem cell transplantation was reported by Kenyon and Tseng[16], as an autograft in cases with unilateral diffuse involvement or a focal limbal involvement in unilateral or bilateral cases. Their results were encouraging. There was an improvement in visual acuity, rapid surface healing, stable epithelial adhesion without recurrent erosion or PED and arrest or regression of corneal neovascularization.
Enthused by reports on the role of limbal and conjunctival transplants in the management of PED, we adopted a similar approach and achieved considerable success as depicted by our case reports. Our first case when initially seen had chemical injury with a PED with underlying clear stroma and superficial vascularization at the limbus. Since there was limbal stem cell loss and the central stroma was not significantly scarred or vascularized, we were optimistic about improvement in visual acuity with a limbal transplant from the fellow eye. Postoperative results were encouraging with a totally re-epithelialized cornea and a significant improvement in vision (from counting finger at 2 meters to 6/18) eight weeks after surgery. It is likely that a further improvement in visual acuity will occur with the passage of time.
The second case had PED secondary to alkali burn with significant stromal thinning and scarring, with both superficial and deep vascularization. The clinical picture here again was suggestive of a limbal stem cell loss. This and the presence of dense vascularization made the patient a poor candidate for penetrating keratoplasty. We were considering the option of doing an autologous limbal graft so that the corneal phenotype would be established, followed by a penetrating keratoplasty at a later date. However, considering the risk-benefit ratio and the fact that the other eye had good vision, the patient opted for autologous conjunctival transplantation, since his prime intention was to get rid of a chronically inflamed eye. Accordingly, the conjunctival transplantation was done and we achieved our objective of begetting a well healed and smooth ocular surface.
Limbal grafting holds considerable promise in reconstruction of the corneal surface in recalcitrant epithelial defects. The current debate is whether to use an autograft or an allograft. Since data is not available regarding the critical amount of stem cell population required for a healthy corneal surface, it is unclear how much of limbal tissue can be removed from the donor eye, without compromising it's safety. Thus, there is a theoretical possibility of inadvertent depletion of the stem cell pool in the donor eye in cases of autografts. It is therefore difficult to predict the long-term results in these patients. The use of an allograft obviates these problems, but brings in the risk of allograft rejection. To reduce the risk of rejection, cyclosporin - A postoperatively[17] or allograft transplantation using a HLA-matched limbal tissue either from a close relative or a cadaver eye are the proposed options. Present clinical experience is preliminary and prospective randomised studies evaluating these measures are necessary to actually determine their benefits. It is also required to understand the transplantation biology of limbal tissue so that allograft limbal transplantation may be widely used.
| References | | |
| 1. | Tseng SCG. Classification of conjunctival surgeries for corneal diseases based on the stem cell concept. Ophthalmol Clin North Am 3:595-610,1990.  |
| 2. | Pfitser RR. Corneal stem cell disease. concepts, categorisation and treatment by auto and homotrans-plantation of limbal stem cells. CLAO J, 20:64-72,1994. |
| 3. | Pfitser RR. Chemical injuries of the eye. Ophthalmology 90:1246-1253, 1983. |
| 4. | Hughes WF. Alkali burns of the cornea. II:Clinical and pathologic course. Arch Ophthalmol 36:189, 1946. |
| 5. | Vogt A. Textbook and Atlas of Slit Lamp Microscopy of the Living Eye. 3ed. (Volume 1).Bonn-Bad Godesberg, Germany, Wayenborgh, 1921, pp 52-53. |
| 6. | Davanger M, Evensen A. Role of the pericorneal papillary structure in renewal of corneal epithelium. Nature 229: 560-561,1971. |
| 7. | Schermer A, Gavlin S, Sun TT. Differentiation - related expression of a major 64 K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells J. Cell Biol. 103:49-62, 1986. |
| 8. | Cotsarelis G, Dong G, Sun T-T,et al. Differential response of limbal and corneal epithelia to phorbol myristate acetate (TPA). Invest Ophthalmol Vis Sci. 28(Suppl):l, 1987. |
| 9. | Lavker RW, Dong G, Costarelis G, et al. Limbal basal epithelial cells display characteristics consistent with stem cells from various stratifying epithelia. Invest Ophthalmol Vis Sci. 29 (Suppl):191, 1988. |
| 10. | Zieske JD, Bukusoglu G. Characterisation of a potential marker of corneal epithelial stem cells. Invest Ophthalmol Vis Sci 33:143-152, 1992. |
| 11. | Sharma A, Coles WH. Kinetics of corneal epithelial maintenance and graft loss. Invest Ophthalmol Vis Sci 30:1962-1971,1989. |
| 12. | Thoft RA. Conjunctival transplantation. Arch Ophthalmol 95:1425-1427, 1977. |
| 13. | Thoft RA. Keratoepithelioplasty. Am J Ophthalmol 97:1 -6,1984. |
| 14. | Turgeon PW, Neuheim RC, Roat MI, et al. Indications for Keratoepithelioplasty. Arch Ophthalmol 108:233-236,1990. |
| 15. | Gunderson T. Conjunctival flaps in the treatment of corneal disease with reference to a new technique of application. Arch Ophthalmol 60:888, 1958. |
| 16. | Kenyon KR, Tseng SCG. Limbal autograft transplantation for ocular surface disorders. Ophthalmology 96:709-723, 1989. |
| 17. | Tseng SCG, Tsai RJF. Human allograft limbal transplantation for corneal surface reconstruction. Cornea. 13:389-400,1994. |
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5]
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