Figure 1. Patient 11. A, Left eye that received alkali burns 22 months earlier had undergone symblepharon release, amniotic membrane transplantation, and limbal autograft transplantation 9 months previously. The eye showed inferior corneal stem cell deficiency, conjunctival ingrowth, and moderate symblepharon. B, Fifteen months after living-related limbal allograft transplantation and amniotic membrane transplantation, the eye demonstrated stable epithelia, improved corneal transparency, and absence of symblepharon.
Figure 2. Patient 7. A, Right eye that received alkali burns 20 months earlier had undergone symblepharon release, amniotic membrane transplantation, and limbal autograft transplantation 12 months previously. The eye showed superior corneal stem cell deficiency, conjunctival ingrowth, and moderate symblepharon. B, Twelve months after living-related limbal allograft transplantation and amniotic membrane transplantation, the eye demonstrated stable epithelia, decreased neovascularization and ocular inflammation, improved corneal transparency, and mild symblepharon.
Figure 3. Patient 15. A, Right eye that received acid burns 14 months earlier. The eye showed inferior nasal conjunctival ingrowth, pseudopterygium formation, and severe neovascularization. B, Twelve months after living-related limbal allograft transplantation and amniotic membrane transplantation, the eye demonstrated stable epithelia, improved corneal transparency, decreased neovascularization, and absence of symblepharon.
Huang T, Wang Y, Zhang H, Gao N, Hu A. Limbal Allografting From Living-Related Donors to Treat Partial Limbal Deficiency Secondary to Ocular Chemical Burns. Arch Ophthalmol. 2011;129(10):1267-1273. doi:10.1001/archophthalmol.2011.251
Author Affiliations: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Objective To evaluate outcomes of limbal allograft transplantation from living-related donors to treat partial limbal deficiency secondary to ocular chemical burns.
Methods Retrospective noncomparative case series comprising 17 patients (17 eyes) with partial limbal deficiency (≤50%) secondary to ocular alkali burns (11 eyes) or acid burns (6 eyes). Recipient eyes were treated by removing the conjunctivalized pannus. Superior limbal grafts (mean length, 3-5 clock hours) from HLA antigen–matched living-related donors were transplanted into deficient areas of recipient eyes. No recipients received systemic cyclosporin A therapy. Main outcome measures included corneal reepithelialization, reduction in vascularity, improved corneal clarity, and best-corrected visual acuity.
Results All eyes achieved epithelialization a mean (SD) of 10.1 (1.9) days after surgery. Corneal reepithelialization, reduction in vascularity, and improved corneal opacity were seen in all eyes. No eyes demonstrated recurrent epithelial defects or fibrovascular tissue, but gradual recurrence of peripheral corneal vascularization was observed in 7 eyes during the follow-up period. Allograft rejection developed in 3 eyes (17.6%), all of which were successfully treated. Best-corrected visual acuity improved in all eyes, and 10 eyes (58.8%) had achieved best-corrected visual acuity of 0.5 or better (≥20/10 Snellen) at the last follow-up visit.
Conclusions Transplantation of limbal tissue from live-related donors successfully reconstructed the ocular surface. Long-term graft survival in patients with partial limbal deficiency secondary to ocular chemical burns can be accomplished without the use of systemic immunosuppression.
Limbal stem cells are located at the base of the limbal epithelium and are responsible for repopulation of cells in the corneal epithelium and for inhibition of conjunctival growth over the cornea.1- 4 Limbal stem cell dysfunction or limbal deficiency can lead to ocular surface abnormalities, which are characterized by chronic epithelial defects, stromal inflammation, corneal vascularization, conjunctival epithelial ingrowth (conjunctivalization), and corneal opacification.5- 7 Ocular chemical burns are the primary cause of limbal stem cell deficiency.8,9
Stem cell transplantation is effective to treat limbal stem cell deficiency.10- 14 For patients with unilateral limbal deficiency, the contralateral eye can provide healthy limbal tissue for limbal autograft transplantation,15- 18 whereas for patients with severe bilateral limbal deficiency, only homologous limbal tissue can be used for keratolimbal allograft transplantation.7,10,12,13,19 Unfortunately, the graft rejection rate is high in allograft transplantation, and long-term systemic immunosuppressive treatment is required for these patients.10,12,19
Theoretically, limbal allograft transplantation from an HLA antigen–matched living-related donor is a means to achieve long-term graft survival.12,20 However, to minimize damage to the cornea of the healthy donor, a limited amount of limbal tissue is obtained, and this is insufficient to reconstruct the ocular surface of patients with severe total limbal deficiency.21,22 Moreover, graft rejection can occur even in eyes with limbal allograft transplantation from HLA antigen–matched living-related donors.22 Among eyes with severe total limbal deficiency, previous studies22,23 have shown that long-term efficacy of limbal allograft transplantation from living-related donors is low. Herein, we propose an alternative procedure of limbal allografting from living-related donors to treat partial limbal deficiency secondary to chemical ocular burns.
The study adhered to tenets of the Declaration of Helsinki. Approval of the study was obtained from the Institutional Review Board of Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China. Seventeen eyes of 17 patients undergoing living-related limbal allograft transplantation at Zhongshan Ophthalmic Center between February 4, 2005, and September 27, 2007, were retrospectively reviewed. All the involved eyes had partial limbal deficiency (≤50%) secondary to ocular alkali burns (11 eyes) or acid burns (6 eyes). Among the patients, 5 had unilateral injury, and 12 had bilateral injury; in all the patients, transplantation was performed in only 1 eye. Information obtained from patient medical records included age, sex, nature and duration of injury, best-corrected visual acuity, and prior medical management. Preoperative and postoperative states of the involved eyes were recorded using slitlamp photographs. Variables examined included degree of conjunctivalization or pannus, corneal vascularization, corneal opacification, epithelial defect status, and symblepharon formation. Degree of severity in all the patients was judged by one of us (T.H.). Preoperative patient data are given in Table 1 and Table 2.
Donors were evaluated before surgery to exclude the possibility of stem cell compromise. The nondominant eye of the donor was chosen for limbal tissue donation. Information obtained from donor records included reepithelialization time of the donor site, postoperative ocular discomfort, corneal vascularization, and visual acuity.
Indication for surgery was noted in all the patients. These included conjunctivalization of the corneal surface and corneal neovascularization (n = 17), persistent epithelial defect (n = 4), pseudopterygium combined with amniotic membrane transplantation (n = 3), and symblepharon formation (n =3).
The nature of the surgical procedure was explained to all the patients and donors. Informed consent forms were signed by all the patients and donors before surgery. In 13 patients (76.5%), the donors were their mothers, and in 4 patients (23.5%), the donors were their fathers. HLA antigen typing was performed for all surgical procedures. Criteria for HLA antigen typing were similar to those adopted for high-risk penetrating keratoplasty, with some modifications,12 and patients and donors with a maximum of 2 mismatches on HLA-A, HLA-B, and HLA-DR loci were considered HLA antigen matched.
All the procedures were performed by one of us (T.H.). Adult donors and recipients were operated on using retrobulbar anesthesia, and child recipients were operated on using general anesthesia. Surgery on the recipient was performed following surgery on the donor. Donor limbal tissue was harvested from the superior corneal limbus. The size of the donor area varied among eyes. The mean length was 3 to 5 clock hours and extended 2 mm on the conjunctival surface and 1 mm on the corneal epithelium. The donor site was left unsutured. A conjunctival peritomy adjacent to the deficient area in the recipient cornea was performed, and the limbal conjunctiva was undermined and allowed to retract posteriorly. The donor graft was secured in the recipient bed using a 10-0 nylon suture after the pannus was dissected and cauterized. The conjunctival portion of the graft was sutured to the underlying episcleral tissue, and the corneal edge of the graft was left unsutured. At the end of the procedure, the eye was patched to maintain pressure of the graft on the recipient bed.
Patients were given oral prednisone (30 mg/d) for 1 week before surgery and intravenous dexamethasone sodium phosphate (10 mg/d) combined with ceftizoxime sodium (1.5 mg/d) for 3 days after surgery. When the cornea was reepithelialized, the recipient eye was treated with topical dexamethasone sodium phosphate (0.1%) every 2 hours, topical tobramycin sulfate (0.3%) 4 times daily, and topical tear substitutes 4 times daily. Topical corticosteroids were discontinued 3 to 4 months after surgery. No patients received systemic cyclosporin A or other systemic immunosuppressants.
Study data are given as means (SDs). The mean interval between ocular trauma and surgery was 15.5 (4.0) months (range, 9-22 months).
Before living-related limbal transplantation, patients in the study had undergone the following procedures: symblepharon release, amniotic membrane transplantation, and limbal transplantation from cadaver donor tissue (4 eyes [patients 1, 4, 7, and 11 in Table 1]); removal of the conjunctivalized pannus and limbal transplantation from cadaver donor tissue (6 eyes [patients 2, 5, 6, 9, 13, and 16 in Table 1]); removal of the conjunctivalized pannus, amniotic membrane transplantation, and limbal transplantation from cadaver donor tissue (2 eyes [patients 8 and 12 in Table 1]); and correction for lower eyelid ectropion (2 eyes [patients 10 and 14 in Table 1]). The mean follow-up period was 16.0 (4.2) months (range, 12-26 months).
After surgery, complete corneal reepithelialization was seen in all the patients, and no epithelial defects were observed during the follow-up period (Figure 1). The mean to complete epithelialization was 10.1 (1.9) days (range, 7-14 days). Fibrovascular tissue did not recur in any eye. Preexisting peripheral neovascularization partially regressed at 1 to 3 months after surgery. However, gradual recurrence of corneal neovascularization occurred 7 to 10 months after surgery in 7 eyes, and mild symblepharon was seen in 3 eyes (Figure 2). Corneal opacification improved in all eyes (Figure 3).
After surgery, 3 eyes developed presumed allograft rejection at 3½ (patient 9), 5 (patient 4), and 6 months (patient 14), and these patients exhibited sectoral conjunctival injections, local epithelial defects, graft edema, and ocular pain. All 3 eyes were successfully treated with intensive topical corticosteroids and with high-dose intravenous dexamethasone sodium phosphate (10 mg/d for 3 days), followed by oral prednisone (1 mg/d/kg) for 2 to 3 months.
Best-corrected visual acuity improved in all the eyes during the follow-up period after surgery. Visual acuity in 5 eyes decreased gradually because of recurring peripheral neovascularization but was still better compared with preoperative visual acuity. No eyes required secondary penetrating or lamellar keratoplasty for improvement of visual acuity.
Donor eyes were followed up for 3 months after surgery. All the eyes achieved reepithelialization within 5 days. No donors experienced vision loss, and postoperative courses were unremarkable.
Thirteen of 17 eyes had a maximum of 2 mismatches on HLA-A, HLA-B, or HLA-DR loci (ie, were HLA antigen matched). The other 4 eyes had more than 2 mismatches, and 3 of these eyes developed presumed graft rejection. Outcomes of living-related limbal allograft transplantation are summarized in Table 3.
Although limbal autografting provides excellent results in unilateral disease,15- 18 ocular surface reconstruction is challenging in patients with bilateral limbal deficiency.11 Treatment of bilateral disease, which resulted from ocular chemical burns among patients in this study, requires allograft tissue transplantation. Heterologous limbal tissue can be obtained from cadaveric donors or from living-related donors.10,12,13,19
Although the cornea is considered an “immune privileged” site,24- 26 transplantation of tissue into a vascularized limbus theoretically increases the risk of rejection.24,27 Previous studies12,20,22 have shown that HLA antigen matching is important in graft survival. However, the lengthy preservation time needed for HLA antigen matching results in limbal stem cell dropout.27 Furthermore, complete immunohistocompatibility between cadaveric donors and recipients is rarely obtained. As an alternative option, limbal allografting from living-related donors provides an approach for stem cell transplantation with some degree of histocompatibility.
In previous studies,12,27- 29 surgeons reported successful ocular surface reconstruction using limbal tissue from HLA antigen–matched living-related donors. However, there are obvious disadvantages in using living-related limbal allografts because limbal allografting is limited by the amount of tissue that can be transplanted. Clinically, a superior limbal graft length of 5 or 6 clock hours is the limit to protect a donor's eyes and keep them healthy,22 whereas more limbal tissue and stem cells can be obtained from a cadaveric donor to restore the barrier to the entire limbus.
In severe total limbal stem cell deficiency secondary to severe chemical burns and Stevens-Johnson syndrome, the disadvantages of living-related limbal allograft transplantation, such as limited stem cell transplantation, may outweigh the benefits. Rao et al22 reported that 7 of 9 eyes achieved initial reepithelialization after living-related limbal allografting but that vascularization recurred in all the eyes after surgery. In another study, Shimazaki et al21 observed recurrence of conjunctivalization in eyes with limbal autografting or cadaveric limbal allografting. This indicates that transplanted limbal stem cells were unable to sustain sufficient long-term epithelial cell production for the entire limbus. Therefore, we performed living-related limbal allograft transplantation only in eyes with partial limbal deficiency (≤50%) secondary to chemical burns.
Controversy exists about surgical strategies in the case of partial stem cell deficiency. For partial stem cell deficiency with conjunctivalized epithelium on the cornea, sequential-sector conjunctival epitheliectomy should be considered first.30- 32 Dua30 reported that removal of the conjunctival epithelium from the cornea allowed cells of the corneal epithelial phenotype to cover the denuded area, alleviating symptoms and improving visual acuity. Therefore, it is not always necessary to undertake partial limbal or stem cell transplantation to restore the corneal surface in patients with partial stem cell deficiency.30 However, in partial stem cell deficiency with a fibrovascular pannus covering the cornea (such as in our case series), the pathologic entity tends to involve the stroma, except for the corneal epithelium. In contrast to conjunctivalized epithelium, fibrovascular pannus is difficult to scrape or brush off completely using topical anesthesia, and abnormal epithelium may remain on the denuded corneal surface. Abnormal epithelium influences contact inhibition of the corneal epithelium by the conjunctiva-derived epithelium.31 In partial stem cell deficiency with a fibrovascular pannus covering the cornea, limbal transplant may be required.32
Furthermore, sequential-sector conjunctival epitheliectomy cannot remove superficial neovascularization that preexists on the recipient cornea; this may aggravate inflammation of the host cornea after surgery. Limbal transplantation can remove neovascularization, recover the normal corneal architecture (rather than stem cells), and improve the microenvironment of the ocular surface, alleviating inflammation, reducing graft rejection, and improving graft survival after surgery.
All 17 eyes herein achieved successful ocular surface reconstruction, with complete corneal epithelialization, reduced neovascularization, and improved corneal opacification. In our series, the mean time to complete reepithelialization of 10.1 (1.9) days was shorter than the 13.4 (6.4) days reported by Rao et al.22 A possible explanation for this is that our patients had partial limbal deficiency instead of total limbal deficiency, as in the study by Rao et al, although the surgical procedures and amount of transplanted limbal tissue were similar between the 2 studies.
The rejection rate in our series was less than that in previous studies. In eyes with a presumed graft rejection, all grafts survived following the use of topical and systemic corticosteroids, and no systemic immunosuppressive agents, such as cyclosporin A, were administered. Variations in residual amounts of limbal stem cells among recipient eyes may have contributed to different outcomes herein vs previous studies. Eyes in this study had partial stem cell deficiency, with 50% or more healthy stem cells. Even if graft rejection after living-related limbal allograft transplantation resulted in stem cell dropout, the amount of residual stem cells was able to sustain sufficient epithelial cell production. Compared with rejection rates reported by Kwitko et al20 (25%), Rao et al22 (33.3%), and Daya and Ilari27 (25%), our rejection rate of 17.6% (3 of 17 eyes) was lower, despite not administering cyclosporin A, and overall graft survival seemed better.
Ocular status of the recipient may influence overall graft survival. Rao et al22 demonstrated that unsatisfactory surgical outcomes after living-related limbal allografting can result from limbal ischemia due to advanced alkali burns. In their study of living-related conjunctival limbal allografting, Daya and Ilari27 reported that 2 of 10 eyes did not achieve initial epithelialization because of severe inflammation. That study indicated that severe inflammation may be a major prognostic indicator of early graft failure. In our case series, all eyes of recipients were in a stable late stage of chemical burns, and most patients underwent prior surgical procedures to correct ocular structure abnormalities. All patients received systemic corticosteroids before and after limbal allografting to combat inflammation. These strategic procedures may partly account for the differences in outcomes between this and previous studies.
In our series, graft rejection occurred in 3 of 17 eyes, and the use of topical and systemic corticosteroids alone (without cyclosporin A) successfully inhibited rejection and prolonged graft survival. Our experience suggests that limbal allografting from living-related donors may not require systemic immunosuppression. In contrast, Rao et al22 and Shimazaki et al21 indicated that systemic immunosuppression is necessary even in HLA antigen–matched limbal allografting. In their series, limbal allografting in severely vascularized eyes was considered a high-risk transplantation. The Collaborative Corneal Transplantation Studies research group reported that HLA antigen compatibility is irrelevant to survival of high-risk corneal grafts,24,31 and other researchers state that patients require postoperative systemic immunosuppression even if their donors were HLA antigen matched.21,22 In comparison, the patients in our series were considered low risk because all the eyes were in the stable late stage of chemical burns and had only partial limbal stem cell deficiency. Previous studies24,31 have shown that corneal allografting may result in approximately a 90% survival rate in uncomplicated and low-risk eyes without the use of systemic immunosuppressive drugs. Some study6,12,20 findings have reported that a good HLA antigen match, in which 2 or more class I antigens were matched, provided long-term graft survival in corneal allografting. In developing nations, systemic immunosuppressive drugs, such as cyclosporin A, are costly, and long-term use seems impossible. Our study provides encouraging findings for patients with partial stem cell deficiency.
In summary, limbal allograft transplantation from living-related donors is effective for eyes with partial (≤50%) limbal stem cell deficiencies resulting from chemical burns. Partial stem cell deficiency may be the best indication for living-related limbal allografting. Moreover, the graft failure rate herein was low even without application of systemic immunosuppressants. Limbal allograft transplantation from living-related donors is a good option for individuals with bilateral partial stem cell deficiency.
Correspondence: Ting Huang, MD, PhD, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 Xianlie Rd, Guangzhou 510060, China (firstname.lastname@example.org).
Submitted for Publication: August 24, 2010; final revision received January 10, 2011; accepted January 17, 2011.
Author Contributions: Dr Huang had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Financial Disclosure: None reported.