Coculture of autologous limbal and conjunctival epithelial cells to treat severe ocular surface disorders: long-term survival analysis.

BACKGROUND: Cultivated limbal epithelium for reconstruction of corneal surface is a well-established procedure; however, it is not adequate for damage which also extensively involves the conjunctiva. In severe cases of ocular surface damage that warrant additional conjunctival transplantation apart from cultivated limbal stem cell transplantation, we describe the long-term survival of a novel method of cocultivating autologous limbal and conjunctival epithelium on a single substrate. MATERIALS AND METHODS: Forty eyes of 39 patients with severe limbal stem cell deficiency and conjunctival scarring or symblepharon underwent transplantation of autologous cocultivated epithelium on human amniotic membrane. A ring barrier was used to segregate the central limbal and peripheral conjunctival epithelia in vitro. Patients were followed up at regular intervals to assess stability of the ocular surface, defined by absence of conjunctivalization into the central 4 mm of the cornea and absence of diffuse fluorescein staining. Penetrating keratoplasty (PKP) was subsequently performed, where indicated, in patients with surface stability. RESULTS: The cumulative survival probability was 60% at 1 year and 45% at 4 years by Kaplan-Meier analysis (mean follow-up duration: 33 ± 29 months, range: 1-87 months). Best-corrected visual acuity improved to greater than 20/200 in 38% eyes at the last follow-up, compared with 5% eyes before surgery. Immunohistochemistry in five of the corneal buttons excised for PKP showed an epithelial phenotype similar to cornea in all five. CONCLUSIONS: Synchronous use of cultured limbal and conjunctival epithelium offers a feasible alternative and a simpler one-step surgical approach to treat severe ocular surface disorders involving limbus and conjunctiva.

Presentation, diagnosis and management of limbal stem cell deficiency.

The human corneal surface epithelium is continuously repopulated by the limbal stem cells (LSCs). Limbal Stem Cell Deficiency (LSCD) can lead to corneal opacity and vascularization, with consequent visual impairment or blindness. Many acquired and congenital diseases can lead to LCSD by direct injury to the LSCs, destruction of LSC niche, or both. Based on the severity of the disease, LSCD can present with various symptoms and signs. Although LSCD can be detected clinically, laboratory tests are necessary to confirm the diagnosis and monitor the disease progression. This article concisely reviews the clinical presentation, techniques for diagnosis and management of LSCD.

Cultivated limbal epithelial transplantation in children with ocular surface burns.

IMPORTANCE: Although several reports are available on the use of conventional and cultured limbal epithelium using various substrates in the treatment of limbal stem cell deficiency (LSCD), the patient populations studied have been largely adults. Thus, to our knowledge, the outcomes of this procedure exclusively in a pediatric population have not been reported previously. OBJECTIVE: To report the outcomes of autologous ex vivo cultivated limbal epithelial transplantation (CLET) in pediatric patients with LSCD after ocular burns. DESIGN AND SETTING: A retrospective, interventional case series of patients treated at the L. V. Prasad Eye Institute, Hyderabad, India. PARTICIPANTS: Children up to 15 years with LSCD secondary to chemical or thermal injury who underwent CLET from April 1, 2001, through June 31, 2010, with a follow-up of at least 1 year, were included in the study. INTERVENTION: After a limbal biopsy specimen obtained from a healthy area of the limbus, the limbal epithelial cells were cultured on a denuded human amniotic membrane substrate using a xeno-free explant culture technique. A monolayer of cultivated epithelial cells along with the amniotic membrane was transplanted on the patient's affected eye after pannus excision. In cases of failure, the same procedure was repeated. MAIN OUTCOMES AND MEASURES: Ocular surface stability and visual improvement were the primary and secondary outcome measures, respectively. Success was defined as a stable corneal epithelium without conjunctivalization. Eyes with conjunctivalization and persistent epithelial defects were classified as failures. RESULTS: Of the 107 eyes of 107 patients included in this study, 73 eyes (68.2%) underwent 1 and 34 eyes (31.8%) underwent 2 autologous CLET procedures. At a mean follow-up of 3.4 years, 50 eyes (46.7%) achieved completely epithelialized, avascular, and stable ocular surfaces. At the final visit, 58 eyes (54.2%) had improvement in visual acuity of 0.2 or more logMAR units. CONCLUSIONS: Autologous CLET was successful in restoring the ocular surface and improving vision in almost half of the children blinded by ocular burns.

In vivo confocal microscopic findings in Lisch corneal dystrophy.

PURPOSE: To describe the in vivo confocal microscopic and clinicopathologic correlations in Lisch corneal dystrophy. METHODS: This is a retrospective case series of 2 patients with Lisch corneal dystrophy. The diagnosis was made based on clinical findings in both cases and was confirmed histopathologically following epithelial debridement in case 1. In vivo laser scanning confocal microscopy using the Heidelberg Retina Tomograph III with the Rostock Cornea Module was carried out in both cases. RESULTS: Clinical examination of the corneas revealed areas of epithelial opacification that were sharply demarcated in juxtaposition with normal corneal epithelium. The gray feathery appearance of the epithelial lesions in both cases was characteristic of Lisch corneal dystrophy. The central visual axis was involved in case 1, and corneal topography showed irregular astigmatism. Histological analysis of the epithelial cells in this case showed intracytoplasmic vacuoles, confirming the diagnosis of Lisch corneal dystrophy. In vivo confocal microscopy in both cases demonstrated highly hyperreflective epithelial cytoplasm with hypo-reflective nuclei. There was involvement of all epithelial layers extending to the limbus and findings on imaging were confined to the clinically observed areas of corneal opacity. The lesion in case 1 recurred after epitheliectomy of the central cornea without removal of affected limbal cells. CONCLUSIONS: The unique features on in vivo confocal microscopy correlated with the clinical and histopathologic features of Lisch corneal dystrophy may be used to distinguish this disorder from other corneal epithelial conditions. The affected epithelial cells appear to originate from abnormal limbal stem cells.

Characterization of limbal stem cell deficiency by in vivo laser scanning confocal microscopy: a microstructural approach.

OBJECTIVE: To evaluate the cellular changes in the corneal epithelium and surrounding structures in limbal stem cell deficiency (LSCD) by using in vivo laser scanning confocal microscopy. METHODS: This was a prospective comparative study that included 27 eyes of 20 patients with LSCD and 12 eyes of 10 healthy subjects. All subjects underwent slitlamp examination, and LSCD was classified into 3 groups on the basis of clinical presentation. Confocal imaging of the central cornea and 4 locations of limbus was performed. Morphologic characteristics of the corneal epithelium were studied. The basal epithelial cell density and subbasal nerve density in the central cornea were calculated, and a potential correlation between the decrease in basal epithelial cell density and subbasal nerve density in LSCD was investigated. RESULTS: The wing and basal epithelial cells became progressively metaplastic, and the basal epithelial cell density and subbasal nerve density in the early and intermittent stages decreased significantly compared with controls (all P < .01). Normal basal epithelial cell morphology was completely lost and subbasal nerves were absent in the late stage of LSCD. The decrease in basal cell density correlated with the decrease in subbasal nerve density in patients with LSCD (P = .03). CONCLUSIONS: There are significant microstructural changes associated with early LSCD. These cellular changes could help to understand the disease process and classify and monitor limbal stem cell dysfunction.

Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study.

PURPOSE: Ocular burns can damage the corneal epithelial stem cells located at the limbus. This study evaluated the efficacy of xeno-free autologous cell-based treatment of limbal stem cell deficiency. METHODS: This retrospective study included 200 patients, above 8 years of age, with clinically diagnosed unilateral total limbal stem cell deficiency due to ocular surface burns treated between 2001 and 2010. A small limbal biopsy was obtained from the unaffected eye. The limbal epithelial cells were expanded ex vivo on human amniotic membrane for 10-14 days using a xeno-free explant culture system. The resulting cultured epithelial monolayer and amniotic membrane substrate were transplanted on to the patient's affected eye. Postoperative corneal surface stability, visual improvement and complications were objectively analysed. RESULTS: A completely epithelised, avascular and clinically stable corneal surface was seen in 142 of 200 (71%) eyes at a mean follow-up of 3 ± 1.6 (range: 1-7.6) years. A two-line improvement in visual acuity, without further surgical intervention, was seen in 60.5% of eyes. All donor eyes remained healthy. CONCLUSIONS: Autologous cultivated limbal epithelial transplantation using a xeno-free explant culture technique was effective in long-term restoration of corneal epithelial stability and improvement of vision in eyes with ocular surface burns.

The Boston keratoprosthesis in the management of corneal limbal stem cell deficiency.

PURPOSE: To report the outcomes of the Boston type I keratoprosthesis (Massachusetts Eye and Ear Infirmary, Boston, MA) in the management of corneal limbal stem cell deficiency (LSCD). METHODS: A retrospective review of all cases of keratoprosthesis implantation performed by a single surgeon (A.J.A.) between May 1, 2004, and March 31, 2010. Procedures performed for the management of LSCD were identified, and data were collected regarding the patients' ocular history, surgical procedure(s) performed, and postoperative outcomes, including interval visual acuities, retention, complications, and required surgical procedures. The outcome data from patients with LSCD were compared with those of individuals who underwent keratoprosthesis implantation for indications other than LSCD. RESULTS: Twenty-eight keratoprostheses were performed in 23 eyes of 22 patients with LSCD. The most common indications for surgery were chemical injury (7 eyes) and Stevens-Johnson syndrome (6 eyes). Preoperative corrected distance visual acuity (CDVA) was better than counting fingers in 4% of eyes with LSCD and in 9% of eyes without LSCD. Postoperative CDVA was 20/50 or better in 69%, 88%, and 67% of eyes with LSCD at 1, 2, and 3 years after keratoprosthesis implantation, respectively. Each of these percentages is higher than the percentage of eyes without LSCD obtaining CDVA of 20/50 or better at each time point. Whereas the retention failure rate in eyes with LSCD (0.148/eye-year) was higher than the rate in eyes without LSCD (0.114/eye-year), when eyes with Stevens-Johnson syndrome were excluded from the LSCD group, the non-Stevens-Johnson syndrome LSCD retention failure rate (0.056/eye-year) was half that of the non-LSCD group. The most common postoperative complications in eyes with LSCD were persistent corneal epithelial defect (PED) formation (56.5% of eyes) and sterile corneal necrosis (30%), whereas retroprosthetic membrane formation (46%) was the most common postoperative complication in eyes without LSCD, followed by PED formation (23%). The development of a PED was found to be a significant risk factor for sterile corneal stromal necrosis and infectious keratitis. CONCLUSION: Boston type I keratoprosthesis implantation results in a significant improvement in CDVA in the majority of patients with LSCD, with CDVA of 20/50 or better in more than two-thirds of eyes up to 3 years after surgery. PED formation is the most common postoperative complication in eyes with LSCD and is associated with an increased rate of sterile stromal necrosis and a lower retention rate in eyes undergoing keratoprosthesis implantation for immune-mediated LSCD. These results support the use of the Boston type I keratoprosthesis in managing bilateral, non-immune-mediated LSCD.

Clinical outcomes of penetrating keratoplasty after autologous cultivated limbal epithelial transplantation for ocular surface burns.

PURPOSE: To report the clinical outcomes of penetrating keratoplasty (PK) after autologous cultivated limbal epithelial transplantation in eyes with limbal stem cell deficiency (LSCD) after ocular surface burns. DESIGN: Retrospective case series. METHODS: This study included 47 patients with unilateral LSCD treated by autologous cultivated limbal epithelial transplantation and PK between 2001 and 2010. PK was performed either along with (single-stage; n = 12) or at least 6 weeks after (2-stage; n = 35) limbal transplantation. The primary outcome measure was corneal allograft survival, and failure was defined clinically as loss of central graft clarity. Secondary outcomes were postoperative Snellen visual acuity and complications. RESULTS: Most patients were young (mean age, 18 ± 11.4 years) males (76.6%) with LSCD resulting from alkali burns (78.7%) and with visual acuity less than 20/200 (91.5%). The mean follow-up was 4.2 ± 1.9 years. Kaplan-Meier corneal allograft survival rate at 1 year was significantly greater in eyes undergoing 2-stage limbal and corneal transplantation (80 ± 6%; median survival, 4 years) compared with single-stage limbal and corneal transplantation (25 ± 13%; median survival, 6 months; P = .0003). Visual acuity of 20/40 or better was attained by 71.4% of eyes with clear corneal grafts. Allograft failure occurred in 26 (60.5%) eyes as a result of graft rejection (57.7%), graft infiltrate (26.9%), or persistent epithelial defects (15.4%). Recurrence of LSCD was more common after single-stage (58.3%) than 2-stage (14.3%) surgery (P = .008). CONCLUSIONS: The 2-stage approach of autologous cultivated limbal epithelial transplantation followed by PK successfully restores ocular surface stability and vision in eyes with chronic ocular burns. The single-stage approach is associated with poorer clinical outcomes and should be avoided.

New endothelial keratoplasty, phacoemulsification, and intraocular lens implantation triple procedure: comparison with conventional triple procedure.

PURPOSE: To compare the clinical outcomes of a new triple procedure comprising endothelial keratoplasty, phacoemulsification, and intraocular lens (IOL) implantation and a conventional triple procedure comprising penetrating keratoplasty, extracapsular cataract extraction, and IOL implantation. SETTING: Cornea Services, Medical Research Foundation, Chennai, Tamil Nadu, India. METHODS: In this prospective nonrandomized study, the new triple procedure was performed in eyes with predominant endothelial dysfunction and coexisting cataract and the conventional triple procedure, in eyes with opacities affecting other layers of the cornea. Outcome measures were intraoperative and postoperative complications, 3-month postoperative distance visual acuity, refractive status, mean corneal power, corneal topography, and endothelial cell loss. The outcomes in the 2 groups were compared. RESULTS: The new procedure group comprised 54 eyes and the conventional procedure group, 26 eyes. All grafts in both groups were clear 3 months postoperatively. The IOL was fixated in the capsular bag in all eyes in the new procedure group and 23% of eyes in the conventional procedure group. Postoperatively, persistent epithelial defects, uveitis, glaucoma, and posterior capsule opacification were significantly more frequent in the conventional procedure group. Visual acuity was significantly better and the postoperative refractive status more predictable in the new procedure group. Endothelial cell loss was greater in the new procedure group than in the conventional group, although the difference was not statistically significant. CONCLUSION: The new triple procedure was safer than the conventional triple procedure and resulted in better visual outcomes, a more predictable refractive error, a smoother corneal contour, and a tectonically stronger globe. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.