Edgar M. Espana

How Does Amniotic Membrane Work

Transplantation of amniotic membrane as a temporary or permanent graft promotes epithelial wound healing and exerts potent anti-inflammatory and anti-scarring effects on the ocular surface. These actions depend on the killing of allogeneic amniotic cells and preservation of the cytokine-containing matrix during the preparation of the amniotic membrane. This review describes how these actions inherently operate in utero and how amniotic membrane transplantation aims to recreate such a fetal environment to exert these actions by insulating the surgical site from the host environment. These actions also render the amniotic membrane a unique niche capable of expanding both epithelial and mesenchymal progenitor cells ex vivo, while maintaining their normal cell phenotypes. As a result, the amniotic membrane becomes an ideal substrate for engineering different types of ocular surface tissues for transplantation. Further studies investigating the exact molecular mechanism by which the amniotic membrane works will undoubtedly unravel additional applications in reconstruction and engineering of both ocular and nonocular tissues in the burgeoning field of regenerative medicine.

PHENOTYPIC STUDY OF A CASE WITH SUCCESSFUL TRANSPLANTATION OF EX VIVO EXPANDED HUMAN LIMBAL EPITHELIUM FOR UNILATERAL TOTAL LIMBAL STEM CELL DEFICIENCY

OBJECTIVE To minimize the risk to the donor eye when a conjunctival limbal autograft is performed for unilateral total limbal stem cell deficiency (LSCD), a new approach has been reported of expanding limbal epithelial progenitor cells from a small limbal biopsy cultured on amniotic membrane (AM). Herein, we present for the first time the morphologic and phenotypic outcome of one such patient. DESIGN Interventional case report. METHODS A 31-year-old male with a severe acid burn to his left eye received AM transplantation at the acute stage and a keratolimbal allograft (KLAL) at the chronic stage for total LSCD. As an alternative to combat the failed KLAL, the above-mentioned new surgical procedure was performed. The corneal button, obtained after a penetrating keratoplasty performed 5.5 months later, and a normal corneal button as a control were submitted to hematoxylin-eosin and immunofluorescence staining for keratin K3, connexin 43, goblet-cell mucin MUC 5AC, laminin 5, and integrins alpha3beta1 and alpha6beta4. MAIN OUTCOME MEASURES Clinical and immunohistologic features. RESULTS The resultant epithelium was stratified with five to six cell layers and anchored to laminin 5 of the amniotic basement membrane via integrins alpha3beta1 and alpha6beta4 in a manner similar to the normal corneal epithelium. Intriguingly, the epithelial phenotype was limbal and not corneal, based on the negative expression of keratin K3 and connexin 43 of the basal epithelium. CONCLUSIONS The technique described ensures the preservation of amniotic basement membrane, which allows formation of adhesion complexes and maintains normal corneal architecture. The preservation of a limbal epithelial phenotype on the reconstructed corneal surface indicates that AM provides a unique stromal environment conducive to the preservation and expansion of limbal epithelial progenitor cells.

The challenge of dry eye diagnosis

The currently available methods for the diagnosis of dry eye are still far from being perfect for a variety of reasons. This review attempts to highlight the advantages and disadvantages of both traditional tests (such as Schirmer’s test, break-up time and ocular surface staining) and innovative noninvasive procedures, including tear meniscus height measurement, corneal topography, functional visual acuity, tear interferometry, tear evaporimetry and tear osmolarity assessment.

Amniotic membrane transplantation for reconstruction of the conjunctival fornices

PURPOSE To describe the clinical outcome of amniotic membrane transplantation (AMT) for fornix reconstruction in a variety of ocular surface disorders. DESIGN Noncomparative interventional case series. PARTICIPANTS Seventeen eyes in 15 patients with symblepharon. Four eyes had ocular-cicatricial pemphigoid, two eyes had symblepharon after pterygium excision, four eyes had chemical or mechanical trauma, two eyes had strabismus surgery, two eyes (one patient) had Stevens-Johnson syndrome, one eye had toxic epidermal necrolysis, and two eyes (one patient) had chronic allergic conjunctivitis. INTERVENTION The subconjunctival scar tissue was dissected from the episclera, and the freed conjunctival flap was recessed to the fornix. A layer of amniotic membrane (AM) was applied to cover the exposed episclera. The fornical edge of the membrane was anchored with sutures passing through the full thickness of the lid. MAIN OUTCOME MEASURES A deep conjunctival fornix, lack of motility restriction. RESULTS The mean follow-up period was 37 +/- 24 months (range, 9-84 months). Complete fornix reconstruction was demonstrated in 12 of 17 eyes (70.6%), whereas 2 eyes had a partial success, and 3 eyes (3 patients) had recurrence of symblepharon with restricted motility. In eyes that demonstrated partial success or failure, the underlying etiology was either an autoimmune disorder or a recurrent pterygium. The most successful outcome was observed in eyes with symblepharon associated with trauma. CONCLUSIONS AMT is an effective method of fornix reconstruction for the repair of symblepharon in a variety of ocular surface disorders. Future modifications, including an epithelial cellular component on the AM (conjunctival autograft or ex vivo expanded epithelial stem cells) may improve the outcome of this surgical procedure.

Clinical characteristics of conjunctivochalasis with or without aqueous tear deficiency

Aim: To show characteristic ocular surface findings caused by conjunctivochalasis (CCh) in dry eye patients with or without aqueous tear deficiency (ATD). Design: Comparative non-interventional cases. Patients and methods: Clinical data of five ATD patients without CCh (group A), eight CCh patients with ATD (group B), and eight CCh patients without ATD (group C) were retrospectively reviewed. Presence or absence of CCh was determined by fluorescein staining to outline tear meniscus and conjunctival folds with an enhancing filter. Dry eye symptoms, history of subconjunctival haemorrhage, meibum expression, tear break up time, fluorescein and rose bengal staining, and fluorescein clearance test, and other abnormal ocular surface findings were measured. Results: CCh patients were significantly older (p = 0.001). In pure ATD, the principal symptom of dryness became worse as the day progressed. In contrast, blurry vision, burning sensation, and dryness became worse during reading in all CCh patients (p = 0.0008) or worse in the morning upon awakening in the majority patients with CCh only (p = 0.02). Besides the interpalpebral exposure, which was noted in ATD, positive fluorescein or rose bengal staining was noted in the redundant conjunctival folds and the non-exposure zone in CCh (p = 0.0008). Redundant conjunctival folds were present in both lower and upper bulbar conjunctiva, obliterating both lower and upper tear meniscuses, and spatially correlated with anterior migration of the mucocutaneous junction in CCh. Delayed tear clearance was significantly more prevalent in CCh than ATD (p = 0.0008). Vigorous blinking worsened in CCh but not in ATD (p = 0.0008). Lacrimal puncta were swollen in groups B and C, but not in group A (p = 0.04). Conclusions: CCh is not restricted to the lower bulbar conjunctiva, and contributes to pathogenesis of dry eye by obliterating both lower and upper tear meniscus, causing unstable tear film and by creating delayed tear clearance. Dry eye symptoms were worsened by downgaze during reading and by vigorous blinking. Other characteristic signs including subconjunctival haemorrhage, swollen puncta, anterior migration of the mucocutaneous junction, and patterns of dye staining also help distinguish dry eye associated with CCh from that caused by ATD alone.

Amniotic membrane transplantation for reconstruction after excision of large ocular surface neoplasias

Aim: To evaluate the clinical outcome of patients in whom ocular surface reconstruction was performed using amniotic membrane transplantation (AMT) after the excision of large (>20 mm square) ocular surface neoplasias (OSN). Methods: A non-comparative interventional case series. In 16 eyes of 16 patients, excision of large OSN including conjunctival intraepithelial neoplasia (CIN), primary acquired melanosis, and malignant melanoma was followed by adjunctive cryotherapy and suturing of a single layer of amniotic membrane (AM) with the basement membrane side facing up to the healthy bordering tissue. Epithelial healing, complications, and tumour recurrences were analysed. Results: During a mean follow up of 23.7 (SD 11, range 11–43) months, ocular surface healing was rapid and complete in all cases. One complication of pyogenic granuloma was noted. Tumour recurrence occurred in one out of 10 CIN cases (10%), no recurrences were observed in the patients with melanotic lesions. Conclusions: AMT in lieu of conjunctival or mucosal autograft is an effective substrate for reconstructing the ocular surface following excision of large OSN. AMT is effective in managing large OSN by avoiding the complications that may be associated with conventional removal, specifically in cases where the limbal architecture is destroyed by surgical resection or adjuvant therapies.

Amniotic membrane transplantation with conjunctival limbal autograft for total limbal stem cell deficiency.

PURPOSE To evaluate the outcomes of corneal surface reconstruction with conjunctival limbal autograft when combined with amniotic membrane transplantation on both the donor and recipient eyes. DESIGN Retrospective, noncomparative, interventional small case series. PARTICIPANTS Five eyes of five patients with total limbal stem cell deficiency (LSCD) resulting from pseudopemphigoid (n = 1), chemical burns (n = 3), and extensive removal of conjunctival intraepithelial neoplasia (n = 1) were operated on by one surgeon (SCGT). INTERVENTION After the removal of fibrovascular pannus from the corneal surface, two conjunctival limbal free grafts were harvested from the fellow eyes in all five patients with unilateral LSCD. Amniotic membrane, with the basement membrane side up, was grafted onto the defect created at the donor site and onto the recipient corneal and limbal sclera before placement of conjunctival limbal grafts. MAIN OUTCOME MEASURES Symptomatic relief, improvement in visual acuity, fornix deepening, and rapid healing and restoration of normal cornea and limbus in the recipient and donor eyes were assessed. RESULTS During the mean follow-up of 22 months (range, 11-48 months), all eyes experienced symptomatic relief. All recipient eyes had a mean improvement in visual acuity of nine lines (range, 7-12). The three eyes with stromal vascularization showed regression, and all recipient eyes had marked improvement in corneal clarity. Three eyes receiving simultaneous symblepharon lysis and fornix reconstruction successfully regained deep, stable fornices. The donor eyes showed rapid healing and restoration of the normal limbal landmark, even in one eye where nearly the entire limbus was removed. CONCLUSIONS Limbal conjunctival transplantation is an effective procedure for restoring the corneal surface integrity in eyes with total LSCD. The additional use of amniotic membrane may contribute to a higher rate of success in the recipient eye and a lower rate of complications in the donor eye, as well as allow the simultaneous correction of concomitant cicatricial abnormalities.

Intrastromal Invasion by Limbal Epithelial Cells Is Mediated by Epithelial-Mesenchymal Transition Activated by Air Exposure

Corneal epithelial stem cells are located in the basal layer of the limbus between the cornea and the conjunctiva. Regulation of these limbal epithelial progenitor cells by the stromal niche dictates corneal surface health. To further characterize this process, limbal explants were cultured at the air-fluid interface, termed air-lifting, to stimulate the niche. As compared to submerged cultures, air-lifting significantly promoted epithelial stratification, migration, proliferation, and intrastromal invasion by limbal epithelial cells. Epithelial intrastromal invasion was noted when the limbal, but not corneal, epithelium was recombined with the limbal stroma containing live, but not dead, cells. Invading limbal basal cells displayed up-regulated nuclear expression of p63 and Ki67, down-regulated E-cadherin and cornea-specific keratin 3, and switched expression of beta-catenin from intercellular junctions to the nucleus and cytoplasm, indicating the activation of the Wnt/beta-catenin pathway. Invaded cells isolated by collagenase from the stroma of air-lifted, but not submerged, explants showed vivid clonal growth on 3T3 fibroblast feeder layers and complete epithelial-mesenchymal transition by expressing nuclear p63 and cytoplasmic S100A4. These findings collectively suggest that epithelial-mesenchymal transition via the Wnt/beta-catenin pathway influences the fate of limbal epithelial cells, likely to be progenitor cells, between regeneration and fibrosis when the stromal niche is activated.

Keratolimbal allograft in corneal reconstruction

AbstractThe replenishment of corneal epithelial SC is a crucial step for reconstructing the ocular surface in patients suffering from devastating ocular surface diseases manifesting with total LSCD. KLAL is one of such procedures and has a long track record and a long follow-up for patients with bilateral total LSCD. This review summarizes the literature experiences and outline new strategies that are important to enhance the success of this procedure. Further research is needed to fully understand the biological processes involved in allogeneic tissue transplantation for preserving epithelial SC adhesion, migration, and survival.

Idiopathic limbal stem cell deficiency.

PURPOSE To describe and to characterize the clinical findings and prognosis of patients with idiopathic limbal stem cell deficiency (LSCD). DESIGN Retrospective case series. PARTICIPANTS/METHODS We reviewed records from seven patients whose LSCD had been diagnosed clinically and confirmed by impression cytology and in whom the cause of LSCD was never identified. A detailed history, clinical records, and results of slit-lamp biomicroscopy, photography, vital staining, and impression cytology were evaluated. RESULTS Six of seven patients (86%) were women, indicating a female predominance. Two patients were from the same family, whereas one other had a positive family history. Severe photophobia was noted in all patients and reduced vision in three patients. The main clinical findings included superficial vascularization, worse in the superior followed by the inferior and nasal cornea. The limbal regions showed a loss of limbal palisades of Vogt, and the adjacent peripheral cornea revealed an irregular and hazy epithelium with positive late fluorescein staining and the presence of conjunctival goblet cells by impression cytology. LSCD was bilateral in all patients but asymmetric in four. During a mean follow-up of 6.1+/- 3.8 years, the visual acuity decreased in both eyes of one patient after cataract extraction and in both eyes of two other patients without surgery. The process of conjunctivalization advanced in four patients (57%) and remained stable in three (43%) without surgical intervention. CONCLUSIONS Idiopathic LSCD is a rare and as yet poorly recognized clinical entity, and the findings reported herein may help explore how progressive loss of limbal stem cells occurs. Correct diagnosis of idiopathic LSCD is important so that the patient will not be subjected to unnecessary surgeries, which may actually severely worsen the clinical course.