Andre A.M. Torricelli

The corneal epithelial basement membrane: structure, function, and disease.

The corneal epithelial basement membrane (BM) is positioned between basal epithelial cells and the stroma. This highly specialized extracellular matrix functions not only to anchor epithelial cells to the stroma and provide scaffolding during embryonic development but also during migration, differentiation, and maintenance of the differentiated epithelial phenotype. Basement membranes are composed of a diverse assemblage of extracellular molecules, some of which are likely specific to the tissue where they function; but in general they are composed of four primary components--collagens, laminins, heparan sulfate proteoglycans, and nidogens--in addition to other components such as thrombospondin-1, matrilin-2, and matrilin-4 and even fibronectin in some BM. Many studies have focused on characterizing BM due to their potential roles in normal tissue function and disease, and these structures have been well characterized in many tissues. Comparatively few studies, however, have focused on the function of the epithelial BM in corneal physiology. Since the normal corneal stroma is avascular and has relatively low keratocyte density, it is expected that the corneal BM would be different from the BM in other tissues. One function that appears critical in homeostasis and wound healing is the barrier function to penetration of cytokines from the epithelium to stroma (such as transforming growth factor β-1), and possibly from stroma to epithelium (such as keratinocyte growth factor). The corneal epithelial BM is also involved in many inherited and acquired corneal diseases. This review examines this structure in detail and discusses the importance of corneal epithelial BM in homeostasis, wound healing, and disease.

Correlation between signs and symptoms of ocular surface dysfunction and tear osmolarity with ambient levels of air pollution in a large metropolitan area.

Purpose: To evaluate the effect of high levels of environmental air pollution on tear osmolarity and its possible correlation with clinical signs and symptoms. Methods: This was a panel study involving 71 taxi drivers and traffic controllers from São Paulo, Brazil. Mean individual levels of 24-hour exposure to nitrogen dioxide (NO2) and particulate matter smaller than 2.5 &mgr;m (PM2.5) were assessed on 4 different occasions. On the first and third visits, subjects were submitted to clinical evaluations including the administration of the Ocular Surface Disease Index questionnaire, slit-lamp examination, estimation of tear breakup time (BUT), the Schirmer test, and vital staining of the cornea and conjunctiva. On the second and fourth visits, tear samples were collected for osmolarity assays. Statistical analysis was performed using generalized estimating equations. Results: Although the taxi drivers and traffic controllers in our sample were exposed to high levels of NO2 and PM2.5, few symptoms were reported on the Ocular Surface Disease Index questionnaire. BUT values were reduced, whereas vital staining and Schirmer test mean results were within normal limits, despite considerable variability. A significant and negative correlation was found between PM2.5 levels and tear film osmolarity levels (P < 0.05). An increase of 10 &mgr;g/m3 in PM2.5 was associated with a 10.9 mOsm/kg decrease in tear osmolarity. There also was a negative correlation, although not statistically significant, between NO2 and tear osmolarity. Conclusions: Exposure to air pollution reduces tear film stability and influences tear film osmolarity. Combining clinical examination with the assessment of tear osmolarity may help understand ocular surface response to high levels of air pollution.

Transmission electron microscopy analysis of epithelial basement membrane repair in rabbit corneas with haze.

PURPOSE To assess the ultrastructure of the epithelial basement membrane using transmission electron microscopy (TEM) in rabbit corneas with and without subepithelial stroma opacity (haze). METHODS Two groups of eight rabbits each were included in this study. Photorefractive keratectomy (PRK) was performed using an excimer laser. The first group had -4.5-diopter (-4.5D) PRK and the second group had -9.0D PRK. Contralateral eyes were unwounded controls. Rabbits were sacrificed at 4 weeks after surgery. Immunohistochemical analysis was performed to detect the myofibroblast marker α-smooth muscle actin (SMA). TEM was performed to analyze the ultrastructure of the epithelial basement membrane and stroma. RESULTS At 4 weeks after PRK, α-SMA+ myofibroblasts were present at high density in the subepithelial stroma of rabbit eyes that had -9.0D PRK, along with prominent disorganized extracellular matrix, whereas few myofibroblasts and little disorganized extracellular matrix were noted in eyes that had -4.5D PRK. The epithelial basement membrane was irregular and discontinuous and lacking typical morphology in all corneas at 1 month after -9D PRK compared to corneas at 1 month in the -4.5D PRK group. CONCLUSIONS The epithelial basement membrane acts as a critical modulator of corneal wound healing. Structural and functional defects in the epithelial basement membrane correlate to both stromal myofibroblast development from precursor cells and continued myofibroblast viability, likely through the modulation of epithelial-stromal interactions mediated by cytokines. Prolonged stromal haze in the cornea is associated with abnormal regeneration of the epithelial basement membrane.

Cellular and extracellular matrix modulation of corneal stromal opacity

Stromal transparency is a critical factor contributing to normal function of the visual system. Corneal injury, surgery, disease and infection elicit complex wound healing responses that serve to protect against insults and maintain the integrity of the cornea, and subsequently to restore corneal structure and transparency. However, in some cases these processes result in prolonged loss of corneal transparency and resulting diminished vision. Corneal opacity is mediated by the complex actions of many cytokines, growth factors, and chemokines produced by the epithelial cells, stromal cells, bone marrow-derived cells, lacrimal tissues, and nerves. Myofibroblasts, and the disorganized extracellular matrix produced by these cells, are critical determinants of the level and persistence of stromal opacity after corneal injury. Decreases in corneal crystallins in myofibroblasts and corneal fibroblasts contribute to cellular opacity in the stroma. Regeneration of a fully functional epithelial basement membrane (BM) appears to have a critical role in the maintenance of corneal stromal transparency after mild injuries and recovery of transparency when opacity is generated after severe injuries. The epithelial BM likely has a regulatory function whereby it modulates epithelium-derived growth factors such as transforming growth factor (TGF) β and platelet-derived growth factor (PDGF) that drive the development and persistence of myofibroblasts from precursor cells. The purpose of this article is to review the factors involved in the maintenance of corneal transparency and to highlight the mechanisms involved in the appearance, persistency and regression of corneal opacity after stromal injury.

Ultrastructure of the Posterior Corneal Stroma

PURPOSE To reinvestigate the ultrastructure of the posterior stroma of the human cornea and to correlate the findings with the stromal behavior after big-bubble creation. DESIGN Observational consecutive 3-center case series. SPECIMENS Fresh corneoscleral buttons from human donors (n = 19) and organ-cultured corneoscleral buttons (n = 10) obtained after Descemets membrane endothelial keratoplasty. METHODS Corneal specimens were divided into central (3 mm), mid peripheral (8 mm), and peripheral parts by trephination and processed for transmission electron microscopic and immunohistochemical analyses. A big bubble was created by air injection into the stroma of organ-cultured corneas before fixation. MAIN OUTCOME MEASURES The distance of keratocytes to Descemets membrane, number of collagen lamellae between keratocytes and Descemets membrane, diameter and arrangement of collagen fibrils, thickness of stromal lamella created by air injection, and immunopositivity for collagen types III, IV, and VI. RESULTS Stromal keratocytes were observed at variable distances from Descemets membrane, increasing from 1.5 to 12 μm (mean, 4.97±2.19 μm) in the central, 3.5 to 14 μm (mean, 8.03±2.47 μm) in the midperipheral, and 4.5 to 18 μm (mean, 9.77±2.90 μm) in the peripheral regions. The differences in mean distances were significant (P < 0.0001). The number of collagen lamellae between Descemets membrane and most posterior keratocytes varied from 2 to 10 and the diameter of collagen fibrils averaged 23.5±1.8 nm and corresponded with that of the remaining stroma. A thin layer (0.5-1.0 μm thick) of randomly arranged, unaligned collagen fibers, which was positive for collagen types III and VI, was observed at the Descemet-stroma interface. The residual stromal sheet separated by air injection in 8 of 10 donor corneas varied in thickness from 4.5 to 27.5 μm, even within individual corneas (≤3-fold), and was composed of 5 to 11 collagen lamellae that revealed keratocytes on their anterior surface and in between. CONCLUSIONS Barring an anchoring zone of interwoven collagen fibers at the Descemet-stroma interface, the findings did not provide any evidence for the existence of a distinctive acellular pre-Descemets stromal layer in the human cornea. The intrastromal cleavage plane after pneumodissection seems to be nonreproducibly determined by the intraindividually and interindividually variable distances of keratocytes to Descemets membrane.

Surgical management of presbyopia.

Presbyopia, the gradual loss of accommodation that becomes clinically significant during the fifth decade of life, is a physiologic inevitability. Different technologies are being pursued to achieve surgical correction of this disability; however, a number of limitations have prevented widespread acceptance of surgical presbyopia correction, such as optical and visual distortion, induced corneal ectasia, haze, anisometropy with monovision, regression of effect, decline in uncorrected distance vision, and the inherent risks with invasive techniques, limiting the development of an ideal solution. The correction of the presbyopia and the restoration of accommodation are considered the final frontier of refractive surgery. The purpose of this paper is to provide an update about current procedures available for presbyopia correction, their advantages, and disadvantages.

Accelerated Corneal Collagen Cross-linking for Postoperative LASIK Ectasia: Two-Year Outcomes

PURPOSE To evaluate the effectiveness and safety of accelerated corneal collagen cross-linking for postoperative LASIK ectasia after 2 years. METHODS A prospective, single-center case series was performed with patients treated for postoperative LASIK ectasia. All eyes underwent accelerated corneal collagen cross-linking (CCL-Vario Crosslinking; Peschke Meditrade GmbH, Zurich, Switzerland) at 9 mW/cm(2) for 10 minutes. The main outcome measures were changes in uncorrected distance visual acuity, corrected distance visual acuity, central corneal thickness, corneal topography, and endothelial cell density. These parameters were assessed at baseline and at the 6-month and 1- and 2-year follow-up visit. RESULTS The study enrolled 40 eyes of 24 patients (15 male and 9 female) with a mean age of 33.8 ± 7.5 years (range: 24 to 52 years) that attained at least 2 years of follow-up. The surgical procedure was uneventful in all cases. All eyes stabilized after treatment without any further signs of progression and no statistically significant changes in the mean uncorrected distance visual acuity (P = .649), corrected distance visual acuity (P = .616), mean keratometry (P =.837), steep keratometry (P = .956), ultrasonic pachymetry (P = .135), slit-scanning pachymetry (P = .276), and endothelial cell density (P = .523). In addition, 72.5% of the patients presented stable or gains of Snellen lines over time. CONCLUSIONS Accelerated corneal collagen cross-linking seems to be safe and effective in halting postoperative LASIK ectasia progression after 2 years of follow-up. However, a longer follow-up period with a larger cohort is needed to validate these findings.

Epithelial basement membrane proteins perlecan and nidogen-2 are up-regulated in stromal cells after epithelial injury in human corneas

The epithelial basement membrane (BM) is a specialized extracellular matrix that has been shown to have a critical role in corneal development, wound healing, and disease. Although the epithelial BM contributes to corneal homeostasis, relatively little is know about non-epithelial production of its components that may be important in defective regeneration of the epithelial basement membrane associated with opacity after photorefractive keratectomy. The purpose of the current study was to investigate stromal production of corneal epithelial BM proteins in wounded human corneas using immunohistochemistry. A total of five unwounded control eyes and five 30-min epithelial-wounded corneas were obtained from fresh corneoscleral buttons removed from human eyes enucleated due to choroidal melanoma with normal anterior segments. In the wounded corneas, an eight mm patch of central corneal epithelium and epithelial BM was removed with a Beaver blade when the patient was under general anesthesia. Immunohistochemical analyses were performed to detect perlecan and nidogen-2 proteins-important components of the epithelial BM lamina lucida and lamina densa zones. Perlecan and nidogen-2 proteins were detected in the BM itself and at low levels in keratocytes in all unwounded corneas. After epithelial injury, both perlecan and nidogen-2 were expressed at high levels in stromal keratocytes, including superficial keratocytes in the early phases of apoptosis. Thus, after epithelial and epithelial BM injury, stromal keratocytes contribute important perlecan and nidogen-2 components to the regenerating epithelial BM.

Corneal Collagen Cross-linking in Advanced Keratoconus: A 4-Year Follow-up Study.

PURPOSE To analyze the safety and efficacy of standard corneal collagen cross-linking (CXL) in advanced cases of progressive keratoconus after 4 years of follow-up. METHODS A retrospective case series of patients with advanced progressive keratoconus (stages 3 and 4 of Amsler-Krumeich classification) underwent standard CXL treatment. The parameters examined were changes in uncorrected visual acuity (UDVA), corrected visual acuity (CDVA), keratometry values (mean, flat, steep, and apical), pachymetry, and endothelial cell count at the baseline and at 12, 24, and 48 months postoperatively. RESULTS Forty eyes of 40 patients were enrolled in the study. The mean patient age was 22.5 years (range: 15 to 37 years). Both mean UDVA and CDVA remained stable during the time points; no statistically significant change was noted. Although a slight reduction was observed in all keratometric readings, a statistically significant reduction was only reached in the apical keratometry (P = .037) at 4 years after CXL. A significant reduction in the corneal thickness was also found (ultrasonic: 388 ± 49 to 379 ± 48 μm; slit-scanning: 362 ± 48 to 353 ± 51 μm); however, this change was likely not clinically meaningful. Endothelial cell count was not significantly different at the end of the study. Treatment failure or progression was noted in two patients (5%) over the follow-up period. CONCLUSIONS Standard CXL treatment was safe and able to stabilize both visual acuity and topographic parameters at 4 years of follow-up in eyes with advanced keratoconus. [J Refract Surg. 2016;32(7):459-464.].

Codeine Plus Acetaminophen for Pain After Photorefractive Keratectomy: A Randomized, Double-blind, Placebo-controlled Add-on Trial

Background: Pain after photorefractive keratectomy (PRK) is significant, and the analgesic efficacy and safety of oral opioids in combination with acetaminophen has not been fully investigated in PRK trials. Purpose: To assess the efficacy and safety of the combination of codeine plus acetaminophen (paracetamol) versus placebo as an add-on therapy for pain control after PRK. Study design: Randomized, double-blind, placebo-controlled trial. Setting: Single tertiary center. Methods: One eye was randomly allocated to the intervention, whereas the fellow eye was treated with a placebo. Eyes were operated 2 weeks apart. The participants were adults older than 20 years with refractive stability for ≥1 year, who underwent PRK for correction of myopia or myopic astigmatism. Codeine (30 mg) plus acetaminophen (500 mg) was given orally 4 times per day for 4 days after PRK. The follow-up duration was 4 months. The study outcomes included pain scores at 1 to 72 hours, as measured by the visual analog scale, McGill Pain Questionnaire, and Brief Pain Inventory, as well as adverse events and corneal wound healing. Results: Of the initial 82 eyes, 80 completed the trial (40 intervention, 40 placebo). Median (interquartile range) pain scores as measured by the visual analog scale were statistically and clinically lower during treatment with codeine/acetaminophen compared with the placebo: 1 hour: 4 (2–4) versus 6 (3–6), P < 0.001; 24 hours: 4 (3–6) versus 7 (6–9), P < 0.001; 48 hours: 1 (0–2) versus 3 (2–5), P < 0.001; and 72 hours: 0 (0–0) versus 0 (0–2), P = 0.001. Virtually identical results were obtained by the McGill Pain Questionnaire and Brief Pain Inventory scales. The most common adverse events with codeine/acetaminophen were drowsiness (42%), nausea (18%), and constipation (5%). No case of delayed epithelial healing was observed in both treatment arms. Conclusions: When added to the usual care therapy, the oral combination of codeine/acetaminophen was safe and significantly superior to the placebo for pain control after PRK. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02625753.