Preoperative Predictors for Acute Pain After Photorefractive Keratectomy.

PURPOSE: The aim of this study was to identify preoperative predictors for the occurrence of early severe postoperative pain in patients undergoing photorefractive keratectomy (PRK). The implementation of preoperative screening methods may facilitate more specific or aggressive pain therapies specifically targeted to individuals at a high risk of experiencing severe postoperative pain. METHODS: This was exploratory research that included patients who underwent PRK. Before PRK, patients were administered a sociodemographic questionnaire, the Pain Catastrophizing Scale, and the State-Trait Anxiety Inventory and underwent corneal sensitivity and conditioned pain modulation (CPM) tests. Post-PRK pain was assessed using a pain intensity visual analog scale (VAS), and the short-form McGill Pain Questionnaire (SF-MPQ) was completed 21 days before PRK and 1, 24, 48, and 72 hours after PRK. Spearman correlations were calculated for pain scores and preoperative predictors. RESULTS: This research included 34 eyes of 34 patients. Preoperative corneal sensitivity was positively correlated with post-PRK pain scores as assessed by VAS and SF-MPQ (rho = 0.39 and rho = 0.41, respectively, P < 0.05). No correlations were found between Pain Catastrophizing Scale, State-Trait Anxiety Inventory, and CPM scores and post-PRK pain scores ( P > 0.05). CONCLUSIONS: Abnormal presurgical corneal sensitivity was a protective marker for severe pain after PRK, while scores as assessed by VAS and SF-MPQ and CPM were not related to postoperative pain.

Corneal epithelial basement membrane: Structure, function and regeneration.

Basement membranes are highly specialized extracellular matrices. More than providing scaffolds, basement membranes are recognized as dynamic and versatile structures that modulate cellular responses to regulate tissue development, function, and repair. Increasing evidence suggests that, in addition to providing structural support to adjacent cells, basement membranes serve as reservoirs and modulators of growth factors that direct and fine-tune cellular functions. Since the corneal stroma is avascular and has a relatively low keratocyte density, it's likely that the corneal BM is different in composition from the BMs in other tissues. BMs 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 fibronectin. Severe injuries to the cornea, including infection, surgery, and trauma, may trigger the development of myofibroblasts and fibrosis in the normally transparent connective tissue stroma. Ultrastructural studies have demonstrated that defective epithelial basement membrane (EBM) regeneration after injury to the cornea underlies the development of myofibroblasts from both bone marrow- and keratocyte-derived precursor cells. Defective EBM permits epithelium-derived and tear-derived transforming growth factor beta (TGF-ß), platelet-derived growth factor (PDGF), and possibly other modulators, to penetrate the stroma at sustained levels necessary to drive the development and persistence of vimentin + alpha-smooth muscle actin + desmin+ (V + A + D+) mature myofibroblasts. A recent discovery that has contributed to our understanding of haze development is that keratocytes and corneal fibroblasts produce critical EBM components, such as nidogen-1, nidogen-2 and perlecan, that are essential for complete regeneration of a normal EBM once laminin secreted by epithelial cells self-polymerizes into a nascent EBM. Mature myofibroblasts that become established in the anterior stroma are a barrier to keratocyte/corneal fibroblast contributions to the nascent EBM. These myofibroblasts, and the opacity they produce, often persist for months or years after the injury. Transparency is subsequently restored if the EBM is fully regenerated, myofibroblasts are deprived of TGF-ß and undergo apoptosis, and keratocytes reoccupy the anterior stroma and reabsorb the disordered extracellular matrix.

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.

Injury and defective regeneration of the epithelial basement membrane in corneal fibrosis: A paradigm for fibrosis in other organs?

Myofibroblast-mediated fibrosis is important in the pathophysiology of diseases in most organs. The cornea, the transparent anterior wall of the eye that functions to focus light on the retina, is commonly affected by fibrosis and provides an optimal model due to its simplicity and accessibility. Severe injuries to the cornea, including infection, surgery, and trauma, may trigger the development of myofibroblasts and fibrosis in the normally transparent connective tissue stroma. Ultrastructural studies have demonstrated that defective epithelial basement membrane (EBM) regeneration after injury underlies the development of myofibroblasts from both bone marrow- and keratocyte-derived precursor cells in the cornea. Defective EBM permits epithelium-derived transforming growth factor beta, platelet-derived growth factor, and likely other modulators, to penetrate the stroma at sustained levels necessary to drive the development of vimentin+ alpha-smooth muscle actin+ desmin+ (V+A+D+) mature myofibroblasts and promote their persistence. Defective versus normal EBM regeneration likely relates to the severity of the stromal injury and a resulting decrease in fibroblasts (keratocytes) and their contribution of EBM components, including laminin alpha-3 and nidogen-2. Corneal fibrosis may resolve over a period of months to years if the inciting injury is eliminated through keratocyte-facilitated regeneration of normal EBM, ensuing apoptosis of myofibroblasts, and reorganization of disordered extracellular matrix by repopulating keratocytes. We hypothesize the corneal model of fibrosis associated with defective BM regeneration and myofibroblast development after epithelial or parenchymal injury may be a paradigm for the development of fibrosis in other organs where chronic injury or defective BM underlies the pathophysiology of disease.

Regeneration of Defective Epithelial Basement Membrane and Restoration of Corneal Transparency After Photorefractive Keratectomy.

PURPOSE: To study regeneration of the normal ultrastructure of the epithelial basement membrane (EBM) in rabbit corneas that had -9.00 D photorefractive keratectomy (PRK) and developed late haze (fibrosis) with restoration of transparency over 1 to 4 months after surgery and in corneas that had incisional wounds. METHODS: Twenty-four rabbits had one of their eyes included in one of the two procedure groups (-9.00 D PRK or nearly full-thickness incisional wounds), whereas the opposite eyes served as the unwounded control group. All corneas were evaluated with slit-lamp photographs, transmission electron microscopy, and immunohistochemistry for the myofibroblast marker alpha-smooth muscle actin and collagen type III. RESULTS: In the -9.00 D PRK group, corneas at 1 month after surgery had dense corneal haze and no evidence of regenerated EBM ultrastructure. However, by 2 months after surgery small areas of stromal clearing began to appear within the confluent opacity (lacunae), and these corresponded to small islands of normally regenerated EBM detected within a larger area of the excimer laser-ablated zone with no evidence of normal EBM. By 4 months after surgery, the EBM was fully regenerated and the corneal transparency was completely restored in the ablated zone. In the incisional wound group, the two dense, linear corneal opacities were observed at 1 month after surgery and progressively faded by 2 and 3 months after surgery. The EBM ultrastructure was fully regenerated at the site of the incisions, including around epithelial plugs that extended into the stroma, by 1 month after surgery in all eyes. CONCLUSIONS: In the rabbit model, spontaneous resolution of corneal fibrosis (haze) after high correction PRK is triggered by regeneration of EBM with normal ultrastructure in the excimer laser-ablated zone. Conversely, incisional wounds heal in rabbit corneas without the development of myofibroblasts because the EBM regenerates normally by 1 month after surgery. [J Refract Surg. 2017:33(5):337-346.].

EBM regeneration and changes in EBM component mRNA expression in stromal cells after corneal injury.

PURPOSE: To investigate the production of the epithelial basement membrane (EBM) component mRNAs at time points before lamina lucida and lamina densa regeneration in anterior stromal cells after corneal injury that would heal with and without fibrosis. METHODS: Rabbit corneas were removed from 2 to 19 days after -4.5D or -9.0D photorefractive keratectomy (PRK) with the VISX S4 IR laser. Corneas were evaluated with transmission electron microscopy (TEM) for full regeneration of the lamina lucida and the lamina densa. Laser capture microdissection (LCM) based quantitative real-time (RT)-PCR was used to quantitate the expression of mRNAs for laminin α-3 (LAMA3), perlecan, nidogen-1, and nidogen-2 in the anterior stroma. RESULTS: After -4.5D PRK, EBM was found to be fully regenerated at 8 to 10 days after surgery. At 4 days after PRK, the nidogen-2 and LAMA3 mRNAs levels were detected at statistically significantly lower levels in the anterior stroma of the -9.0D PRK corneas (where the EBM would not fully regenerate) compared to the -4.5D PRK corneas (where the EBM was destined to fully regenerate). At 7 days after PRK, nidogen-2 and LAMA3 mRNAs continued to be statistically significantly lower in the anterior stroma of the -9.0D PRK corneas compared to their expression in the anterior stroma of the -4.5D PRK corneas. CONCLUSIONS: Key EBM components LAMA3 and nidogen-2 mRNAs are expressed at higher levels in the anterior stroma during EBM regeneration in the -4.5D PRK corneas where the EBM is destined to fully regenerate and no haze developed compared to the -9.0D PRK corneas where the EBM will not fully regenerate and myofibroblast-related stromal fibrosis (haze) will develop.

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.].

Corneal Molecular and Cellular Biology for the Refractive Surgeon: The Critical Role of the Epithelial Basement Membrane.

PURPOSE: To provide an overview of the recent advances concerning the corneal molecular and cellular biology processes involved in the wound healing response after excimer laser surface ablation and LASIK surgery. METHODS: Literature review. RESULTS: The corneal wound healing response is a complex cascade of events that impacts the predictability and stability of keratorefractive surgical procedures such as photorefractive keratectomy and LASIK. The generation and persistence of corneal myofibroblasts (contractile cells with reduced transparency) arise from the interaction of cytokines and growth factors such as transforming growth factor beta and interleukin 1 produced by epithelial and stromal cells in response to the corneal injury. Myofibroblasts, and the opaque extracellular matrix they secrete into the stroma, disturb the precise distribution and spacing of collagen fibers related to corneal transparency and lead to the development of vision-limiting corneal opacity (haze). The intact epithelial basement membrane has a pivotal role as a structure that regulates corneal epithelial-stromal interactions. Thus, defective regeneration of the epithelial basement membrane after surgery, trauma, or infection leads to the development of stromal haze. The apoptotic process following laser stromal ablation, which is proportional to the level of attempted correction, leads to an early decrease in anterior keratocyte density and the diminished contribution of these non-epithelial cells of components such as perlecan and nidogen-2 required for normal regeneration of the epithelial basement membrane. Haze persists until late repair of the defective epithelial basement membrane. CONCLUSIONS: Defective regeneration of the epithelial basement membrane has a critical role in determining whether a cornea heals with late haze after photorefractive keratectomy or with scarring at the flap edge in LASIK.

Femtosecond Laser-Assisted LASIK Flap Complications.

PURPOSE: To discuss intraoperative and postoperative femtosecond laser-assisted LASIK flap complications and their management. METHODS: Review of published literature. RESULTS: Flap creation is a critical step in LASIK. The femtosecond laser has improved the overall predictability and safety of the lamellar incision, but complications can still occur during or after flap creation. Although many complications (eg, epithelial ingrowth and flap striae) were reduced with the femtosecond laser application, other specific complications have emerged, such as vertical gas breakthrough, opaque bubble layer, and transient light-sensitivity syndrome. CONCLUSIONS: The application of femtosecond laser technology to LASIK flap creation has increased greatly since its introduction. These lasers have improved the safety and predictability of the lamellar incision step. The majority of the femtosecond laser-assisted flap complications can be well managed without significant effects on refractive outcomes.

The corneal fibrosis response to epithelial-stromal injury.

The corneal wound healing response, including the development of stromal opacity in some eyes, is a process that often leads to scarring that occurs after injury, surgery or infection to the cornea. Immediately after epithelial and stromal injury, a complex sequence of processes contributes to wound repair and regeneration of normal corneal structure and function. In some corneas, however, often depending on the type and extent of injury, the response may also lead to the development of mature vimentin+ α-smooth muscle actin+ desmin+ myofibroblasts. Myofibroblasts are specialized fibroblastic cells generated in the cornea from keratocyte-derived or bone marrow-derived precursor cells. The disorganized extracellular matrix components secreted by myofibroblasts, in addition to decreased expression of corneal crystallins in these cells, are central biological processes that result in corneal stromal fibrosis associated with opacity or "haze". Several factors are associated with myofibroblast generation and haze development after PRK surgery in rabbits, a reproducible model of scarring, including the amount of tissue ablated, which may relate to the extent of keratocyte apoptosis in the early response to injury, irregularity of stromal surface after surgery, and changes in corneal stromal proteoglycans, but normal regeneration of the epithelial basement membrane (EBM) appears to be a critical factor determining whether a cornea heals with relative transparency or vision-limiting stromal opacity. Structural and functional abnormalities of the regenerated EBM facilitate prolonged entry of epithelium-derived growth factors such as transforming growth factor ß (TGF-ß) and platelet-derived growth factor (PDGF) into the stroma that both drive development of mature myofibroblasts from precursor cells and lead to persistence of the cells in the anterior stroma. A major discovery that has contributed to our understanding of haze development is that keratocytes and corneal fibroblasts produce critical EBM components, such as nidogen-1, nidogen-2 and perlecan, that are essential for complete regeneration of a normal EBM once laminin secreted by epithelial cells self-polymerizes into a nascent EBM. Mature myofibroblasts that become established in the anterior stroma are a barrier to keratocyte/corneal fibroblast contributions to the nascent EBM. These myofibroblasts, and the opacity they produce, often persist for months or years after the injury. Transparency is subsequently restored when the EBM is completely regenerated, myofibroblasts are deprived of TGFß and undergo apoptosis, and the keratocytes re-occupy the anterior stroma and reabsorb disordered extracellular matrix. The aim of this review is to highlight factors involved in the generation of stromal haze and its subsequent removal.

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.

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 Descemet's 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 Descemet's membrane, number of collagen lamellae between keratocytes and Descemet's 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 Descemet's 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 Descemet's 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-Descemet's 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 Descemet's membrane.

Differential expression of epithelial basement membrane components nidogens and perlecan in corneal stromal cells in vitro.

PURPOSE: The purpose of this study was to examine the expression of corneal epithelial basement membrane (EBM) components in different corneal stromal cell types. In vitro model systems were used to explore the expression of EBM components nidogen-1, nidogen-2, and perlecan that are the primary components in the lamina lucida and the lamina densa that defectively regenerate in corneas with stromal opacity after in -9.0 D photorefractive keratectomy (PRK). METHODS: Primary rabbit corneal stromal cells were cultured using varying serum concentrations and exogenous growth factors, including fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-ß1, to optimize the growth of each cell type of interest. The expression of the keratocyte-specific marker keratocan and the myofibroblast-specific marker α-smooth muscle actin (α-SMA) were analyzed with real-time PCR, western blot, and immunocytochemical staining to evaluate the specificity of the cell types and select optimal conditions (high keratocan and low α-SMA for keratocytes; low keratocan and high α-SMA for myofibroblasts; low keratocan and low α-SMA for corneal fibroblasts). The expression of the EBM components nidogen-1, nidogen-2, and perlecan was evaluated in each corneal cell type using real-time PCR, immunostaining, and western blotting. In agreement with previous studies, serum-free DMEM was found to be optimal for keratocytes, DMEM with 10% serum and 40 ng/ml FGF-2 yielded the best marker profile for corneal fibroblasts, and DMEM with 1% serum and 2 ng/ml TGF-ß1 was found to be optimal for myofibroblasts. RESULTS: Nidogen-1 and nidogen-2 mRNAs were highly expressed in keratocytes, whereas perlecan was highly expressed in myofibroblasts. In keratocytes, nidogen-2 and perlecan proteins were expressed predominantly in intracellular compartments, whereas in myofibroblasts expression of both EBM components was observed diffusely throughout the cell. Although the perlecan mRNA levels were high in the myofibroblasts, the qualitative protein expression was different from that of the keratocytes. Corneal fibroblasts produced a low amount of each EBM component. CONCLUSIONS: We have demonstrated qualitative and quantitative differences in the expression of nidogen-1, nidogen-2, and perlecan by keratocytes compared to myofibroblasts that may contribute to defective regeneration of the lamina lucida and the lamina densa of the EBM associated with late stromal haze after high-correction PRK.

Topical cyclosporine a treatment in corneal refractive surgery and patients with dry eye.

PURPOSE: To evaluate preoperative and postoperative dry eye and the effect of cyclosporine A treatment in patients screened for corneal refractive surgery and treated with photorefractive keratectomy (PRK) or LASIK. METHODS: A consecutive case series of 1,056 patients screened for corneal refractive surgery from 2007 to 2012 was retrospectively analyzed. The level of preoperative and postoperative dry eye and the responsiveness to topical cyclosporine A treatment were assessed. RESULTS: One eye of each patient was randomly selected. A total of 642 eyes progressed to surgery: 524 (81.6%) and 118 (18.4%) underwent LASIK and PRK, respectively. Of 81 (7.7%) diagnosed as having dry eye, 55 were deemed potential candidates and optimized for refractive surgery. Thirty-seven patients with moderate dry eye were treated with topical cyclosporine A prior to surgery (mean duration: 3.2 ± 2.1 months; range: 1 to 12 months). After cyclosporine A treatment, 28 (75.7%) eyes underwent LASIK, 4 (10.8%) eyes underwent PRK, and 5 (13.5%) eyes were not operated on due to failed treatment of dry eye. Postoperative refractive surgery-induced neurotrophic epitheliopathy (LINE in LASIK) was noted in 132 (27.3%) and 12 (11.1%) eyes that underwent LASIK and PRK, respectively. Topical cyclosporine A was prescribed in 79 LASIK-induced and 3 PRK-induced dry eyes. After 12 months or more of cyclosporine A treatment, 5 (6.1%) eyes continued to have dry eye symptoms or signs. CONCLUSIONS: Topical cyclosporine A treatment is effective therapy for optimizing patients for refractive surgery and treatment of new onset or worsened dry eye after surgery.

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) ß 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.

Screening of refractive surgery candidates for LASIK and PRK.

PURPOSE: The aim of this study was to evaluate exclusion criteria in screening patients for refractive surgery. METHODS: Patients screened for initial refractive surgery by a single surgeon at the Cole Eye Institute (Cleveland Clinic) between 2007 and 2012 were reviewed. Exclusion criteria for patients who were not offered refractive surgery based on history and/or examination parameters were analyzed. RESULTS: A total of 1067 refractive candidates were enrolled in the study. Five hundred nineteen (48.6%) were male and 548 (51.4%) were female with a mean age of 39 ± 12 (range, 17-78) years. Refractive surgery was performed in 657 (61.6%) patients, and photorefractive keratectomy was considered the best option for 106 (9.9%) patients. Four hundred ten (38.4%) of all screened patients did not have refractive surgery, and 134 of these patients (12.6%) were considered to have contraindications for laser in situ keratomileusis and photorefractive keratectomy. Among the excluded patients, 69 (51.5%) were male and 65 (48.5%) were female with a mean age of 40 ± 14 (range, 18-78) years. Abnormal corneal topography (34.3%) and low or insufficient corneal thickness (23.1%) were the most common reasons for exclusion. High myopia (10.5%) and (insipient or definite) cataract (9.7%) were also common reasons for exclusion. Other common factors for exclusion were high hyperopia (3.7%), need to wear reading glasses after surgery (3.7%), and severe dry eye unresponsive to treatment (3.7%). CONCLUSIONS: Abnormal corneal topography and low, or insufficient, corneal thickness remain the most common exclusion factors for corneal refractive surgery. Factors such as cataract, too high of correction, and severe dry eye are also common reasons for exclusion of patients.

BAC-EDTA transepithelial riboflavin-UVA crosslinking has greater biomechanical stiffening effect than standard epithelium-off in rabbit corneas.

Studies suggest that standard corneal collagen crosslinking (CXL) is a safe and effective treatment to stiffen the cornea for keratoconus and other ectatic corneal disorders. The purpose of the present study was to compare the biomechanical effects of transepithelial benzalkonium chloride-EDTA (BAC-EDTA) riboflavin-UVA crosslinking to standard epithelium-off riboflavin-UVA crosslinking in a rabbit model. Corneal stiffness was quantified using optical coherence elastography at two months after treatment. The mean lateral-to-axial displacement ratio for the BAC-EDTA transepithelial CXL group was lower (greater stiffness) [0.062 ± 0.042, mean ± SD] than epithelium-off CXL (mean ± SD: 0.065 ± 0.045) or untreated control eyes (0.069 ± 0.044). Using ANOVA with Tukey correction, a statistically significant difference was found between the BAC-EDTA transepithelial CXL group and standard epithelium-off CXL (p = 0.0019) or the untreated control (p < 0.0001) groups. A graph of the probability density functions for biomechanical stiffness also showed a greater shift in stiffening in the BAC-EDTA transepithelial CXL group than the standard epithelium-off CXL or untreated control group. These results demonstrated that the biomechanical stiffening effect produced by BAC-EDTA transepithelial CXL was greater than that produced by standard epithelium-off CXL in a rabbit model.

TGFß and PDGF-B signaling blockade inhibits myofibroblast development from both bone marrow-derived and keratocyte-derived precursor cells in vivo.

Myofibroblasts, the primary cells associated with corneal stromal haze (opacity), can be derived from both cornea-derived and bone marrow-derived precursor cells. In the present study, the role of TGFß or PDGF blockage on bone marrow-derived myofibroblast development was investigated using a green fluorescent protein (GFP) chimeric bone marrow mouse model and plasmid vectors that blocked TGFß or PDGF signaling. At the peak of corneal haze one month after irregular phototherapeutic keratectomy the central stroma had significantly less alpha-smooth muscle actin (α-SMA)-positive cells derived from GFP+ bone marrow-derived cells or GFP- keratocyte/corneal fibroblast-derived cells when corneas were treated with the TGFß blocking vector pGFPC1.TGFRBKDEL or the PDGF blocking vector pCMV.PDGFRB.23KDEL compared with the corresponding empty vector treated or untreated control groups. In individual animals, 30-60% of myofibroblasts were derived from bone marrow-derived precursor cells and 40-70% of myofibroblasts were derived from keratocyte-derived precursor cells. TGFß and PDGF regulate corneal myofibroblast development from bone marrow-derived precursor cells and keratocyte/corneal fibroblast-derived precursor cells.

Transforming growth factor ß and platelet-derived growth factor modulation of myofibroblast development from corneal fibroblasts in vitro.

The purpose of this study was to test the hypotheses that development of mature vimentin+/α-smooth muscle actin+/desmin+ (V+A+D+) myofibroblasts from corneal fibroblasts is regulated by transforming growth factor (TGF) ß and platelet-derived growth factor (PDGF); and that myofibroblast development in vitro follows a similar developmental pathway as it does in vivo. Mouse corneal stromal fibroblasts (MSF) were isolated from the corneas of Swiss Webster mice and cultured in serum-free media augmented with DMEM/F12 and varying doses of TGFß (0.1-2.0 ng/ml), with and without mouse PDGF-AA and/or PDGF-BB (2.0 ng/ml), to study the transition of the MSF to V+A+D+ myofibroblasts. The mean percentage of vimentin+, α-SMA+ and desmin+ cells was determined at each time point (2-15 days), with each growth factor concentration. MSF in vitro were noted to undergo the same developmental transition from V+A-D- to V+A+D- to V+A+D+ myofibroblasts as precursors undergo in vivo. TGFß at a dose of 0.5 ng/ml and 1.0 ng/ml with 2.0 ng/ml PDGF-AA and 2.0 ng/ml PDGF-BB in DMEM/F12 serum-free media was optimal for the development of V+A+D+ myofibroblasts. This study defines optimal in vitro conditions to monitor the development of MSF into myofibroblasts. The combined effects of TGFß and PDGF promote the full development of V+A+D+ myofibroblasts from MSF.