Corneal Buttons Research Articles

Regulatory T cells promote corneal endothelial cell survival following transplantation via interleukin-10.

The functional competence of corneal endothelial cells (CEnCs) is critical for survival of corneal allografts, but these cells are often targets of the immune response mediated by graft-attacking effector T cells. Although regulatory T cells (Tregs) have been studied for their role in regulating the host's alloimmune response towards the graft, the cytoprotective function of these cells on CEnCs has not been investigated. The aim of this study was to determine whether Tregs suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death, and to elucidate the mechanism by which this cytoprotection occurs. Using 2 well-established models of corneal transplantation (low-risk and high-risk models), we show that Tregs derived from low-risk graft recipients have a superior capacity in protecting CEnCs against effector T cell-mediated and interferon-γ and tumor necrosis factor-α-induced cell death compared to Tregs derived from high-risk hosts. We further demonstrate that the cytoprotective function of Tregs derived from low-risk hosts occurs independently of direct cell-cell contact and is mediated by the immunoregulatory cytokine IL-10. Our study is the first to report that Tregs provide cytoprotection for CEnCs through secretion of IL-10, indicating potentially novel therapeutic targets for enhancing CEnC survival following corneal transplantation.

Mycobacterium tuberculosis does not show evidence of molecular DNA in human cadaveric ocular tissues in an endemic setting.

Mycobacterium tuberculosis (MTB) in latent infection has been demonstrated in pulmonary/extra-pulmonary locations (lung, spleen, liver, kidney, adipose tissue) in autopsy studies, but its presence in ocular tissues in the latent state is not known. We conducted molecular and histopathological study of 100 cadaver eyes (50 humans) who died from causes other than tuberculosis (TB) (and were potential candidates for corneal transplantation) to detect MTB in ocular tissues in an endemic setting. After removal of the corneal button, an 8 to 10 mm block of tissue (choroid, retina and part of the vitreous) was excised from the remaining globe for DNA isolation. Gel-based IS6110 and devR3 polymerase chain reaction (PCR) assays were done, followed by real-time PCR using beta actin gene as an internal control. Sixteen randomly selected DNA samples were double checked using a commercial kit for MTB and non-tuberculous mycobacteria (NTM) DNA. The remaining larger part of the globe was subjected to histopathology. The mean age was 65.14 ± 18 years. All 100 samples were negative for both IS6110 and devR, and all 16 samples were negative with NTM MTB commercial kit. All samples were negative with Ziehl-Neelsen stain for acid fast bacilli and none showed any inflammation or granulomatous pathology. MTB could not be detected in human ocular tissues in latent state in India, a TB-endemic country. This may suggest the inability of MTB to seed ocular tissues in the latent state, unlike other organs which serve as reservoirs for the bacilli in the absence of manifest disease.

Increased NF-κB and iNOS Expression in Keratoconus Keratocytes - Hints for an Inflammatory Component?

In recent years, there has been increasing evidence of an inflammatory component in keratoconus. A key gene in inflammatory processes is the nuclear factor kappa B (NF-κB). NF-κB is a transcription factor for the enzyme nitric oxide synthase (NOS), which is involved with the competing enzyme arginase (Arg) in inflammatory processes. The aim of this study was to analyze the isotypes of NOS and arginase, the expression of NF-κB, NOS and arginase, and the regulatory mechanism of NOS and arginase in keratocytes of keratoconus patients using the inhibitor 1400W in vitro. Human keratocytes were isolated from surgically removed corneas of 8 KC patients and 8 normal human corneal buttons and were cultured to confluence, in vitro. Quantitative PCR and Western blot analysis were performed to examine NF-κB, NOS and arginase expression in keratocytes. Nitrite and urea concentrations in the supernatant of the cells were analyzed using 0 - 40 µM 1400W iNOS inhibitor concentrations. Only the isotypes iNOS and Arg-II were detected in the keratocytes. The mRNA expression of NF-κB and iNOS were higher in KC keratocytes than in normal cells (p = 0.0135 and p = 0.0001), whereas no differences were measurable in Arg-II expression. In the WB, a higher band intensity was measurable in NF-κB (p = 0.0012), and in iNOS, no differences in band intensity could be detected. In the supernatant of the KC keratocytes, lower concentrations of nitrite and urea were measured after the addition of the inhibitor 1400W (p ≤ 0.014), but not in normal cells (p ≥ 0.178). Due to the increased expression of NF-κB and iNOS, an inflammatory component in keratoconus must be assumed. The different regulation of the KC keratocytes by the iNOS inhibitor 1400W suggests an altered metabolic activity which can be caused by inflammatory processes.

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet's membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Corneal Hydration Control during Ex Vivo Experimentation Using Poloxamers

ABSTRACTPurpose: The purpose of this study was to develop an effective treatment method using poloxamers to restore and maintain physiological hydration in postmortem porcine and human corneas during ex vivo experimentation, and to compare corneal inflation response with or without treatment.Materials and Methods: Corneal buttons obtained from whole globes (n = 30 porcine, n = 8 human) were treated with various concentrations of poloxamer 188 (P188, a synthetic macromolecule surfactant) for 24 hrs to identify the concentration that would return the cornea to near-physiological hydration (i.e. H = 3.2). Whole globes (n = 12 porcine, n = 16 human) were also used to monitor central corneal thickness (CCT) during deswelling treatment. Inflation testing from 5 to 30 mmHg was performed in the porcine globes and a subset of human globes to characterize the mechanical response of the cornea after treatment.Results: Physiological hydration was obtained after 24 hrs immersion in 3.25% P188 for porcine corneas and 4.25% P188 treatment for human corneas. CCT was stabilized and returned to physiological levels after 24 hrs of treatment in 3.25% P188 in porcine (891 ± 66 µm) and 4.25% P188 in human (574 ± 34 µm) whole globes. Corneal axial strains at 30 mmHg were significantly larger at physiological hydration than in swollen cornea in both porcine (−6.42%±1.50% vs. −3.64%±1.05%, p = .004) and human (−2.85%±0.09% in vs. −1.53%±0.27%, p = .031) eyes.Conclusions: Our results suggest that P188 treatment was effective in restoring and maintaining near physiological corneal hydration during ex vivo testing, and hydration appeared to significantly impact corneal inflation response in both porcine and human eyes.

Histological Corneal Alterations in Keratoconus After Crosslinking-Expansion of Findings.

To investigate histopathologic, immunohistochemical, and electron microscopic findings in 8 keratoplasty specimens with a history of corneal collagen crosslinking (CXL) for keratoconus. Five new (hitherto unreported) and 3 previously published specimens were analyzed. Corneal buttons of 8 keratoconus corneas (5-114 months after CXL) were compared with 5 keratoconus specimens without CXL and 5 normal corneas for morphological alterations. Corneal buttons were evaluated by light microscopy and immunohistochemistry using antibodies against CD34, PGP 9.5, nestin, telomerase reverse transcriptase, and Ki67 as well as by transmission electron microscopy. Keratoconus corneas after CXL showed a significant keratocyte loss (except 1 specimen with an increased keratocyte number), whereas keratoconus corneas without CXL revealed a higher keratocyte density compared with healthy controls. Keratocyte loss could be clinically correlated with corneal opacification and corneal perforation. In corneas after CXL, the remaining keratocytes appeared more polymorphic and revealed a different expression of surface markers similar to keratocytes in corneal scars. The presence of proteoglycans, nerves, and endothelial cells was unaffected by CXL. CXL may cause permanent keratocyte loss or repopulation of altered keratocytes, resulting in clinical complications such as corneal opacification or perforation. Despite its good safety profile and high effectiveness in progressive keratoconus, CXL should be performed in accordance with current guidelines strictly adhering to protocol and safety standards.

Effects of ultrasound energy on the porcine corneal endothelium - Establishment of a phacoemulsification damage model.

The purpose of this study was to establish a standardized in vitro phacoemulsification damage model for future investigations of the effects of phacoemulsification, surgical devices, protective ophthalmic viscoelastic devices (OVDs), irrigation solutions and other aspects related to cataract phacoemulsification surgery on the corneal endothelium using porcine eyes. Thirty-four porcine eyes were randomly assigned to three groups (phacoemulsification (n=13), irrigation (n=9), control (n=12)). A total of 5min of ultrasound energy with intermittent irrigation/aspiration was applied in the eyes of the phacoemulsification group. The eyes of the irrigation group received the identical treatment, but without the application of ultrasound energy. The control group was left untreated. All eyes were then prepared to split corneal buttons followed by 15days of cultivation. Endothelial cell density (ECD) was assessed blinded on day 15. Endothelial cell density declined significantly more until day 15 in the phacoemulsification group (2567±317/267 cells/mm² (median±25%/75%-quartiles), -32.5±7.0/6.4%) compared to the irrigation (3450±350/383 cells/mm², -11.8±5.3/2.6%; p<0.001) and the control group (3650±288/258 cells/mm², -10.2±3.2/4.6%; p<0.001). The phacoemulsification damage model presented in this study is sensitive to phacoemulsification energy and may reliably be used to investigate various factors involved in phacoemulsification with regard to their influence on corneal endothelial cells. This method is able to replace animal experiments or in vitro cell culture experiments that often do not translate well to the in vivo situation in humans.

Quantification of Double Stranded DNA Breaks and Telomere Length as Proxies for Corneal Damage and Replicative Stress in Human Keratoconus Corneas.

PurposeThe pathogenesis of keratoconus (KC) is multifactorial, and associated with oxidative stress and subsequent DNA damage. We investigate differences in DNA damage and replicative stress in patients with KC, and in healthy and diseased controls.MethodsWe obtained 64 corneal buttons from 27 patients with KC after corneal transplant surgery, 21 with a decompensated graft (DG), and 16 healthy controls (HC). The amount of intact Alu elements per genome copy as measured by quantitative polymerase chain reaction (qPCR) was used to quantify intact DNA. Telomere length was measured as a proxy for replicative stress. In addition, telomerase reverse transcriptase (hTERT) gene expression level was assessed.ResultsMean (± standard deviation [SD]) DNA damage was similar between the KC (5.56 ± 14.08), DG (3.16 ± 8.22), and HC (3.51 ± 6.66) groups (P = 0.807). No associations were found between DNA damage and patient age (P = 0.523), atopic constitution (P = 0.240), or contact lens wear (P = 0.393). Telomere length differed (P = 0.034), most notably in the KC group, and hTERT was not detected in any corneal sample. Three cross-linked (CXL) KC corneas did not contain significantly more DNA damage (×2.6, P = 0.750).ConclusionsBased on these findings, differences in actual corneal DNA damage in KC could not be identified, and the longer telomere length in KC did not support replicative stress as a major etiologic factor in the pathogenesis of KC. Future longitudinal investigations on KC etiology should assess progressively early cases to better comprehend the cellular and molecular processes preceding the archetypical morphologic changes.Translational RelevanceThe standard treatment for progressive keratoconus promotes the crosslinking of collagen fibers through ultraviolet radiation and the subsequent formation of reactive oxygen species. Our study helps to underline the safety of this treatment approach.

Evaluation of Liposomal and Conventional Amphotericin B in Experimental Fungal Keratitis Rabbit Model.

PurposeWe evaluate the efficacy of liposomal amphotericin (Fungisome) compared to conventional amphotericin (AMB) for the treatment of fungal keratitis (FK) in an experimental rabbit model.MethodsFK was induced in 48 New Zealand White rabbits using Aspergillus flavus and Candida albicans (24 rabbits each). Rabbits were divided into four groups: 0.1% and 0.05% Fungisome-, and 0.1% AMB-treated groups, and one untreated control group. Clinical scores were recorded throughout the study while fungal burden was estimated by corneal button culture on day 19 (study endpoint).ResultsA statistically significant improvement in clinical score was seen on day 11 in the 0.1% and 0.05% Fungisome versus untreated groups (13.91 and 14.4 vs. 19.3; P < 0.001) in the A. flavus model, and on day 9 in the 0.1% Fungisome-treated versus untreated groups (12.96 vs. 14.2; P = 0.006) in the C. albicans model. At endpoint, the mean clinical scores of the untreated controls, and the 0.1% and 0.05% Fungisome-, and 0.1% AMB-treated groups were 20 ± 1.4, 5.33 ± 1.85, 9.66 ± 2.41, and 8.16 ± 1.95, respectively, in the A. flavus model and 15.85 ± 1.87, 3.08 ± 1.31, 4.21 ± 1.370, and 4.13 ± 1.38, respectively, in the C. albicans model. Conjunctival hyperemia score was higher in the 0.1% AMB- versus 0.1% Fungisome-treated groups (1.33 vs. 0.5, P = 0.452). Lowest fungal burden in both models was seen in the 0.1% Fungisome-treated groups.ConclusionsClinical improvement was observed with Fungisome relative to untreated controls. However, no statistically significant differences in outcomes were observed between animals treated with Fungisome and AMB. Although the results are encouraging, future studies in humans are warranted.Translational RelevanceFK is a leading cause of corneal blindness and is on the rise especially in developing countries. Despite the availability of various antifungal agents, heterogeneous treatment outcomes are seen due to lack of a standardized treatment regimen for FK. Although the use of liposomal AMB has been substantiated by clinical evidence in systemic infections, to our knowledge there are no in vivo studies evaluating the role of topical liposomal versus conventional formulation in FK. Our study investigated the efficacy and toxicity profile of liposomal versus conventional formulation of AMB in an experimental rabbit FK model.

Microbiological Screening of Hypothermic Preserved Donor Corneas in Keratoplasty

ABSTRACTPurpose: To determine the prevalence, risk factors and microbial profiles of donor corneal contamination and its association with postoperative infection.Materials and Methods: 1348 hypothermic preserved donor corneas were screened during keratoplasty to assess the impacts of donor age, gender, cause of death and corneal preservation time on the contamination risk. The microbial spectrum and antibiotic sensitivity of causative microorganisms and the prognostic role of corneoscleral rim cultures were analyzed.Results: 111 donor corneas (8.2%) had positive microbial cultures, with 84 contaminated by bacteria, 25 by fungi and 2 by both. Acinetobacter baumannii complex (19.8%) and Candida spp. (9.0%) were the most commonly isolated bacteria and fungi, respectively. Two patients (1.8%) who received contaminated corneal buttons developed postoperative infections. Death due to cardiac disease led to more corneal contaminations than death due to brain disease (odds ratio (OR) = 2.59, P = .009). Longer preservation time was associated with a trend toward increasing contamination rate (from 8.3% to 15.0%). Moreover, fungal-contaminated corneas were preserved longer than bacterial-contaminated corneas (6.6 ± 4.5 versus 10.2 ± 5.4 days, P = .001). Corneas from donors who died from cardiac diseases and trauma showed the highest prevalence of bacterial (10.9%) and fungal (2.6%) contamination, respectively. Antibiotic sensitivity testing revealed that the third-generation fluoroquinolone levofloxacin had high rates of susceptibility to both gram-positive (G+) (60.0%) and gram-negative (G-) (44.6%) bacteria.Conclusions: The causes of donor corneal contamination are multifactorial. The antibiotic resistance rate of contaminating microbes seems to be increasing. Whether antibiotic usage in storage medium and postoperative prophylaxis should be updated accordingly warrants further investigation.

Effects of Perfluorobutylpentane (F4H5) on Corneal Endothelial Cells

ABSTRACTPurpose: To evaluate the effects of perfluorobutylpentane (F4H5) on corneal endothelial cell density (ECD) and morphology using a porcine corneal endothelial organ culture model.Materials and methods: “Split corneal buttons” were cultivated for 15 days (d) after incubation in F4H5 (15, 30, 60, and 120 min) or BSS (controls). ECD was assessed manually on d1, d8, and d15. After histological staining (trypan blue, alizarin red S) on d15 morphological changes (reformation figures, rosette formations, and alizarin red cells) were evaluated.Results: ECD was significantly reduced after incubation in F4H5 for 120 min (median ± 25%/75%-quartile; 3281 ± 43/222 cells/mm2; p = 0.046) on d15 compared to controls (3658 ± 129/296 cells/mm2), but not after shorter incubation times (15, 30, and 60 min). Morphological assessment supports these findings as reformation figures (F4H5 120 min: 10.5 ± 9.3/13.9/mm2 vs. controls: 5.2 ± 2.8/7.2/mm2; p = 0.010), rosette formations (F4H5 120 min 25.566 ± 17.044/36.219/mm2 vs. controls: 8.333 ± 0.000/15.667/mm2; p = 0.002), and alizarin red cells (F4H5 120 min: 38.350 ± 29.827/51.333/mm2 vs. controls: 20.833 ± 10.417/25.000/mm2; p = 0.049) were significantly more prevalent after incubation in F4H5 for 120 min compared to controls. Also, F4H5 60 min showed significantly more rosette formations (25.452 ± 16.968/36.057/mm2; p = 0.006) and alizarin red cells (46.662 ± 42.420/50.903/mm2; p = 0.007), but not reformation figures (7.0 ± 2.2/1.6 %; p = 0.953).Conclusion: Short exposure (≤30 min) of porcine corneal endothelial cells to F4H5 does not have significant effects on ECD or morphological characteristics. Longer exposure times (≥60–120 min) may cause ECD decline and/or induce morphological changes.

Safety and efficacy of collagen cross linking for donor corneal button in therapeutic penetrating keratoplasty

Purpose: To study the role of collagen crosslinking (CXL) of donor corneal tissue in preventing recurrence of infection after therapeutic penetrating keratoplasty (TPK). Materials and Methods: This was a prospective, clinical, interventional case study done from May 2016 to July 2017. 15 cases and 15 controls were enrolled in the study. Patients who had infectious keratitis and needed TPK were included in the study. Pediatric patients, one eyed patients, perforated ulcers, patients with limbal/scleral involvement or posterior segment involvement or any other significant co-morbidity were excluded. In patients who were enrolled as cases, donor cornea was subjected to CXL using modified Dresden protocol just before TPK. TPK was done as per standard protocol. 3 cases and 2 controls had lens removal and required vitrectomy. All cases and controls were assessed post operatively on day 1, day 7 and 1 month post operatively. Digital slit lamp photos were taken at each visit for documentation of recurrence of infection, if any. The patients were followed up for at least 3 months duration. Results: Etiology was fungal in 13 cases and 2 cases were culture and smear negative. Among controls, etiology was fungal in 10, mixed bacterial and fungal in 1, acanthamoeba in 2, microsporidial in 1 and 1 was culture and smear negative. Recurrence of infection was found in 2 cases and in 4 controls within 1 month of TPK (Odd’s ratio 2.36). Conclusion: CXL done on donor corneal tissue before transplantation has got a positive role in reducing recurrence of infection after TPK. Keywords: Collagen crosslinking, Donor cornea, Infectious keratitis, Therapeutic penetrating keratoplasty.

Endogenous fluorescence can differentiate the keratoconic cornea

The purpose of the study was to investigate the endogenous fluorescence of the keratoconic cornea in order to analyze changes in the spectra due to the keratoconic stroma abnormalities.Twenty-two corneal buttons obtained from patients with keratoconus (KC, N = 22) at the time of penetrating keratoplasty were used. As a reference, twelve normal corneas (N = 12): ten from the Eye Bank and two from enucleated eyes due to choroidal melanoma were used. The fluorescence excitation/emission matrices (EEM) in the ranges of 250–400/260–600 nm were recorded.Healthy cornea, keratoconic cornea and sclera showed three main EEM bands, which correspond to the following fluorophores: tryptophan residues in the proteoglycan fraction of corneal/scleral stromas, naturally occurring collagen cross-links and the NAD(P)H fraction present in the metabolically active cells. Relative intensity factors S1, S2 and S3 describing the contribution of each kind of fluorophore to the total fluorescence of the tissue were calculated. Normal and keratoconic corneas show qualitatively similar fluorescence matrices, but the statistically significant differences in the mean values of the S1, S2 and S3 parameters for the KC and normal corneas were observed indicating changes in contribution of different fluorophores to the whole fluorescence of the tissue. Moreover, differences between multidimensional distribution of the relative intensity factors S1, S2 and S3 between these groups were demonstrated (p < 0.001).In conclusions: Differences in the relative intensity factors calculated on a basis of the fluorescence spectra can correspond to the changes found in the KC stroma regarding natural collagen cross-links and the proteoglycan fraction. These parameters well differentiate the KC and normal corneas that could serve as an additional tool for the keratoconus characterization.

The Short-Term Safety Evaluation of Corneal Crosslinking Agent-Genipin

Background: Genipin (GP) is a safe method for corneal crosslinking, even for very thin corneas. However, there have been no reports on the optimal GP concentration range to use in vivo for corneal crosslinking. Objectives: To investigate the safety of corneal crosslinking after a 24-h incubation with different concentrations of GP. Methods: Twenty New Zealand white rabbits were divided into a phosphate-buffered saline (PBS) group, 0.2% GP crosslinking (GP-CXL) group, 0.25% GP-CXL group, and 0.3% GP-CXL group. Before and after surgery, the operated eyes of each group were characterized by confocal microscopy, and corneal buttons were excised for endothelium staining and electron microscopy. Results: The keratocyte structures in each GP group appeared to be similar to those in the PBS group. Through the confocal microscopy, the changes in corneal endothelial cell density also did not significantly differ among groups. There was a significant difference in apoptosis between the 0.3% GP-CXL and PBS groups (p < 0.05) and between the 0.3% GP-CXL and 0.25% GP-CXL groups (p < 0.05), but there were no significant differences between the 0.2 and 0.25% GP-CXL groups compared to the PBS group. Transmission electron microscopy showed endothelial cell damage in the 0.3% GP-CXL group, with minimal endothelial cell damage in the other groups. Conclusions: Treatment of rabbit corneas with ≤0.25% GP resulted in minimal toxicity to keratocytes and endothelial cells, suggesting that it is a safe crosslinking agent at those concentrations.

Correlation between age and corneal edema in pediatric patients with Peters anomaly.

To evaluate corneal edema in different-aged pediatric patients with Peters anomaly and to correlate in vivo with ex vivo histopathologic findings. A retrospective cross-sectional study was performed. The medical records of patients diagnosed with Peters anomaly who underwent examination under anesthesia (EUA) between 2011 and 2015 were reviewed. Eyes in which central corneal thickness (CCT) measurements were taken were included. The thickest point in the CCT pachymetric map was used to objectively quantify corneal edema. Correlation between CCT and age was calculated. Additionally, a retrospective review of histopathologic studies of excised corneal buttons from pediatric eyes with Peters anomaly between 2011 and 2015 was performed. Eighteen eyes of 12 children were included. Mean age was 14 ± 15months, and mean CCT was 842 ± 304µm. A significant inverse correlation was noted between the CCT and the age of the patients, with lower CCT values in older children (r = 0.6; P = 0.01). Seven excised corneal buttons that underwent penetrating keratoplasty were reviewed. All corneal buttons showed absence of Descemet membrane and localized absence of endothelium. However, three specimens showed presence of corneal endothelium in areas of absent or attenuated Descemet membrane. In Peters anomaly, the CCT decreases with age, possibly due to a decrease in corneal edema. Histopathologic studies show cases of endothelial expansion in areas of absent or attenuated Descemet membrane. This may contribute to improved endothelial function and decreased edema with age.

First case of immunohistochemical study of a corneal button after treatment by recombinant Neuronal Growth Factor (cenegermin).

First case of immunohistochemical study of a corneal button after treatment by recombinant Neuronal Growth Factor (cenegermin).

Geometric Properties of Donor Corneas After Mechanical Trephination in Deep Anterior Lamellar Keratoplasty.

To evaluate the geometry of donor corneal buttons after mechanical trephination and to determine whether there were any possible variables that could influence the accuracy of cutting corneal buttons in deep anterior lamellar keratoplasty. This cross-sectional study included 85 sclerocorneal buttons that were transplanted during deep anterior lamellar keratoplasty. Donor corneas were punched from the posterior surface. Photographs that most clearly represented the entire edges of the donor corneas were taken from the punched corneas and systematically analyzed using ImageJ software. The univariate analyses were used to investigate the influence of potential variables on the precision and roundness of the donor cut. The epithelial side of the grafts was significantly larger than the posterior side in diameter, perimeter, and area. The perimeter and area of the donor posterior surface and the trephine used for punching the grafts were the same, whereas the epithelial side had a significantly larger perimeter and area than those of the trephine. Graft roundness varied from 0.78 to 1.0 at the epithelial side and from 0.77 to 1.0 at the posterior side. The roundness of the scleral spur, which represented the shape of the donor cornea, was identified as the main predictor of the roundness of the donor cut (P < 0.001). The donor buttons after mechanical trephination from the posterior surface may not be circular and of the intended diameter; the epithelial surface dimensions were significantly larger than the posterior surface and trephine dimensions. The roundness of the punched graft was primarily affected by the roundness of the cornea before trephination.

Crescentic Tuck in Lamellar Keratoplasty—A Novel Technique in a Case of Advanced Pellucid Marginal Corneal Degeneration

To describe a new surgical technique "crescentic tuck in lamellar keratoplasty" for the management of advanced pellucid marginal corneal degeneration (PMCD). A 45-year-old man with advanced PMCD and maximal thinning in the inferior juxtalimbal periphery underwent a crescentic lamellar keratoplasty in the right eye. Best-corrected visual acuity was 6/60 with no further improvement because of severe irregular astigmatism. Thinnest pachymetry in the peripheral cornea was 280 μm. Peripheral crescent-shaped lamellar corneal dissection of 3.5-mm width was performed, and an intrastromal pocket was fashioned from 4 to 8 O'clock, extending beyond limbus. A crescent-shaped donor corneal button of 3.5-mm width was denuded of its endothelium and beveled in inferior periphery to create a flange. The flange was tucked into the peripheral scleral pocket. The donor graft was secured to the host bed using 10-0 nylon interrupted sutures. Donor cornea integrated with the host bed over time and best spectacle-corrected visual acuity at 1 year improved to 6/9 with -1.25/-3.5 D × 90°. The simK (simulated keratometry) changed from 41.6/56.3 diopters (D) to 45.1/50.6 D. The astigmatism reduced from 14.6 D to 5.5 D. Crescentic tuck in lamellar keratoplasty provides good apposition of the donor lenticule and tectonic support to the peripheral thin cornea while preserving the peripheral limbal stem cells. Additionally, there is a significant reduction in the astigmatism, resulting in visual improvement.

Aniridia-related keratopathy: Structural changes in naïve and transplanted corneal buttons.

BackgroundTo study structural changes in naïve and surgically treated corneas of aniridia patients with advanced aniridia-related keratopathy (ARK).Methods and findingsTwo naïve corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time, one corneal button from an ARK patient that had undergone a keratolimbal allograft (KLAL), two corneal buttons from ARK patients who had previously undergone centered or decentered transplantation and were now retransplanted and two adult healthy donor control corneas were processed for immunohistochemistry. Antibodies against extracellular matrix components in the stroma and in the epithelial basement membrane (collagen I and IV, collagen receptor α11 integrin and laminin α3 chain), markers of fibrosis, wound healing and vascularization (fibronectin, tenascin-C, vimentin, α-SMA and caveolin-1), cell division (Ki-67) and macrophages (CD68) were used. Naïve ARK, KLAL ARK corneas and transplanted corneal buttons presented similar histopathological changes with irregular epithelium and disruption or absence of epithelial basal membrane. There was a loss of the orderly pattern of collagen lamellae and absence of collagen I in all ARK corneas. Vascularization was revealed by the presence of caveolin-1 and collagen IV in the pannus of all ARK aniridia corneas. The changes observed in decentered and centered transplants were analogous.ConclusionsGiven the similar pathological features of all cases, conditions inherent to the host seem to play an important role on the pathophysiology of the ARK in the long run.

Expression of cytokines in aqueous humor from fungal keratitis patients

BackgroundAlthough a series of reports on corneal fungal infection have been published, studies on pathogenic mechanisms and inflammation-associated cytokines remain limited. In this study, aqueous humor samples from fungal keratitis patients were collected to examine cytokine patterns and cellular profile for the pathogenesis of fungal keratitis.MethodsThe aqueous humor samples were collected from ten patients with advanced stage fungal keratitis. Eight aqueous humor samples from patients with keratoconus or corneal dystrophy were taken as control. Approximately 100 μl to 300 μl of aqueous humor in each case were obtained for examination. The aqueous humor samples were centrifuged and the cells were stained and examined under optical microscope. Bacterial and fungal cultures were performed on the aqueous humor and corneal buttons of all patients. Cytokines related to inflammation including IL-1β, IL-6, IL-8, IL-10, TNF-α, and IFN-γ were examined using multiplex bead-based Luminex liquid protein array systems.ResultsFungus infection was confirmed in these ten patients by smear stains and/or fungal cultures. Bacterial and fungal cultures revealed negative results in all aqueous humor specimens. Polymorphonuclear leukocytes were the predominant infiltrating cells in the aqueous humor of fungal keratitis. At the advanced stages of fungal keratitis, the levels of IL-1β, IL-6, IL-8, and IFN-γ in the aqueous humor were significantly increased when compared with control (p<0.01). The levels of IL-10 and TNF-α also showed an ascending trend but with no statistical significance.ConclusionsHigh concentration of IL-1β, IL-6, IL-8, and IFN-γ in the aqueous humor was associated with fungal keratitis.