Nadia Zakaria

Normative Values for Corneal Densitometry Analysis by Scheimpflug Optical Assessment

PURPOSE To describe the normative data for corneal Scheimpflug densitometry based on a cohort of normal participants. METHODS A total of 445 healthy participants were recruited for assessment (794 eyes). Left and right eyes were considered separately. All participants were assessed using the corneal densitometry analysis add-on to the standard software of the Oculus Pentacam. Densitometry measurements were obtained and expressed in standardized grayscale units (GSU). RESULTS All participants were Caucasian; 42% were male and 58% were female. The mean age was 48.0 ± 15.3 years (range, 20.2-84.2 years). Mean corneal densitometry over the 12-mm-diameter area was 19.74 ± 3.89 GSU. When divided by radial zone, densitometry values were lowest in the central zone (16.76 ± 1.87 GSU) and highest in the periphery (27.36 ± 7.47 GSU). There was no difference between central zone and the surrounding 2- to 6-mm annulus (P > 0.05), though the 6- to 10-mm and the 10- to 12-mm zones displayed higher densitometry values (P < 0.001). When divided by depth, the anterior layer displayed the highest densitometry reading of 25.81 ± 5.14 GSU, which was significantly higher than that of both the central (P < 0.001) and the posterior layers (P < 0.001). Changes in corneal densitometry were correlated with age, though not within the central 6-mm-diameter ring. No sex difference was seen within the cohort. CONCLUSIONS This add-on to the standard imaging software allows rapid and objective assessment of the corneal densitometry. We provide normative data that may be used as a reference facilitating research and complementing clinical examination.

Limbal Stem Cell Deficiency: Current Treatment Options and Emerging Therapies.

Severe ocular surface disease can result in limbal stem cell deficiency (LSCD), a condition leading to decreased visual acuity, photophobia, and ocular pain. To restore the ocular surface in advanced stem cell deficient corneas, an autologous or allogenic limbal stem cell transplantation is performed. In recent years, the risk of secondary LSCD due to removal of large limbal grafts has been significantly reduced by the optimization of cultivated limbal epithelial transplantation (CLET). Despite the great successes of CLET, there still is room for improvement as overall success rate is 70% and visual acuity often remains suboptimal after successful transplantation. Simple limbal epithelial transplantation reports higher success rates but has not been performed in as many patients yet. This review focuses on limbal epithelial stem cells and the pathophysiology of LSCD. State-of-the-art therapeutic management of LSCD is described, and new and evolving techniques in ocular surface regeneration are being discussed, in particular, advantages and disadvantages of alternative cell scaffolds and cell sources for cell based ocular surface reconstruction.

Human Tears Reveal Insights into Corneal Neovascularization

Corneal neovascularization results from the encroachment of blood vessels from the surrounding conjunctiva onto the normally avascular cornea. The aim of this study is to identify factors in human tears that are involved in development and/or maintenance of corneal neovascularization in humans. This could allow development of diagnostic tools for monitoring corneal neovascularization and combination monoclonal antibody therapies for its treatment. In an observational case-control study we enrolled a total of 12 patients with corneal neovascularization and 10 healthy volunteers. Basal tears along with reflex tears from the inferior fornix, superior fornix and using a corneal bath were collected along with blood serum samples. From all patients, ocular surface photographs were taken. Concentrations of the pro-angiogenic cytokines interleukin (IL)-6, IL-8, Vascular Endothelial Growth Factor (VEGF), Monocyte Chemoattractant Protein 1 (MCP-1) and Fas Ligand (FasL) were determined in blood and tear samples using a flow cytometric multiplex assay. Our results show that the concentration of pro-angiogenic cytokines in human tears are significantly higher compared to their concentrations in serum, with highest levels found in basal tears. Interestingly, we could detect a significantly higher concentration of IL- 6, IL-8 and VEGF in localized corneal tears of patients with neovascularized corneas when compared to the control group. This is the first study of its kind demonstrating a significant difference of defined factors in tears from patients with neovascularized corneas as compared to healthy controls. These results provide the basis for future research using animal models to further substantiate the role of these cytokines in the establishment and maintenance of corneal neovascularization.

Repeatability and Inter-device Agreement for Three Different Methods of Keratometry: Placido, Scheimpflug, and Color LED Corneal Topography

PURPOSE To determine the repeatability of a color LED corneal topographer (Cassini; iOptics, The Hague, The Netherlands) and compare it with Placido and Scheimpflug based devices (EyeSys 2000; EyeSys Laboratories, Houston, TX, and Pentacam HR; Oculus Optikgeräte GmbH, Wetzlar, Germany). METHODS This prospective study involved 20 healthy volunteers (20 eyes) recruited from the staff of the Antwerp University Hospital. For each eye, three measurements were taken using each device, from which eight parameters describing keratometry and astigmatism were derived. Repeatability was assessed using intrasession, within-subject analysis of variance. Agreement was evaluated between pairs of devices with Bland- Altman plots and 95% confidence intervals. RESULTS The repeatability values were good for all three devices, although slightly worse for the Cassini than for the other two devices for steep, flat, and mean keratometry. The EyeSys showed worse repeatability for the astigmatism axis. EyeSys and Pentacam agreed well with each other in terms of mean keratometry, whereas the Cassini gave consistently higher values by 0.52 diopters (D) when compared with the EyeSys (P < .05, paired t test) and by 0.38 D when compared with the Pentacam (P < .05, paired t test). The Cassini provided similar repeatability values (2.31°) for the astigmatism axis to the Pentacam (2.22°), in contrast to the EyeSys, which produced much more variable axes (9.0°) (P < .05, paired t test). CONCLUSIONS Overall, the three devices display comparable repeatability. The one exception is the astigmatism axis measurement of the EyeSys. The Cassini provides higher keratometry values than the other two devices, but the astigmatism axis agrees well with that provided by the Pentacam.

Safety of Cultivated Limbal Epithelial Stem Cell Transplantation for Human Corneal Regeneration

Ex vivo cultivated limbal stem cell transplantation is a promising technique for the treatment of limbal stem cell deficiency. While the results of the clinical trials have been extensively reported since the introduction of the technique in 1997, little has been reported regarding the potential health risks associated with production processes and transplantation techniques. Culture procedures require the use of animal and/or human-derived products, which carry the potential of introducing toxic or infectious agents through contamination with known or unknown additives. Protocols vary widely, and the risks depend on the local institutional methods. Good manufacturing practice and xeno-free culture protocols could reduce potential health risks but are not yet a common practice worldwide. In this review, we focus on the safety of both autologous- and allogeneic-cultivated limbal stem cell transplantation, with respect to culture processes, surgical approaches, and postoperative strategies.

A method for quantifying limbal stem cell niches using OCT imaging

Aims To evaluate the efficacy of Fourier domain-optical coherence tomography (FD-OCT) in imaging and quantifying the limbal palisades of Vogt and to correlate these images with histological findings. Methods The superior and inferior limbal region of both eyes of 50 healthy volunteers were imaged by FD-OCT. Images were processed and analysed using Matlab software. In vitro immunofluorescent staining of a cadaveric donor limbus was analysed to correlate the presence of stem cells in the visualised structures. Results FD-OCT could successfully visualise limbal crypts and the palisades of Vogt in the limbus region. Fluorescent labelling confirmed the presence of stem cells in these structures. The mean palisade ridge width (ΔPR) and the mean interpalisade epithelial rete peg width (ΔERP) were both of the order of 72 μm, leading to a palisade density (PD) of about 7.4 palisades/mm. A significant difference in ΔPR, ΔERP and PD was seen between the inferior and superior sides of the right eye and the superior sides of the left and right eye(p<0.05.). A significant influence of iris colour on parameters ΔPR, ΔERP and PD was found, and of age on PD and ΔERP (p<0.05). Conclusions In vivo OCT imaging is a safe and effective modality to image the limbus and can be used to visualise the palisades of Vogt. Image processing using Matlab software enabled quantification and density calculation of imaged limbal palisades of Vogt. This technique may enhance targeted limbal biopsies for transplantation.

Evaluation of UVA Cytotoxicity for Human Endothelium in an Ex Vivo Corneal Cross-linking Experimental Setting

PURPOSE To evaluate endothelial cytotoxicity after exposure of human corneas to ultraviolet-A (UVA) (λ = 365 nm; 5.4 J/cm(2)) in an experimental ex vivo corneal cross-linking setting. METHODS Sixteen pairs of human donor corneas were cut into two pieces. One piece of each cornea was treated with 0.025% riboflavin solution prior to UVA irradiation (5 minutes; 18 mW/cm(2)), whereas the other piece was not irradiated but treated with riboflavin (right eye) or preservation medium (left eye). By irradiating from the endothelial side, the UVA dosage applied to endothelial cells exceeded at least eight times the cytotoxic threshold established in animal models (0.65 J/cm(2)). Endothelial cell counts were performed by two independent investigators after storage (4 to 5 days at 31 °C) and staining (trypan blue, alizarin red). Normality (Q-Q plot; Shapiro-Wilk test) and equivalence (mixed-effects modeling with a 10% equivalence threshold) of the endothelial cell counts of the different groups were evaluated. RESULTS Equivalence of mean endothelial cell density between both groups was observed: 2,237 ± 208 cells/mm(2) in UVA-irradiated pieces and 2,290 ± 281 cells/mm(2) in control pieces (mean difference of 53 ± 240 cells/mm(2) between both groups). CONCLUSIONS Despite direct irradiation of human donor endothelium using the clinical dosage for cross-linking, equivalence in endothelial cell counts was observed between irradiated tissues and controls. Ex vivo human corneal endothelial cells seem to be far more resistant to riboflavin-enhanced UVA irradiation than previously estimated by animal experiments.

Corneal regeneration: A review of stromal replacements

Corneal blindness is traditionally treated by transplantation of a donor cornea, or in severe cases by implantation of an artificial cornea or keratoprosthesis. Due to severe donor shortages and the risks of complications that come with artificial corneas, tissue engineering in ophthalmology has become more focused on regenerative strategies using biocompatible materials either with or without cells. The stroma makes up the bulk of the corneal thickness and mainly consists of a tightly interwoven network of collagen type I, making it notoriously difficult to recreate in a laboratory setting. Despite the challenges that come with corneal stromal tissue engineering, there has recently been enormous progress in this field. A large number of research groups are working towards developing the ideal biomimetic, cytocompatible and transplantable stromal replacement. Here we provide an overview of the approaches directed towards tissue engineering the corneal stroma, from classical collagen gels, films and sponges to less traditional components such as silk, fish scales, gelatin and polymers. The perfect stromal replacement has yet to be identified and future research should be directed at combined approaches, in order to not only host native stromal cells but also restore functionality. STATEMENT OF SIGNIFICANCE In the field of tissue engineering and regenerative medicine in ophthalmology the focus has shifted towards a common goal: to restore the corneal stroma and thereby provide a new treatment option for patients who are currently blind due to corneal opacification. Currently the waiting lists for corneal transplantation include more than 10 million patients, due to severe donor shortages. Alternatives to the transplantation of a donor cornea include the use of artificial cornea, but these are by no means biomimetic and therefore do not provide good outcomes. In recent years a lot of work has gone into the development of tissue engineered scaffolds and other biomaterials suitable to replace the native stromal tissue. Looking at all the different approaches separately is a daunting task and up until now there was no review article in which every approach is discussed. This review does include all approaches, from classical tissue engineering with collagen to the use of various alternative biomaterials and even fish scales. Therefore, this review can serve as a reference work for those starting in the field and but also to stimulate collaborative efforts in the future.

Intraocular lens exchange technique for an opacified bag-in-the-lens.

We describe the intraocular lens (IOL) exchange technique that is specific to the bag-in-the-lens IOL. The subsequent IOL analysis displayed a deep granular opacification consisting predominantly of calcium and phosphates that has been described in hydrophilic IOLs.

Xeno-Free Cultivation of Mesenchymal Stem Cells From the Corneal Stroma

Purpose The human cornea has recently been described as a source of corneal stroma-derived mesenchymal stem cells (hMSCs). In vitro expansion of these cells involves basal medium supplemented with fetal bovine serum (FBS). As animal-derived serum can confer a risk of disease transmission and can be subject to considerable lot-to-lot variability, it does not comply with newer Good Manufacturing Practice (GMP)-required animal component-free culture protocols for clinical translation. Methods This study investigated animal-free alternatives to FBS for cultivation of human corneal stromal MSCs. Proliferative capacity was studied for cultures supplemented with different concentrations (2.5%, 5%, and 10%) of FBS, human AB serum, human platelet lysate (HPL), and XerumFree. Unsupplemented basal medium was used as a control. The expression of specific hMSC markers (CD73+, CD90+, CD105+, CD19-, CD34-, CD79α-, CD11b-, CD14-, CD45-, and HLA-DR-) and trilineage differentiation (adipogenesis, osteogenesis, and chondrogenesis) were compared for the two outperforming supplements: 10% FBS and HPL. Results HPL is the only consistent non-xeno supplement where hMSC cultures show significantly higher proliferation than the 10% FBS-supplemented cultures. Both FBS- and HPL-supplemented hMSC cultures showed plastic adherence and trilineage differentiation, and no significant differences were found in the expression of the hMSC marker panel. No significant differences in stemness were detected between FBS and HPL cultures. Conclusions We conclude that HPL is the best supplement for expansion of human corneal stromal MSCs. HPL significantly outperforms human AB serum, the chemically defined XerumFree, and even the gold standard, FBS. The xeno-free nature of HPL additionally confers preferred standing for use in GMP-regulated clinical trials using human corneal stromal MSCs.