Pathological Corneas Research Articles

Light microscopy of the corneal pathologies that we meet in our daily practice as an eye banker

AbstractPurpose Organ culture is the method of choice for the preservation of a human donor cornea for transplantation throughout Europe. This method is often closely connected with the assessment of the cornea using light microscopy. The corneal endothelium, its polymeghatism, pleomorphism, cornea guttata and other endothelial abnormalities as well as epithelial and stromal pathologies will be presented to show the usefulness of light microscopy in the evaluation of corneal pathologies.Methods Photographs from light microscopy (both phase contrast and bright field) taken before and after corneal storage in organ culture will be compared and discussed in relation to changes in endothelial morphology and the swelling of the intercellular spaces. The individual corneal layers (the corneal epithelium, the stroma and the endothelium) of pathological corneas will be compared to those of control corneas.Results Among corneal pathologies, cornea guttata, corneal dystrophies (Fuchs endothelial corneal dystrophy, posterior polymorphous corneal dystrophy) and crystalline keratopathy will be shown using pathological explants. Moreover, the corneal pathologies will be correlated with histological findings. Finally, bacterial and fungal contamination of the cornea during organ culture will be shown.Conclusion Light microscopy is an essential part of qualitative and quantitative corneal assessment, which allows corneas with a variety of pathologies, mostly endothelial in origin, to be excluded from grafting. This work was supported by the project PRVOUK‐P24LF13 of Charles University in Prague.

Recombinant fragment 44–77 of the pigment epithelium-derived factor prevents the development of the pathological cornea neovascularization

Pigment epithelium-derived factor (PEDF), a 50 kDa secreted glycoprotein, is among the most potent endogenous inhibitors of angiogenesis. PEDF-derived fragment (44-77) possesses antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of ocular neovascular diseases. In this study we propose an efficient scalable biotechnological method for the production of PEDF (44-77) as part of a fusion protein with SspDnaB intein. The fusion protein was obtained in bacterial E. coli cells in the form of inclusion bodies, solubilized and subjected to autocatalytic cleavage with the release of PEDF (44-77) (yield, 77%). The target peptide was separated from the intein using tangential ultrafiltration. The final purification of PEDF (44-77) was performed by reversed-phase HPLC. The yield of the target peptide (purity, 99%) was 65 mg per 1 liter of culture. Antiangiogenic activity of the obtained peptide was studied in vitro using murine endothelial cells SVEC-4-10. PEDF (44-77) suppressed proliferation of endothelial cells by 53% and inhibited endothelial cell tube formation at the concentration of 1 nM. The ability of the recombinant PEDF (44-77) to block initial stages of angiogenesis was demonstrated using the model of rabbit corneal neovascularization.

Reproducibility of Cornea Measurements in Anterior Segment OCT Images of Normal Eyes and Eyes with Bullous Keratopathy Analyzed with the Zhongshan Assessment Program

To determine the interobserver and intraobserver measurement reproducibility of cornea parameters of both normal eyes and eyes with bullous keratopathy (BK) obtained with the Zhongshan Assessment Program (ZAP) on anterior segment optical coherence tomography (AS-OCT) images. A comparative study was carried out on 24 healthy volunteers and 25 subjects with BK. AS-OCT images were independently analyzed by two examiners. Parameters examined: anterior chamber depth (ACD), central corneal thickness (CCT), posterior corneal curvature (PCC), and posterior corneal arc length (PCAL). Interobserver and intraobserver reproducibility of these parameters was calculated in terms of limits of agreement (mean of differences ± 1.96SD of differences). In the normal group, both horizontal and vertical ACD were successfully measured in 23 of 24 (96%) images. The mean bias for two measurements by two different observers ranged from 0.003 to 0.117 mm for ACD, PCC, and PCAL measurements and from 0.013 to 2.25 μm for CCT measurements, and there were no differences between the two observers (P > 0.05). Mean bias for two measurements by the same grader ranged from 0.005 to 0.327 mm for ACD, PCC, and PCAL measurements and 1.46 to 2.53 μm for CCT measurements. There was no difference between the two observations (P > 0.05). Similar results were found in the BK group. There was high inter- and intraobserver reproducibility for normal and pathologic corneas using the ZAP software. The ZAP software may serve as a new investigatory tool for accurately evaluating the anterior segment and corneal parameters for corneal procedures.

On-line Mapping kornealer Strukturen mittels In-vivo-Laser-Scanning-Mikroskopie

The aim of this study was to design and evaluate online mapping of human corneal structures by in vivo laser scanning confocal microscopy. Six human corneae (four from healthy volunteers, two after LASIK,) were examined with confocal microscopy based on the confocal laser scanning microscope type Heidelberg Retina Tomograph II and Rostock cornea module. In each case an on-line mapping with max. size up to 3.2 x 3.2 mm (3072 x 3072 pixel) was performed. On-line mapping was performed to demonstrate the structures of the healthy cornea (epithelium, subepithelial nerve plexus, endothelium) as well as postoperative wound healing processes after LASIK. The quality and size of the image are considerably influenced by compliance of the patient and experience of the investigator. On-line mapping of cornea with in vivo confocal microscopy allows one to perform a large area 2D reconstruction and analyses of normal and pathological cornea. The presented method is considerably better than existing off-line reconstruction possibilities in terms of image quality and time consumption and affords an opportunity for further experimental and clinical studies.

In vivo confocal microscopy, an inner vision of the cornea – a major review

The demands of modern ophthalmology have evolved from descriptive findings from the slit lamp to in vivo assessment of cellular level changes. Nowadays, the latter can be provided by in vivo confocal microscopy. This article gives an overview of confocal principles using tandem scanning, scanning slit and laser scanning techniques used in ophthalmology. The main part of the paper describes the clinical applications emphasizing the anatomy of the normal and pathological cornea, and illustrates side-effects of topical medication, contact lens wear, cross-linking and refractive surgery. Finally, a summary about experimental applications, including animal studies, surface characterization and volume rendering as well as future developments, is given.

In vivo three-dimensional confocal laser scanning microscopy of corneal surface and epithelium

Background/aims:To evaluate in vivo three-dimensional (3-D) confocal laser scanning microscopy (CLSM) as a technique for visualising the corneal surface and epithelium.Methods:Ten human corneas (three from healthy volunteers, three with bullous...

Abnormal keratocytes and stromal inflammation in chronic phase of severe ocular surface diseases with stem cell deficiency

Background/Aims:Stevens–Johnson syndrome (SJS), ocular cicatricial pemphigoid (OCP) and alkali burns are associated with chronic, severe inflammation of the ocular surface that occasionally lead to corneal stem cell deficiencies. The corneal...

Extracellular matrix metalloproteinase inducer/CD147 promotes myofibroblast differentiation by inducing α‐smooth muscle actin expression and collagen gel contraction: implications in tissue remodeling

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein enriched on tumor cells and normal epithelia. It is mainly known for its ability to induce matrix metalloproteinase production in fibroblasts following epithelial-stromal interaction. We sought to examine whether EMMPRIN has a broader role promoting fibroblast-to-myofibroblast differentiation. Because alpha-smooth muscle actin (alphaSMA) is considered a marker of this differentiation process, we analyzed the effect of EMMPRIN on its expression in corneal and skin fibroblasts by Western blots, immunocytochemistry, and a functional assay of collagen lattice contraction. Increasing EMMPRIN expression by cDNA transfection or by treatment with exogenously added recombinant EMMPRIN resulted in an up-regulation of alphaSMA expression. EMMPRIN also increased the contractile properties of the treated fibroblasts as demonstrated by the immunohistochemical appearance of stress fibers and by the accelerated contraction of fibroblast-embedded collagen lattices. Blocking EMMPRIN expression by small interfering RNA inhibited alphaSMA and collagen gel contraction induced not only by EMMPRIN but also by transforming growth factor-beta, a major mediator of myofibroblast differentiation that also regulated EMMPRIN expression. These findings, combined with the fact that EMMPRIN and alphaSMA colocalized to the same cells in the stroma of pathological corneas, expand on the mechanism by which EMMPRIN remodels extracellular matrix during wound healing and cancer.

Optical Coherence Tomography (OCT) and Ultrasonic Biomicroscopy in corneal diseases

Abstract Purpose: To present the utility of ultrasound biomicroscopy (UBM) and anterior segment optical coherence tomography Visante (AS‐OCT) in the imaging of the human cornea. Methods: UBM and OCT analysis of healthy and pathological corneas are investigated, with the aim to define morphological/morphometric patterns of clinical relevance. Results: Cross sectional scans provided by UBM and Visante OCT enable morphological investigations of the cornea, ocular surface, and anterior chamber. Particularly AS‐OCT allows morphometric and quantitative analysis of the entire cornea, without invasiveness, together with corneal pachymetric maps. The anatomical relationships between the cornea and the adjacent anterior segment structures can be evaluated also in cases of loss of corneal transparency. Quantitative parameters can be obtained in cases of progressive corneal thinning, in degenerative and ulcerative diseases, descemetoceles, corneal oedema, and in the evaluation of post‐surgical corneal anatomy, such as lamellar interfaces, keratoplasties, ocular surface reconstruction, and amniotic membrane transplantation. Dynamic evaluations, over time, clearly allows clinicians to objectively assess variations of the corneal parameters (i.e. overall and sublayer thicknesses, lesion size, wound healing of stromal and transplanted tissues). Conclusions: UBM and AS‐OCT are complementary analysis to biomicroscopy, and furnish morphometric and objective parameters also when the cornea is opaque. Being non‐contact, and providing a higher resolution, AS‐OCT should be preferred in the investigation of acutely diseased or surgically treated corneas. Cross sectional morphology, tissue reflectivity assessment and precise measurements are the main advantages over traditional slit‐lamp examination.

Rebound Tonometer Compared With Goldmann Tonometer in Normal and Pathologic Corneas

To compare the intraocular pressure (IOP) measurements obtained with the rebound tonometer (RT) and the Goldmann handheld tonometer (GT) in normal and altered corneas. A total of 208 normal corneas and 50 corneas with pathologies were included in this prospective study. All measurements were randomly obtained by 1 observer. The medians and interquartile range (IR) for both tonometers were compared. The median differences were assessed in IOP groups. Agreement between the tonometers was calculated using the Bland-Altman method. The median IOP in all eyes was 17 mm Hg (IR, 13-22 mm Hg) with the RT and 16 mm Hg (IR, 13-21 mm Hg) with the GT (P < 0.001). The correlation was excellent between tonometers (r2 = 0.86; P < 0.001). The minimal differences between the two were obtained from 10 to 20 mm Hg (GT). The Bland-Altman scatterplot obtained good agreement between the instruments. In normal corneas, the median difference was < or =2 mm Hg in 77.4% of cases. In the altered corneas, the median difference was < or =2 mm Hg in 73% of cases (P = 0.21 compared with the normal group). In 10% and 2% of cases, the IOP could not be measured using the GT and RT, respectively. The results were similar for both tonometers. In the altered corneas, the IOP could be difficult to obtain with the GT because of distorted half-circles. The 1-mm-diameter disposable RT tip facilitated obtaining measurements without anesthetic drops, which avoids infections. The RT could be useful in routine clinical settings when measuring IOP in corneas with pathologies.

In situ monitoring of second-harmonic generation in human corneas to compensate for femtosecond laser pulse attenuation in keratoplasty

The application of femtosecond lasers in corneal transplant surgery requires high pulse energies to compensate for the strong optical scattering in pathological corneas. However, excessive energies deteriorate the quality of the incisions. The aim of this study is to demonstrate the dependence of side effects on local radiant exposure, numerical aperture, and tissue properties, to quantify the penetration depth of the laser for individual corneas, and to provide a method for optimizing the energy in the volume of the cornea. We examine histological and ultrastructural sections of clear and edematous corneas with perforating and lamellar incisions performed at different pulse energies. We demonstrate that the augmented energies in edematous corneas may result in unwanted side effects even when using high numerical apertures. The dependence of the laser beam penetration depth on pulse energy is evaluated by histology and an exponential decrease is observed. We show that the penetration length can be determined by evaluating the backscattered second-harmonic emission associated with the nonlinear optical properties of the tissue. This approach represents a noninvasive method for the in situ quantification of the laser beam attenuation, enabling us to adapt the pulse energy accordingly. Experiments using adapted energies show that the side effects are minimized.

Adaptive optics with a magnetic deformable mirror: applications in the human eye

A novel deformable mirror using 52 independent magnetic actuators (MIRAO 52, Imagine Eyes) is presented and characterized for ophthalmic applications. The capabilities of the device to reproduce different surfaces, in particular Zernike polynomials up to the fifth order, are investigated in detail. The study of the influence functions of the deformable mirror reveals a significant linear response with the applied voltage. The correcting device also presents a high fidelity in the generation of surfaces. The ranges of production of Zernike polynomials fully cover those typically found in the human eye, even for the cases of highly aberrated eyes. Data from keratoconic eyes are confronted with the obtained ranges, showing that the deformable mirror is able to compensate for these strong aberrations. Ocular aberration correction with polychromatic light, using a near Gaussian spectrum of 130 nm full width at half maximum centered at 800 nm, in five subjects is accomplished by simultaneously using the deformable mirror and an achromatizing lens, in order to compensate for the monochromatic and chromatic aberrations, respectively. Results from living eyes, including one exhibiting 4.66 D of myopia and a near pathologic cornea with notable high order aberrations, show a practically perfect aberration correction. Benefits and applications of simultaneous monochromatic and chromatic aberration correction are finally discussed in the context of retinal imaging and vision.

Spatial mapping of collagen fibril organisation in primate cornea—an X-ray diffraction investigation

New insights are presented into the collagenous structure of the primate cornea. Wide-angle X-ray diffraction was used to map the fibrillar arrangement and distribution of collagen over three common marmoset corneas. The maps provide a point of reference to help interpret data from pathological corneas or primate models of refractive surgery. The results herein disclose a circum-corneal annulus of highly aligned collagen, 0.5–1.5 mm wide, where the cornea and sclera fuse at the limbus; a feature similar to that observed in human tissue. As in humans, the annulus is not uniform, varying in width, fibril angular spread, and collagen density around its circumference. However, more centrally the marmoset cornea exhibits a preferred lamella orientation in which proportionally more fibrils are oriented along the superior–inferior corneal meridian. This observation is in striking contrast with the situation in human cornea, where there is an orthogonal arrangement of preferentially aligned fibrils. Investigation of a further 16 corneas confirmed that approximately 33% (±1%) ( n=76) of fibrils in the central marmoset cornea lie within a 45° sector of the superior–inferior meridian. Implications for the mechanical and optical properties of the cornea are discussed.

Keratocyte Phenotype Mediates Proteoglycan Structure: A ROLE FOR FIBROBLASTS IN CORNEAL FIBROSIS

In pathological corneas, accumulation of fibrotic extracellular matrix is characterized by proteoglycans with altered glycosaminoglycans that contribute to the reduced transparency of scarred tissue. During wound healing, keratocytes in the corneal stroma transdifferentiate into fibroblasts and myofibroblasts. In this study, molecular markers were developed to identify keratocyte, fibroblast, and myofibroblast phenotypes in primary cultures of corneal stromal cells and the structure of glycosaminoglycans secreted by these cells was characterized. Quiescent primary keratocytes expressed abundant protein and mRNA for keratocan and aldehyde dehydrogenase class 3 and secreted proteoglycans containing macromolecular keratan sulfate. Expression of these marker compounds was reduced in fibroblasts and also in transforming growth factor-beta-induced myofibroblasts, which expressed high levels of alpha-smooth muscle actin, biglycan, and the extra domain A (EDA or EIIIA) form of cellular fibronectin. Collagen types I and III mRNAs were elevated in both fibroblasts and in myofibroblasts. Expression of these molecular markers clearly distinguishes the phenotypic states of stromal cells in vitro. Glycosaminoglycans secreted by fibroblasts and myofibroblasts were qualitatively similar to and differed from those of keratocytes. Chondroitin/dermatan sulfate abundance, chain length, and sulfation were increased as keratocytes became fibroblasts and myofibroblasts. Fluorophore-assisted carbohydrate electrophoresis analysis demonstrated increased N-acetylgalactosamine sulfation at both 4- and 6-carbons. Hyaluronan, absent in keratocytes, was secreted by fibroblasts and myofibroblasts. Keratan sulfate biosynthesis, chain length, and sulfation were significantly reduced in both fibroblasts and myofibroblasts. The qualitatively similar expression of glycosaminoglycans shared by fibroblasts and myofibroblasts suggests a role for fibroblasts in deposition of non-transparent fibrotic tissue in pathological corneas.

Expression of HLA-G in human cornea, an immune-privileged tissue

Human leukocyte antigen (HLA)-G retains the capacity to modulate immune responses, favoring the establishment of tolerance in solid-tissue allotransplants. To better understand the mechanisms that promote corneal allograft survival, we investigated whether HLA-G was an immunoregulatory factor involved in corneal immunology. We therefore sought HLA-G expression in corneal tissues. Corneal transplantation consists in replacing the center of a diseased cornea with normal corneal tissue. Two corneal parts are not used in such surgery: diseased central corneal tissue and peripheral normal cornea. For this study, we used healthy corneas obtained from deceased donors and diseased corneas obtained from patients with pseudophakic bullous keratopathy or keratoconus who had undergone corneal transplantation. Immunohistochemical analysis carried out on the cryopreserved corneas showed a positive immunohistochemical staining with anti–HLA-G, anti–HLA-A, -B, and -C, and anti–HLA class I monoclonal antibodies. Staining was obtained for keratocytes, epithelial cells, and endothelial cells from both healthy and pathologic human corneas, revealing the presence of HLA class I proteins, including HLA-G. HLA-G transcripts were detected in normal cornea by reverse transcriptase–polymerase chain reaction with a classical pattern of alternative splicing. The detection of HLA-G protein in adult corneas leads to the conclusion that this protein may contribute to the maintenance of the privileged immune status of cornea.

Immunohistochemical detection of betaIG-H3 in scarring human corneas.

BetaIG-H3 is a recently described extracellular matrix protein that is present in various organs. In rabbit corneas, increased betaIG (the rabbit form of betaIG-H3) mRNA levels were shown during corneal development and wound healing. In this study, we investigated the localization of betaIG-H3 protein in scarring human corneas. Corneal buttons obtained during keratoplasty were examined. Immunohistological detection using a polyclonal antipeptide antibody against the betaIG-H3 protein was performed on 24 pathological corneas (9 ulcerations, 8 alkali burns, 2 perforating injuries, 5 bullous keratopathy) and 2 normal corneas. In normal corneas, strong staining was present in the basal layer of the epithelium and in the endothelium; the stromal fibers showed faint, uniform immunoreactivity. In all scarring corneas, the epithelium was usually thickened and all of its layers were reactive with the betaIG-H3 antibody. The cytoplasm of the stromal fibroblasts, as well as the stromal fibers around them also showed staining with the antibody. These changes were present in all scarring corneas, irrespective of the pathological process leading to scar formation. These results prove, at the protein level, the presence of increased amounts of betaIG-H3 at the sites of scarring in human corneas.

Localization of TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B in pathological human corneas

PURPOSE. Determine the tissue distribution patterns for tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3), gelatinase A and gelatinase B in normal and pathologic corneas. METHOD. Corneas were examined by immunohistochemistry, using antibodies to TIMP-1, TIMP-2, TIMP-3, gelatinase A or gelatinase B. RESULTS. In normal corneas, TIMP-1 antibody stained the epithelium and endothelium. TIMP-2 and TIMP-3 stained the epithelium, keratocytes and endothelium. Gelatinase A staining was weak and restricted to the epithelial cells. Radial keratotomy scars showed increased staining for TIMP-1 and TIMP-2 around the epithelial cell plug and along the incision. Bullous keratopathy corneas showed TIMP staining patterns similar to normal corneas and increased gelatinase A staining in regions of subepithelial fibrosis. Stromal scars of keratoconus corneas also had increased staining with TIMP-1 and TIMP-2 antibodies. In many keratoconus corneas, the TIMP-3 staining pattern was similar to normal corneas. However, in some keratoconus corneas, when Bowman's layer was missing, the stroma beneath was completely devoid of TIMP-3 antibody staining. No gelatinase B was seen in either the normal or diseased corneas. CONCLUSIONS. These data suggest that TIMP-1 and TIMP-2 are important for scar formation and corneal remodeling, since they were found in increased amounts at radial keratotomy incision sites and keratoconus scars. The significance of the focal stromal defects in TIMP-3 staining, associated with absence of Bowman's layer on keratoconus corneas, needs to be elucidated. At the stages of disease examined in this study, gelatinase B may not play a significant role in these pathological processes, since it was not seen in any of the corneas examined.

Tenascin-C expression in normal, inflamed, and scarred human corneas

AIMS/BACKGROUNDIn adult tissues the expression of tenascin-cytotactin (TN-C), an extracellular matrix glycoprotein, is limited to tumours and regions of continuous renewal. It is also transiently expressed in cutaneous and corneal...

Morphometric analysis of the corneal endothelium

Early developments in specular microscopy and quantitative corneal endothelial image analysis have resulted in the publication of material describing the morphometric appearance of the mosaic in health and disease. A broad range of morphometric parameters, including cell area, diameter, perimeter, form and shape, have been used to describe cellular appearances. In may cases the selection of parameters appears inappropriate and uncoordinated. In this study, the corneal endothelium of 300 normal, traumatised and pathological corneas was photographed using the Keeler Konan Pocklington specular microscope. The morphometric characteristics of endothelial cells were assessed using the Kontron Mop II semi‐automated image analysis system. Results indicate that cell density and mean cell area measurements provide the most useful indices of age‐related, post‐traumatic and pathological cell loss. Cell perimeter and cell diameter measurements add value in those cases where cellular migration occurs in response to trauma or localised cell loss. The quantification of intercellular cell area variability (polymegethism), and cell shape variability (pleomorphism), provides additional data on the effects of physiological stress. Cell form factors, which exhibit large intercellular variability, prove unhelpful when applied to global populations of cells. The authors conclude that in performing quantitative analysis of the corneal endothelium, specular microscopists should apply informed restraint when selecting parameters for morphometric analysis. The relative merits of a range of morphometric parameters are discussed.

T-Cell Repertoire of Normal, Rejected, and Pathological Corneas

The specific immune mechanisms of corneal graft rejection are not completely understood. Recently, the technique of growing T-cell lines from rejected allografts using recombinant IL-2 has enabled the cells involved in allograft rejection to be recognized. In the present study, this method was applied for the extraction and propagation of T lymphocytes from rejected, normal, and diseased corneas. T-cell lines could successfully be grown from rejected and normal corneas, but not from corneas with keratoconus or pseudophakic bullous keratopathy. The phenotypic repertoire of the grown cells was studied by FACS scan analysis. Rejected corneas were invaded by a mixture of activated CD4+ and CD8+ T-cell lines, with one population being predominant. In normal corneas only activated CD8+ cells with cytotoxic function were cultured. No cells were obtained from diseased corneas. The in vitro function of cell lines was assessed by primed lymphocyte testing. The present study shows that the technique of propagating invading T-cell lines from organ grafts can be applied to human corneas, offering a new approach to understanding the immunological mechanisms occurring inside this immune tissue.