Investigation into the elastic properties of ex vivo porcine corneas subjected to inflation test after cross-linking treatment.

BACKGROUND: The aim of this work was to evaluate the effect of cross-linking (CXL) on ex vivo porcine corneal elastic properties, using an inflation procedure. METHODS: Twelve corneas were subjected to standard CXL (370 nm, 3 mW/cm2, 30 minutes), while 12 were used as controls. Corneal thickness was measured by Visante optical coherence tomography, before and immediately after treatment, and before inflation test. Both intraocular pressure and radial apical cornea displacement were measured during inflation. Stress-strain curves were obtained by applying the linear shell theory. The elastic modulus was evaluated by calculating the slope of the stress-strain curves. RESULTS: Results showed a statistically significant increase in elastic modulus (p<0.0001), with a mean of 3,868 ± 502 kPa for cross-linked corneas and 2,727 ± 238 kPa for untreated corneas, when subjected to high pressure (40-60 kPa). CXL significantly increased porcine cornea stiffness by about 42%. Findings did not show any significant difference within the physiological range of pressure (2-4 kPa). CONCLUSIONS: The inflation test has been proven to be a valuable tool for the investigation of corneal biomechanics, maintaining both integrity and geometry of corneal tissue.

Early effects of corneal collagen cross-linking by iontophoresis in ex vivo human corneas.

PURPOSE: The purpose was to investigate the early modifications induced by collagen cross-linking by iontophoresis of riboflavin (ionto-CXL) in ex vivo human corneas by evaluating different protocols of UVA irradiation. METHODS: In this experimental study 46 ex vivo human corneas obtained from the Eye Bank of Mestre (Italy) were divided in different groups: six were utilized as control (CTL); eight were treated with ionto-CXL at 3 mW/cm(2) power for 30 min (I-3); eight were treated with ionto-CXL at 10 mW/cm(2) for 9 min (I-10); eight were treated with iontophoretic delivery of riboflavin only (I-0); eight were treated with the standard CXL at 3 mW/cm(2) for 30 min (S-3); and eight were treated with CXL at 10 mW/cm(2) for 9 min (S-10). All samples were evaluated by haematoxylin-eosin staining and immunohistochemical analysis using different markers (Connexin 43, CD34, Collagen I, TUNEL assay). Western blot analysis, utilizing Bax and Ki67 primary antibodies, for detection of keratocyte apoptosis and proliferation, respectively, was also performed. RESULTS: No endothelial damage was evidenced in the treated groups. In I-10 corneas the epithelial layers were not always well-preserved. Anterior stroma showed an uneven distribution and numerical reduction of keratocytes as well as increased apoptosis; a reduced subepithelial interweaving of collagen I fibers was observed. In S-3 and S-10 the changes induced by treatments were similar to I-10. I-3 and I-0 showed no significant changes with respect to the control group. CONCLUSIONS: In the ionto-CXL at 10 mW/cm(2) group occurred the main morphological and biomolecular changes. This experimental study suggests that iontophoresis can be considered a non-invasive potential delivery tool for riboflavin penetration in corneal stroma during CXL.

Transepithelial riboflavin/ultraviolet. a corneal cross-linking in keratoconus: morphologic studies on human corneas.

PURPOSE: To evaluate histologic and molecular changes in human keratoconic corneas after the procedure of transepithelial collagen cross-linking (CXL), without the removal of corneal epithelium. DESIGN: Experimental laboratory investigation. METHODS: Thirty corneal buttons were examined, 18 of which were from patients affected by severe keratoconus and submitted to penetrating keratoplasty (PK). Among these, 8 were analyzed without any treatment, 4 were treated with transepithelial CXL 2 hours before PK, and 6 were treated with transepithelial CXL 3 months before PK. Twelve normal corneal buttons from healthy donors were used as controls. The corneal buttons were then evaluated by hematoxylin-eosin staining and by immunostaining with markers of epithelial junction proteins (ß-catenin and connexin 43), of stromal keratocytes (CD34), of apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] assay), and of collagen type I fibers. RESULTS: The analysis of epithelial markers showed a clear defective expression in keratoconic corneas before and soon after the transepithelial CXL treatment, returning to normal in corneas analyzed 3 months after transepithelial CXL. The analysis of stroma components indicated a loss of keratocytes in the upper stroma of keratoconic corneas and a trend toward a normal situation 3 months after transepithelial CXL; similarly, collagen fibers appeared disorganized in keratoconus, while their pattern appears to be close to normal 3 months after treatment. CONCLUSIONS: Histologic and immunohistochemical findings on human keratoconic corneas showed the presence of biochemical and morphologic alterations in the epithelium and the upper stroma that are significantly improved 3 months after transepithelial CXL. However, further studies are necessary to assess to what extent these results correlate with measurable biomechanical effects.

Keratoconus corneal architecture after riboflavin/ultraviolet A cross-linking: ultrastructural studies.

PURPOSE: Study to investigate the effects of collagen cross-linking on the ultrastructural organization of the corneal stroma in the human keratoconus cornea (KC). METHODS: Three normal, three keratoconus (KC1, KC2, KC3), and three cross-linked keratoconus (CXL1, CXL2, CXL3) corneas were analyzed. The KC corneas were treated with a riboflavin-ultraviolet A (UVA) treatment (CXL) method described by Wollensak et al. Penetrating keratoplasty (PKP) was performed 6 months after treatment. All samples were processed for electron microscopy. RESULTS: The riboflavin-UVA-treated CXL corneal stroma showed interlacing lamellae in the anterior stroma followed by well-organized parallel running lamellae. The lamellae contained uniformly distributed collagen fibrils (CFs) decorated with normal proteoglycans (PGs). The CF diameter and interfibrillar spacing in the CXL cornea were significantly increased compared to those in the KC cornea. The PG area in the CXL corneas were significantly smaller than the PGs in the KC cornea. The epithelium and Bowman's layer were also normal. On rare occasions, a thick basement membrane and collagenous pannus were also observed. CONCLUSIONS: Corneal cross-linking leads to modifications of the cornea stroma. The KC corneal structure showed a modification in the CF diameter, interfibrillar spacing, and PG area. This resulted in a more uniform distribution of collagen fibrils, a key feature for corneal transparency.

Azithromycin: assessment of intrinsic cytotoxic effects on corneal epithelial cell cultures.

PURPOSE: To compare the cytotoxic effects of preservative-free azithromycin on corneal epithelial cells in vivo with those of preservative-free netilmicin and levofloxacin, and the preservative benzalkonium chloride (BAK). METHODS: Rabbit corneal epithelial cells in vitro were incubated for 15 minutes or 6 hours with commercially available ophthalmic preservative-free netilmicin 0.3%, levofloxacin 0.3%, or azithromycin 1.5% preparations or different concentrations of unpreserved azithromycin and different concentrations of BAK. Qualitative analysis was undertaken using phase-contrast optics to examine the morphological aspects of cell cultures and quantitative analysis was undertaken by measuring the release of the cytoplasmic enzyme lactate dehydrogenase into the medium immediately and 24 hours after exposure to drugs. Finally, we observed the wound-healing rate of mechanically injured corneal epithelial cells exposed to each antibiotic ophthalmic preparation for 48 hours. RESULTS: Our results show that both the commercially available unpreserved mono-dose preparation of azithromycin and ophthalmic preparations of azithromycin up to a concentration of 1.5% were virtually devoid of harmful effects under our experimental conditions. This was not significantly different from the results obtained for the other antibiotic preparations (P > 0.05) tested, but was unlike the results obtained for BAK. Azithromycin 1.5% also showed good recovery properties after a mechanical wound test. CONCLUSION: Under our experimental conditions, unpreserved azithromycin 1.5% showed a much lower toxicity than BAK and did not interfere with the wound-healing process.

In vivo confocal microscopy in chloroquine-induced keratopathy.

In vivo confocal microscopy is becoming a mandatory examination to study corneal abnormalities such as drug deposits in systemic disease. A female diagnosed with fibromyalgia on systemic chloroquine for 9 months presented for an ophthalmic examination. Confocal microscopy was performed using the Confoscan 4 (Nidek Co. Ltd., Gamagori, Japan) and multiple highly reflective deposits in the epithelial basal cells were found, that were consistent with choloquine. Deposits were also present in the wing cell layer. In the anterior stroma these deposits were rare. Atypically shaped and branched nerves were also present in the anterior stroma. Corneal deposits of chloroquine can be evaluated by confocal microscopy. Confocal microscopy provides information on corneal metabolism and physiology. Chloroquine keratopathy can affect the anterior stroma in addition to the epithelium.

Crystalline corneal deposits in monoclonal gammopathy: in-vivo confocal microscopy.

PURPOSE: To describe the in-vivo confocal microscopy corneal findings in a patient with bilateral corneal deposits caused by an underlying monoclonal gammopathy. METHODS: A 68-year-old man came to our center for an ophthalmologic examination. Besides visual acuity, the examination included slit-lamp biomicroscopy, intraocular pressure, and fundoscopy. Confocal microscopy was performed using Confoscan 4 (Nidek Technologies Padova, Italy) with a 40× lens because of the presence of bilateral crystalline corneal deposits. Serological tests were also performed. RESULTS: Every layer of the cornea is interested by deposits with high reflectivity,especially the epithelium and anterior stroma. The emathological tests evidenced a monoclonal gammopathy of undetermined significance with high levels of Immunoglobulin M. CONCLUSION: Crystalline corneal deposits in monoclonal gammopathycan be usefully evaluated by confocal microscopy. These manifestations may be evaluated long before systemic signs of the pathology appear, so the early diagnosis is mandatory.

Corneal thickness measurements using time-domain anterior segment OCT, ultrasound, and Scheimpflug tomographer pachymetry before and after corneal cross-linking for keratoconus.

PURPOSE: To compare minimum corneal pachymetry assessment using three measurement methods in eyes before and after corneal collagen cross-linking (CXL) for keratoconus. METHODS: Fifty patients (54 eyes) who underwent CXL for keratoconus were evaluated with the Visante (Carl Zeiss Meditec), Pentacam (Oculus Optikgeräte GmbH), and ultrasound pachymetry (USP) (Optikon Pacline) to assess corneal thickness at baseline and 1, 3, 6, and 12 months after treatment. RESULTS: Using USP, mean thickness was 456 µm at baseline, decreased by approximately 8 µm at 1 month, and then recovered to initial values. The mean difference between Visante and USP was statistically significant, but not clinically significant, and was similar at baseline and after CXL (-1 to -2 µm, P<.05 except for 12 months). Pentacam had similar readings at baseline (-2 µm vs USP), but lower corneal thickness after CXL (-12 to -20 µm throughout follow-up, P<.001). The width of the Bland-Altman 95% agreement interval of Visante and Pentacam with USP was approximately 5 µm and 15 µm, respectively. CONCLUSIONS: Visante pachymetry shows better agreement with USP compared to Pentacam after CXL, which may be due to the inhomogeneous reflectivity of the postoperative cross-linked cornea and possibly altered refractive index and acoustic impedance that may influence the observed differences among techniques.

In vitro comparison of the cytotoxic effects of clinically available ophthalmic solutions of fluoroquinolones on human keratocytes.

OBJECTIVE: To compare the cytotoxic effects of preserved versus unpreserved commercially available ophthalmic preparations of fluoroquinolones on human keratocytes in vitro. DESIGN: Experimental study. METHODS: Human keratocytes in vitro were incubated for 15 or 60 minutes with commercially available preparations containing different types of fluoroquinolones, with or without benzalkonium chloride. We examined the morphologic aspects of the cultures by an inverted-phase contrast microscope and the release of cytoplasmic enzyme lactate dehydrogenase into the medium immediately or 24 hours after exposure to drugs. RESULTS: Whereas preparations of ofloxacin, norfloxacin, and gatifloxacin, all containing benzalkonium chloride, and moxifloxacin, which is preservative-free, displayed various degrees of cytotoxicity in our model, the unpreserved monodose preparation of norfloxacin was virtually devoid of harmful effects under our experimental conditions. CONCLUSIONS: Our in vitro results indicated the cytotoxic role of preservatives in commercial preparations of fluoroquinolones and the relative nontoxicity of monodose unpreserved norfloxacin, even when keratocytes were incubated with this formulation for 6 hours.

Lectin binding in normal, keratoconus and cross-linked human corneas.

In this study the characterization of various types of sugar residues in normal, keratoconus and cross-linked human corneas was performed using immunohistochemical localization with lectins. Corneal samples were collected and divided into three groups: (1) normal corneas from cadavers; (2) keratoconic corneal buttons; (3) keratoconic corneal buttons treated with cross-linking. A series of lectins including: DBA, SBA, PNA, ConA, WGA, UEA I, GNA, DSA, MAA, SNA, were used in combination with chemical and enzymatic treatments. Compared with the normal corneas, N-acetyl-D-glucosamine increased in the keratoconus corneas. L-fucose increased and/or appeared in the keratoconus and the cross-linked corneas. N-acetyl-D-galactosamine was more abundant in the epithelium of keratoconus corneas, but was lacking in the keratoconus and cross-linked endothelium. D-galactose-(ß1-4)-N-acetyl-D-glucosamine was absent in the whole stroma of the keratoconus corneas and in the deep layers of the cross-linked ones. Sialic acids increased in the keratoconus corneas and decreased in the cross-linked ones. These results showed altered glycosylation in the keratoconic corneas and partially similar glycosylation in the cross-linked corneas, compared to the normal ones. This suggests a role played by sugar residues in maintaining the corneal structure. The changes could be related to structural alterations in keratoconus. The present findings contribute to our understanding of the effect of cross-linking treatment of keratoconic corneas in therapeutic attempts to re-establish the normal corneal structure.

Alcohol delamination in the treatment of recurrent corneal erosion: an electron microscopic study.

AIM: To investigate by electron microscopy the plane of separation of the epithelial sheet from its substratum in the procedure of alcohol delamination (ALD) in patients with recurrent corneal erosion syndrome. METHODS: Ten cases of recurrent corneal erosions (RCE) secondary to trauma and seven cases related to map-dot-fingerprint dystrophy (MDFP) were treated with ALD. The epithelial sheets obtained from these patients were examined by transmission electron microscopy. Similarly sheets obtained from 20 patients undergoing photorefractive keratectomy (10 by mechanical removal and 10 by ALD) were also examined as control group. Five further corneal buttons obtained at keratoplasty were treated with ALD and the epithelial sheet and corresponding stroma were both examined. RESULTS: In all specimens, whether removed mechanically or by ALD, the intercellular surfaces did not show any disruption and desmosomes were preserved. In patients with traumatic RCE and in corneal buttons obtained at keratoplasty, tissue separation occurred along the lamina lucida, whereas in patients with MDFP the whole basal lamina was removed along with the epithelium. Focal areas of basal cell degeneration and epithelial detachment from the basal lamina were also noted. CONCLUSIONS: ALD enables efficient removal of the epithelium with an almost complete preservation of the lamina densa in traumatic RCE. In RCE due to MDFP the epithelium separates from the stroma below the basal lamina and may reflect the pathology of the condition.

Effects of riboflavin/UVA corneal cross-linking on keratocytes and collagen fibres in human cornea.

PURPOSE: To evaluate the effects of corneal cross-linking on keratocytes and collagen fibres in human corneas. METHODS: Fifteen corneal buttons were examined. Ten were from patients with keratoconus submitted to penetrating keratoplasty and five of them were treated with cross-linking 6 months before penetrating keratoplasty. Five normal corneal buttons from healthy donors were used as controls. All samples were prepared for TUNEL assay and Western blot analysis for the detection of keratocyte apoptosis and immunohistochemical analysis for the morphological evaluation of keratocytes and collagen fibre diameter. RESULTS: Normal corneas exhibited no TUNEL-positive keratocytes and keratoconic and cross-linked corneas showed moderate apoptotic cells mainly in the anterior part of the stroma. This apoptotic trend was confirmed by the cleavage of poly (ADP-ribose) polymerase assessed using Western blot. The Ki-67 staining showed a significant increase in the keratocyte proliferation in cross-linked corneas compared with normal and keratoconus. In cross-linked corneas CD34-positive keratocytes were regularly distributed throughout the whole corneal stroma as in the control, and keratoconus was associated with patchy loss of immunoreactivity. The immunohistochemical analysis of collagen type I showed a significant increase in fibre diameter of cross-linked corneas compared with control and keratoconus. CONCLUSION: Corneal cross-linking leads to keratocyte damage; after 6 months a repopulation by proliferating cells, a distribution of CD34-positive keratocytes as in control and an increase in collagen fibre diameter were observed. These modifications are the morphological correlate of the process leading to an increase in biomechanical stability.

Corneal chrysiasis: in vivo confocal microscopy analysis.

PURPOSE: To describe the in vivo confocal microscopy corneal findings in a patient treated with gold sodium thiomalate. METHODS: A woman with rheumatoid arthritis who had been treated with gold sodium thiomalate for 32 years came to our center for an ophthalmologic examination about 5 years ago. Besides visual acuity, the examination included slit-lamp biomicroscopy, intraocular pressure, and funduscopy. Confocal microscopy was performed using Confoscan 4 (Nidek Technologies, Padova, Italy) with a 40x lens. RESULTS: Every layer of the cornea is affected by gold deposits with high reflectivity, especially in the anterior stroma, where they have a larger dimension. CONCLUSIONS: Corneal chrysiasis can be evaluated by confocal microscopy, giving information on corneal metabolism and physiology.