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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.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.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.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.

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,...

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 P 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.

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.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 showed a statistically significant increase in elastic modulus (plt;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).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.

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-(β1-4)-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.

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.

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 (s-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.

Purpose To evaluate the effects of transepithelial corneal crosslinking (TE-CXL) on epithelium and stroma in human corneas Methods Fifteen corneal buttons were examined. Ten were from patients with keratoconus submitted to penetrating keratoplasty (PKP). Five of them were treated with TE-CXL 2 hours before PKP, five of them were treated with TE-CXL 3 months before PKP. Five normal corneal buttons from healthy donors were used as controls. TE-CXL was performed with two different time of imbibition: 30 minutes and 2 hours.All samples were prepared for the detection of keratocyte apoptosis by TUNEL assay and for the morphological evaluation of epithelium and stroma by immunohistochemical analysis (Connexin 43, CD34). Results Normal corneas exhibited no TUNEL positive keratocytes while keratoconus and crosslinked samples showed moderate apoptotic cells in the anterior part of the stroma. Moreover, the samples treated with TE-CXL 2 hours before PKP showed also an almost completely deteriorated epithelium with TUNEL positive cells. The epithelial positivity for connexin43 (transmembrane protein that forms gap junction channels) was similar in the control and in the corneas with crosslinking 3 months before PKP, while seemed more scattered in the keratoconus. In the samples treated with TE-CXL 2 hours before PKP the positivity was patchy in the few remained epithelial cells. Conclusion The treatment with TE-CXL leads to epithelial damage and a reduction of keratocytes in the sub-epithelial region in the corneas treated 2 hours before PKP. In the samples treated with TE-CXL 3 months before PKP the positivity of both CD34 keratocytes and connexin-43 epithelial cells is similar to control.

Purpose To compare the cytotoxic effects of preservative free azithromycin versus preservative free netilmycin and levofloxacin upon corneal epithelial cells in vitro. Methods Corneal epithelial cells in vitro were incubated for 15 min and 6 hours with preservative free netilmycin or levofloxacin and different concentrations of unpreserved azithromycin. Qualitative analysis was performed by using phase contrast optics and examining the morphological aspects of cell cultures. Quantitative analysis was performed by measuring the release of cytoplasmic enzyme lactate dehydrogenase (LDH) into the medium immediately and 24 h after exposure to drugs. We also observed the wound healing rate of mechanically injured corneal epithelial cells cultured in each antibiotic preparation for 24 and 48 hours. Results The unpreserved preparation of azithromycin up to a concentration of 1.5% showed a low cell toxicity, not significantly different from the other antibiotic preparations (p>0.05). Azithromycin did not inhibit the wound healing process after the mechanical injury. Conclusion Under our experimental conditions, unpreserved azithromycin 1.5 % showed a low cytotoxicity and did not interphere with wound healing process.

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.

International audience; Purpose: To investigate if amniotic membrane (AM) incubated with antivirals can inhibit viral growth in vitro. Methods: AM samples were incubated with a solution of acyclovir (ACV) or trifluridine (TFU). AM treated was placed onto a monolayers of Vero cells, a continuous cell line from monkey kidney, infected with Herpes Simplex Virus (HSV). Viral growth was assessed in comparison to control infected cells by direct examination with an inverted microscope at low magnification for the presence and extension of the typical cytopathic effect, or by estimation of viral genomes. Results: AM soaked in ACV or TFU inhibited significantly the development of HSV in cell cultures, based on the viral growth compared to controls. Non-treated AM did not significantly affect viral replication. Conclusions: Our preliminary in vitro data show that antiviral-treated amniotic membrane can inhibit viral replication. Therefore, it could be taken into consideration the possibility to combine the previously published anti-inflammatory properties of AM with the capability to absorb antivirals and sustain drug release.

Purpose In the present studies we assess the effects of collagen cross-linking on ultrastructure organisation of the corneal stroma of keratoconus human corneas. Methods One normal, one keratoconus (KC) and three cross-linked keratoconus corneas were analysed. One was treated with standard cross linking (SXL) and two with trans-epithelial collagen cross linking (TEXL). Penetrating keratoplasty was performed three months after treatment. All samples were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. Results The structure of SXL corneas was very similar to normal corneas in their hemidesmosomes, basement membrane (BM), Bowman’s layer (BW) and stromal lamellae that were not undulated. The architecture of TEXL corneas presented some differences. The BM was thick with degenerated hemidesmosomes. Bowman’s layer was disorganised at some places and replaced by thin filaments forming pannus. There were thin undulating lamellae in anterior, middle and posterior stroma. The keratocytes were embedded between undulating lamellae. Large amounts of abnormal PGs were attached around collagen fibrils. The parallel running lamellae were very thin. In some parts of the anterior stroma collagen fibrils were oriented (running) in random directions instead of running parallel. There were some parts of the stroma which showed a normal appearance. Conclusion The present studies demonstrate that corneal cross-linking leads to modifications in keratocytes and in the organisation of collagen fibril. The morphological changes might be correlated to the process of increase in biomechanical stability although there are differences between stromal structures treated by standard and trans

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.

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 40× 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.

Purpose To performed an electron microscopy study to investigate the cleavage plane and the efficacy of alcohol delamination in recurrent corneal erosion (RCE). Methods By electron microscopy we analysed the epithelium of: seven controls treated with mechanical debridment, seven controls treated with alcohol delamination, ten cases of traumatic RCE and seven RCE due to MDFP treated with alcohol delamination, with special regard to the epithelial cells and the cleavage plane. Moreover we analysed four corneas from penetrating keratoplastys that were treated by alcohol delamination on the bench and both the epithelium and stroma were studied. Results In traumatic RCE the basement membrane remained in situ, a precondition for quick epithelial healing . In MDFP the whole basement membrane was detached from the stroma and remained adherent to the epithelium, therefore after alcohol delamination the healing process should be different between MDFP and traumatic RCE. Conclusion The present findings give strenght to alcohol delamination as a promising treatment for RCE

Purpose To investigate if amniotic membrane soaked with antivirals could inhibit viral growth in vitro Methods Amniotic membrane fragments were incubated with antiviral solutions of acyclovir and trifluridine . After having inoculated vero cells with a viral suspension of HSV1 and added with the AM fragments we tested them for the antiviral activity by recording the cytophatic effect and DNA assessment by Quantitative-PCR. Results AM soaked in ACV 50 mg/ml was capable to inhibit the development of HSV viral growth in cell cultures. Non-treated AM did not significantly affect viral replication. Conclusion Our preliminary in vitro data show that amniotic membrane can absorb the antivirals

Rita Mencucci,1 Domenico E Pellegrini-Giampietro,2 Iacopo Paladini,1 Eleonora Favuzza,1 Ugo Menchini,1 Tania Scartabelli21Department of Specialized Surgical Science – Eye Clinic, 2Department of Health Science Section of Clinical Pharmacology and Oncology, University of Florence, Florence, ItalyPurpose: 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.Keywords: macrolides, toxicity, corneal epithelial cell culture, wound healing, preservative, benzalkonium chloride

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.