Iacopo Paladini

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.

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.

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.

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-(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.

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.

Inhibition of viral replication in vitro by antiviral-treated amniotic membrane. Possible use of amniotic membrane as drug-delivering tool

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 or trifluridine. The treated AM was placed onto monolayers of Vero cells, a continuous cell line from monkey kidney, infected with herpes simplex virus. 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 acyclovir or trifluridine inhibited significantly the development of herpes simplex virus in cell cultures, based on the viral growth compared with 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, the possibility to combine the previously published anti-inflammatory properties of AM with the capability to absorb antivirals and sustain drug release could be taken into consideration.

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.

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 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.

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.

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.