Claudio Traversi

Parasurgical therapy for keratoconus by riboflavin–ultraviolet type A rays induced cross-linking of corneal collagen : Preliminary refractive results in an Italian study

PURPOSE: To assess the effectiveness of riboflavin–ultraviolet type A rays induced cross‐linking of corneal collagen in reducing progression of keratoconus and in improving visual acuity in patients with progressive keratoconus. SETTING: Department of Ophthalmology, Siena University, Siena, Italy. METHODS: This was a second‐phase prospective nonrandomized open study. Starting in September 2004, 10 eyes of 10 patients (mean age 31.4 years) with bilateral keratoconus were treated by combined riboflavin–ultraviolet type A rays (UVA) collagen cross‐linking. Radiant energy was 3 mW/cm2 or 5.4 joule/cm2 for a 30‐minute exposure at 1 cm from the corneal apex. A complete ophthalmologic examination (uncorrected visual acuity [UCVA], sphere spectacles corrected visual acuity (SSCVA), best spectacle‐corrected visual acuity [BSCVA]) was performed. Patients had corneal computerized topographic examination, linear scan optical tomography, endothelial cell count, ultrasound pachometry, intraocular pressure (IOP) evaluation, and HRT II system confocal microscopy at 1, 2, 3, and 6 months. After treatment, eyes were medicated and dressed with a soft contact lens. RESULTS: Comparative preoperative and postoperative results showed increases of 3.6 lines for UCVA (P = .0000112), 1.85 lines for SSCVA (P = .00065), and 1.66 lines for BSCVA (P = .00071). Topographic analysis showed a mean K reduction of 2.1 ± 0.13 diopters (D) in the central 3.0 mm. Statistical analysis of IOP and endothelial cell count did not show significant differences. Topo‐aberrometric analysis findings of corneal symmetry showed a trend toward increasing corneal symmetry with a major reduction in asymmetry between vertical hemimeridians. CONCLUSIONS: Refractive results showed a reduction of about 2.5 D in the mean spherical equivalent, topographically confirmed by the reduction in mean K. Results of surface aberrometric analysis showed improvement in morphologic symmetry with a significant reduction in comatic aberrations.

Treatment of progressive keratoconus by riboflavin-UVA-induced cross-linking of corneal collagen: ultrastructural analysis by Heidelberg Retinal Tomograph II in vivo confocal microscopy in humans.

Purpose: To assess ultrastructural stromal modifications after riboflavin-UVA-induced cross-linking of corneal collagen in patients with progressive keratoconus. Methods: This was a second-phase prospective nonrandomized open study in 10 patients with progressive keratoconus treated by riboflavin-UVA-induced cross-linking of corneal collagen and assessed by means of Heidelberg Retinal Tomograph II Rostock Corneal Module (HRT II-RCM) in vivo confocal microscopy. The eye in the worst clinical condition was treated for each patient. Treatment under topical anesthesia included corneal deepithelization (9-mm diameter) and instillation of 0.1% riboflavin phosphate-20% dextran T 500 solution at 5 minutes before UVA irradiation and every 5 minutes for a total of 30 minutes. UVA irradiation was 7 mm in diameter. Patients were assessed by HRT II-RCM confocal microscopy in vivo at 1, 3, and 6 months after treatment. Results: Rarefaction of keratocytes in the anterior and intermediate stroma, associated with stromal edema, was observed immediately after treatment. The observation at 3 months after the operation detected keratocyte repopulation in the central treated area, whereas the edema had disappeared. Cell density increased progressively over the postoperative period. At ∼6 months, keratocyte repopulation was complete, accompanied by increased density of stromal fibers. No endothelial damage was observed at any time. Conclusions: Reduction in anterior and intermediate stromal keratocytes followed by gradual repopulation has been confirmed directly in vivo in humans by HRT II-RCM confocal microscopy after riboflavin-UVA-induced corneal collagen cross-linking.

Corneal Healing After Riboflavin Ultraviolet-A Collagen Cross-Linking Determined by Confocal Laser Scanning Microscopy In Vivo: Early and Late Modifications

PURPOSE To assess early and late micromorphological modifications of cross-linked corneas in vivo by means of Heidelberg Retinal Tomography (HRT) II confocal microscopy. DESIGN Prospective nonrandomized open trial. METHODS Micromorphological examination of 44 cross-linked keratoconic corneas was performed in vivo by HRT II confocal laser scanning microscopy. Riboflavin ultraviolet (UV)-A-induced corneal collagen cross-linking (CXL) was performed according to the Siena protocol: pilocarpin 1% drops 30 minutes before, topical anesthesia with lidocaine 4% drops 15 minutes before irradiation, mechanical scraping of epithelium (9-mm-diameter area), preirradiation soaking for 10 minutes in riboflavin solution 0.1% (Ricrolin, Sooft, Italy) applied every 2.5 minutes for 30 minutes, 30 minutes exposure to solid-state UVA illuminator (Caporossi; Baiocchi; Mazzotta, X-linker, CSO, Italy), 8-mm-diameter irradiated area, energy delivered 3 mW/cm(2). All patients were examined by confocal scans preoperatively and at the following times after treatment: one, three, and six months, and one, two, and three years. RESULTS No damage to the limbal region was observed. Epithelial regrowth was complete after four days of soft contact lens bandage. The anatomy of the subepithelial plexus was restored one year after the operation with full corneal sensitivity. Increased density of extracellular matrix in late postoperative period indicated cross-linked collagen to a depth of 340 microm expressed by a late demarcation line. CONCLUSION In vivo confocal microscopy showed early and late modification of corneal microstructure after the treatment. The three-year stability of CXL recorded could be related to increased cross-links formation, synthesis of well-structured collagen and new lamellar interconnections.

Stromal haze after combined riboflavin-UVA corneal collagen cross-linking in keratoconus : in vivo confocal microscopic evaluation

The technique of corneal collagen cross‐linking consists of photopolymerization of stromal fibres by the combined action of a photosensitizing substance (riboflavin or vitamin B2) and ultraviolet light from a solid state UVA source. Photopolymerization increases the rigidity of corneal collagen and its resistance to keratectasia. In this report we present two cases, studied through in vivo confocal microscopy, with stage III keratoconus that developed stromal haze after the cross‐linking treatment.

An In Vivo Confocal Microscopy Analysis of Effects of Topical Antiglaucoma Therapy With Preservative on Corneal Innervation and Morphology

PURPOSE To evaluate the long-term effects of preservative-free and preservative-containing antiglaucoma eye drops on the tear secretion and ocular surface. DESIGN Comparative retrospective study. METHODS A total of 84 patients with bilateral primary open-angle glaucoma or ocular hypertension divided into 5 groups according to type of topical hypotensive therapy and 20 healthy age-matched volunteers were studied. Clinical tests (corneal sensitivity, Schirmer I test, and lachrymal film break-up time), and in vivo confocal microscopy were performed in all patients. RESULTS A significant reduction of the scores was found between groups on topical hypotensive therapy and the control group in all clinical parameters studied (P < .05). In particular, the clinical scores were significantly lower in the preservative medication groups than in the preservative-free group (P < .05). The density of superficial epithelial cells was reduced in all glaucomatous patients, except for the preservative-free group (P > .05), with respect to control subjects (P < .001). On the contrary, the density of basal epithelial cells of glaucomatous preservative therapy groups was higher than control and preservative-free groups (P < .05). Stromal keratocyte activation and the number of beads were higher in all glaucoma preservative groups (P < .05). The number of sub-basal nerves was lower in all glaucoma groups than in the control group (P < .05) and tortuosity was significantly higher in glaucoma than control groups (P < .05). Reflectivity of fibers did not show any significant difference between the 6 groups (P < .05). CONCLUSIONS Glaucomatous patients with chronic treatment show ocular surface alterations. The development of nontoxic antiglaucoma treatment may reduce damage to the ocular surface and improve the compliance and the adherence in the medical therapy.

Conservative treatment of keratoconus by riboflavin-uva-induced cross-linking of corneal collagen: qualitative investigation.

PURPOSE To assess corneal tissue modifications after riboflavin-UVA-induced cross-linking of corneal collagen in patients with progressive keratoconus as well as regeneration of epithelium and subepithelial nerve plexus by in vivo HRT II system confocal microscopy in humans. METHODS Ten patients with progressive keratoconus were treated by riboflavin-UVA-induced cross-linking of corneal collagen, involving assessment of ultrastructural modifications of the corneal epithelium and subepithelial nerve plexus by HRT II system confocal microscopy. Treatment included instillation of 0.1% riboflavin-20% dextrane solution 5 minutes before UVA irradiation and every 5 minutes for a total of 30 minutes. Radiant energy was 3 mW/cm 2 or 5.4 Joule/cm 2 and the source was dual UVA (370 nm) light-emitting LED. The protocol included the operation followed by antibiotic medication and eye dressing with a soft therapeutic contact lens. Changes in epithelium and subepithelial and stromal nerve plexus were assessed by HRT II system confocal microscopy in vivo. RESULTS After 5 days of soft contact lens wearing, corneal epithelium has a regular morphology and density. Disappearance of subepithelial stromal nerve fibers was observed in the central irradiated area where, 1 month after the operation, initial reinnervation was microscopically observed. No changes in nerve fibers were observed in the peripheral untreated with a clear lateral transition between the two areas. Six months after the operation, the anterior subepithelial stroma was recolonized by nerve fibers with restoration of corneal sensitivity. CONCLUSIONS HRT II system confocal microscopy confirms corneal epithelium restore and re-innervation after riboflavin-UVA-induced collagen cross-linking directly in vivo in humans.

Pulsed vs continuous light accelerated corneal collagen crosslinking: in vivo qualitative investigation by confocal microscopy and corneal OCT

PurposeTo assess qualitative corneal changes and penetration of pulsed and continuous light accelerated crosslinking by in vivo confocal microscopy and corneal OCT.MethodsA total of 20 patients affected from progressive keratoconus were enrolled in the study. Ten eyes of 10 patients underwent an epithelium-off pulsed-light accelerated corneal collagen crosslinking (PL-ACXL) by the KXL UV-A source (Avedro Inc.) with 8 min (1 s on/1 s off) of UV-A exposure at 30 mW/cm2 and energy dose of 7.2 J/cm2; 10 eyes of 10 patients underwent an epithelium-off continuous-light accelerated corneal collagen crosslinking (CL-ACXL) at 30 mW/cm2 for 4 min. Riboflavin 0.1% dextran-free plus hydroxyl-propyl-methylcellulose solution (VibeX Rapid, Avedro Inc.) was used for a 10-min corneal soaking. Treated eyes were examined by in vivo scanning laser confocal analysis and spectral anterior segment OCT at 1, 3, and 6 months.ResultsEpithelial stratification and nerves regeneration improved in time, being complete at month 6 in both groups without endothelial damage. Keratocyte apoptosis in PL-ACXL was estimated at a mean depth of ∼200 μm, whereas an uneven demarcation line was detectable by confocal microscopy at a mean depth of 160 μm in CL-ACXL.ConclusionIn vivo confocal microscopy and corneal OCT allowed a precise qualitative analysis of the cornea after epithelium-off PL-ACXL and CL-ACXL treatments. Apoptotic effect was higher in pulsed than in continuous light treatments, exceeding 200 μm in corneal stroma. According to different morphological data, the clinical efficacy of ACXL needs to be determined in a long-term follow-up and large cohort of patients.

Pulsed Light Accelerated Crosslinking versus Continuous Light Accelerated Crosslinking: One-Year Results

Purpose. To compare functional results in two cohorts of patients undergoing epithelium-off pulsed (pl-ACXL) and continuous light accelerated corneal collagen crosslinking (cl-ACXL) with dextran-free riboflavin solution and high-fluence ultraviolet A irradiation. Design. It is a prospective, comparative, and interventional clinical study. Methods. 20 patients affected by progressive keratoconus were enrolled in the study. 10 eyes of 10 patients underwent an epithelium-off pl-ACXL by the KXL UV-A source (Avedro Inc., Waltham, MS, USA) with 8 minutes (1 sec. on/1 sec. off) of UV-A exposure at 30 mW/cm2 and energy dose of 7.2 J/cm2; 10 eyes of 10 patients underwent an epithelium-off cl-ACXL at 30 mW/cm2 for 4 minutes. Riboflavin 0.1% dextran-free solution was used for a 10-minutes corneal soaking. Patients underwent clinical examination of uncorrected distance visual acuity and corrected distance visual acuity (UDVA and CDVA), corneal topography and aberrometry (CSO EyeTop, Florence, Italy), corneal OCT optical pachymetry (Cirrus OCT, Zeiss Meditec, Jena, Germany), endothelial cells count (I-Conan Non Co Robot), and in vivo scanning laser confocal microscopy (Heidelberg, Germany) at 1, 3, 6, and 12 months of follow-up. Results. Functional results one year after cl-ACXL and pl-ACXL demonstrated keratoconus stability in both groups. Functional outcomes were found to be better in epithelium-off pulsed light accelerated treatment together with showing a deeper stromal penetration. No endothelial damage was recorded during the follow-up in both groups. Conclusions. The study confirmed that oxygen represents the main driver of collagen crosslinking reaction. Pulsed light treatment optimized intraoperative oxygen availability improving postoperative functional outcomes compared with continuous light treatment.

In Vivo Confocal Microscopy after Corneal Collagen Crosslinking.

In vivo confocal microscopy (IVCM) findings of 84 patients who had undergone conventional epithelium-off corneal collagen crosslinking (CXL) and accelerated CXL (ACXL) were retrospectively reviewed. Analysis confirmed that despite a significant decrease in the mean density of anterior keratocytes in the first 6 postoperative months, cell density after CXL and ACXL returned to baseline values at 12 months. The demarcation lines observed after treatments represent an expression of light-scattering (reflectivity changes) through different tissue densities. Temporary haze of the anterior-mid stroma after conventional CXL represents an indirect sign of CXL-induced stromal collagen compaction and remodeling. IVCM showed that treatment penetration varies to some extent, but that the endothelium is not damaged and is correlated with CXL biomechanical effects. IVCM of limbal structures shows no evidence of pathological changes. Regeneration of subepithelial and stromal nerves was complete 12 months after the operation with fully restored corneal sensitivity and no neurodystrophic occurrences. IVCM allowed detailed high magnification in vivo micromorphological analysis of corneal layers, enabling the assessment of early and late corneal modifications induced by conventional and accelerated CXL. IVCM confirms that CXL is a safe procedure, which is still undergoing development and protocol adjustments.

Amniotic membrane graft: Histopathological findings in five cases

Amniotic membrane transplantation (AMT) is an effective treatment for ocular surface reconstruction; however, the mechanisms through which amniotic membrane (AM) exerts its effects as well as its fate after transplantation have not been entirely elucidated and have been investigated only in part. We evaluate the integration of AM in the host cornea in five patients who underwent AMT as the result of Bowens disease, band keratopathy, radio‐ or cryotherapy‐induced keratopathy, chemical burn or post‐herpetic deep corneal ulcer with descemetocele. Due to persistent opacification in four cases and a progressing tumor in one case, penetrating keratoplasty (PK) and enucleation were performed as early as 2 months and up to 20 months after AMT. The corneas were analyzed histopathologically. To evaluate AM remnants, corneas were stained with periodic acid Schiffs reaction (PAS), Alcian blue, and Gomory and Masson trichrome; immunostaining including collagens III and IV antibodies was also performed. None of the corneas showed remnants of AM. In all cases, we observed discontinuity of Bowmans membrane. In three cases, the corneal epithelium was completely restored, ranging from three to six cell layers. In the other two cases, we detected an intense inflammatory reaction with rich neovascularization; the epithelial surface of the central cornea was completely restored, while at the periphery of the cornea goblet mucus‐producing cells were present. Although clinically useful in all cases, restoration of a stable corneal epithelium through AMT is limited by the extent and severity of limbal stem cell deficiency (LSCD). The lack of histologically documented AM remnants in our cases seems to explain the efficacy of AMT more through its biological properties than through its mechanical properties.