Transepithelial corneal crosslinking with oxygen enhancement and pulsed light for progressive ectasia: one-year results.

PURPOSE: To evaluate the efficacy and safety of transepithelial accelerated crosslinking (TE-ACXL) using pulsed light and supplemental oxygen. METHODS: Thirty eyes of 30 consecutive patients with progressive keratoconus or post-LASIK ectasia were enrolled in a prospective non-comparative study conducted at the Magrabi Eye Center (Jeddah, Saudi Arabia). All eyes underwent TE-ACXL with supplemental oxygen. Primary outcome measures were the mean change in corrected distance visual acuity (CDVA) (logMAR) and maximum keratometry (max K) from preoperatively to 12 months postoperatively. Secondary outcome measures included change in manifest refractive spherical equivalent (MRSE), refractive cylinder, keratometry, symmetry index (SI), center-surrounding index (CSI) and ectasia index (EI) of the anterior and posterior corneal surfaces, corneal and epithelial thickness at corneal vertex and thinnest location, corneal densitometry, corneal high order aberrations (HOA) and endothelial cell density (ECD). RESULTS: Mean age was 29.6 ± 8.2 years. At 1 year, the follow up rate was 93.3%. CDVA improved statistically significantly at 12 months (p = 0.027). Measures of corneal keratometry or pachymetry did not change significantly (p < 0.05). Postoperatively, a demarcation line was documented in 78.6% eyes at 1 month, and in 12 (42.9%) eyes at 12 months. The mean depth of the demarcation line was 341.9 ± 49.4 µm. Corneal densitometry increased significantly at 1- and 3-months (p < 0.05) and returned to normal levels at 6- and 12-months postoperatively. CONCLUSION: TE-ACXL with oxygen supplement is effective at halting the progression of corneal ectasia for at least 1 year and can be a refractive neutral procedure.

Iontophoresis Corneal Cross-linking With Oxygen Supplementation in Ovine Eyes.

PURPOSE: To evaluate the biomechanical changes and advanced oxidation protein products (AOPP) production after different corneal cross-linking (CXL) protocols with or without oxygen supplementation. METHODS: Ovine eyes in the study were equally distributed to five groups as control, standard Dresden protocol, diluted alcohol- and iontophoresis-assisted CXL (DAI-CXL), and 0.1% and 0.2% riboflavin-mediated iontophoresis-assisted CXL with oxygen supplementation (I-CXL). Corneas that received CXL were divided into two equal parts, one part was used for uniaxial tensiometry and one part was used for AOPP measurement. RESULTS: All treatment groups showed higher Young's modulus and stiffness compared to the control group (P < .05). Both oxygen-assisted I-CXL groups with 0.1% and 0.2% riboflavin concentrations had higher corneal Young's modulus (P = .009 and .006, respectively) and stiffness (P = .009) values, whereas the DAI-CXL group had lesser Young's modulus and stiffness values (P = .032) compared to the Dresden protocol group. All treatment groups showed higher AOPP concentrations compared to the control group (P < .05). DAI-CXL and I-CXL groups showed similar AOPP formation compared to the Dresden protocol (P = .673). CONCLUSIONS: When the epithelium is intact, the desired increase in corneal stiffness might not be achieved. However, increasing the oxygen in the environment might provide a sufficient increase in stiffness in cases undergoing epitheliumon I-CXL, which might be promising in terms of shortening the CXL therapy and decreasing the complications. [J Refract Surg. 2022;38(10):674-681.].

The Yin and Yang of Mesenchymal Cells in the Corneal Stromal Fibrosis Response to Injury: The Cornea as a Model of Fibrosis in Other Organs.

Mesenchymal cells (keratocytes, corneal fibroblasts, and myofibroblasts), as well as mesenchymal progenitor bone marrow-derived fibrocytes, are the major cellular contributors to stromal fibrosis after injury to the cornea. Corneal fibroblasts, in addition to being major progenitors to myofibroblasts, also have anti-fibrotic functions in (1) the production of non-basement membrane collagen type IV that binds activated transforming growth factor (TGF) beta-1 and TGF beta-2 to downregulate TGF beta effects on cells in the injured stroma, (2) the production of chemokines that modulate the entry of bone marrow-derived cells into the stroma, (3) the production of hepatocyte growth factor and keratinocyte growth factor to regulate corneal epithelial healing, (4) the cooperation with the epithelium or corneal endothelium in the regeneration of the epithelial basement membrane and Descemet's membrane, and other functions. Fibrocytes also serve as major progenitors to myofibroblasts in the corneal stroma. Thus, mesenchymal cells and mesenchymal cell progenitors serve Yin and Yang functions to inhibit and promote tissue fibrosis depending on the overall regulatory milieu within the injured stroma.

The anti-scarring role of Lycium barbarum polysaccharide on cornea epithelial-stromal injury.

PURPOSE: Cornea epithelial-stromal scarring is related to the differentiation of fibroblasts into opaque myofibroblasts. Our study aims to assess the effectiveness of Lycium barbarum polysaccharide (LBP) solution as a pre-treatment in minimizing corneal scarring. METHODS: Human corneal fibroblasts were cultured in a three-dimensional collagen type I-based hydrogel in an eye-on-a-chip model. Fibroblasts were pre-treated with 2 mg/mL LBP for 24 h, followed by another 24-h incubation with 10 ng/mL transforming growth factor-beta 1 (TGF-ß1) to induce relevant physiological events after stromal injury. Intracellular pro-fibrotic proteins, extracellular matrix proteins, and pro-inflammatory cytokines that involved in fibrosis, were assessed using immunocytochemistry and enzyme-linked immunosorbent assays. RESULTS: Compared to the positive control TGF-ß1 group, LBP pre-treated cells had a significantly lower expression of alpha-smooth muscle actin, marker of myofibroblasts, vimentin (p < 0.05), and also extracellular matrix proteins both collagen type II and type III (p < 0.05) that can be found in scar tissues. Moreover, LBP pre-treated cells had a significantly lower secretion of pro-inflammatory cytokines interleukin-6 and interleukin-8 (p < 0.05). The cell-laden hydrogel contraction and stiffness showed no significant difference between LBP pre-treatment and control groups. Fibroblasts pretreated with LBP as well had reduced angiogenic factors expression and suppression of undesired proliferation (p < 0.05). CONCLUSION: Our results showed that LBP reduced both pro-fibrotic proteins and pro-inflammatory cytokines on corneal injury in vitro. We suggest that LBP, as a natural Traditional Chinese Medicine, may potentially be a novel topical pre-treatment option prior to corneal refractive surgeries with an improved prognosis.

Diffuse Lamellar Keratitis in a Patient Undergoing Collagen Corneal Cross-Linking 18 Years After Laser In Situ Keratomileusis Surgery.

PURPOSE: To report a case of diffuse lamellar keratitis (DLK) after corneal collagen cross-linking in an eye with a remote history of laser in situ keratomileusis (LASIK) surgery. METHODS: This is a case report and literature review. RESULTS: This report describes the development of unilateral stage IV DLK in a patient who underwent bilateral corneal cross-linking for corneal ectasia 18 years after LASIK surgery. The patient was treated with high-dose topical steroids that were tapered over 1 month and multiple flap lifts. The ultimate best-corrected visual outcome was 20/60. CONCLUSIONS: DLK is a potential sight-threatening complication of refractive surgery that can occur at any time in the postoperative period, even years after the procedure. Undergoing a subsequent corneal procedure that may disrupt or promote inflammation within the surgical flap-stromal interface, such as corneal collagen cross-linking, is a recognized risk factor for the development of DLK. This case suggests that patients with any history of LASIK surgery undergoing corneal cross-linking or other lamellar corneal surgeries may benefit from closer follow-up (eg, daily) than patients with no history of LASIK.

Comparison of Visual, Tomographic, and Biomechanical Outcomes of 360 Degrees Intracorneal Ring Implantation With and Without Corneal Crosslinking for Progressive Keratoconus: A 5-Year Follow-up.

PURPOSE: To evaluate the long-term (5 years) results of 360 degrees intracorneal ring (ICR) implantation with and without corneal crosslinking (CXL) in patients with progressive keratoconus (KCN). METHOD: This historical cohort study was performed on 35 eyes with progressive KCN, which was randomly divided into 2 groups. Fourteen patients were implanted only with ICR, and 21 patients with KCN were treated with ICR plus CXL simultaneously. Uncorrected distance visual acuity (UCDVA), corrected distance visual acuity (CDVA), and refractive components were collected from patients' medical records. The biomechanical properties of patients were measured using the Corvis ST (Oculus, Inc, Weltzar, Germany). Corneal topography and aberrometry parameters were also recorded from the Pentacam HR device (Oculus). RESULTS: Both UCVA and CDVA increased in both groups after ICR implantation; however, this improvement was more significant in the ICR plus CXL group (P = 0.002 and P = 0.001, respectively). The mean improvement of CDVA in patients with ICR implantation with and without CXL was 0.56 ± 0.67 and 0.33 ± 0.61, respectively. A comparison of the long-term postoperative Tomographic and Biomechanical Index between 2 groups showed a better result in the ICR plus CXL group (P = 0.012). Topographic findings of the anterior corneal surface (flat-K, steep-K, mean-K, and astigmatism) after surgery were significantly better than before surgery in both groups (P < 0.05). CONCLUSIONS: Although ICR implantation alone might halt the KCN progression with acceptable visual, topographic, and biomechanical outcomes, the combination of ICR and CXL has an adjuvant and synergistic effect, especially in long-term follow-up.

Cell sheet technology: Influence of culture conditions on in vitro-cultivated corneal stromal tissue for regenerative therapies of the ocular surface.

The in vitro reconstruction of stromal tissue by long-term cultivation of corneal fibroblasts is a smart approach for regenerative therapies of ocular surface diseases. However, systematic investigations evaluating optimized cultivation protocols for the realization of a biomaterial are lacking. This study investigated the influence of supplements to the culture media of human corneal fibroblasts on the formation of a cell sheet consisting of cells and extracellular matrix. Among the supplements studied are vitamin C, fetal bovine serum, L-glutamine, components of collagen such as L-proline, L-4-hydroxyproline and glycine, and TGF-ß1, bFGF, IGF-2, PDGF-BB and insulin. After long-term cultivation, the proliferation, collagen and glycosaminoglycan content and light transmission of the cell sheets were examined. Biomechanical properties were investigated by tensile tests and the ultrastructure was characterized by electron microscopy, small-angle X-ray scattering, antibody staining and ELISA. The synthesis of extracellular matrix was significantly increased by cultivation with insulin or TGF-ß1, each with vitamin C. The sheets exhibited a high transparency and suitable material properties. The production of a transparent, scaffold-free, potentially autologous, in vitro-generated construct by culturing fibroblasts with extracellular matrix synthesis-stimulating supplements represents a promising approach for a biomaterial that can be used for ocular surface reconstruction in slowly progressing diseases.

Cross-Linking Assisted Infection Reduction (CLAIR): A Randomized Clinical Trial Evaluating the Effect of Adjuvant Cross-Linking on Bacterial Keratitis.

PURPOSE: To determine whether there is a benefit to adjuvant corneal cross-linking (CXL) for bacterial keratitis. METHODS: This is an outcome-masked, randomized controlled clinical trial. Consecutive patients presenting with a smear-positive bacterial ulcer at Aravind Eye Hospitals at Madurai, Pondicherry, and Coimbatore in India were enrolled. Study eyes were randomized to topical moxifloxacin 0.5% or topical moxifloxacin 0.5% plus CXL. The primary outcome of the trial was microbiological cure at 24 hours on repeat culture. Secondary outcomes included best spectacle corrected visual acuity at 3 weeks and 3 months, percentage of study participants with epithelial healing at 3 weeks and 3 months, infiltrate and/or scar size at 3 weeks and 3 months, 3-day smear and culture, and adverse events. RESULTS: Those randomized to CXL had 0.60 decreased odds of culture positivity at 24 hours (95% confidence interval [CI]: 0.10-3.50; P = 0.65), 0.9 logarithm of the minimum angle of resolution lines worse visual acuity (95% CI: -2.8 to 4.6; P = 0.63), and 0.41-mm larger scar size (95% CI: -0.48 to 1.30; P = 0.38) at 3 months. We note fewer corneal perforations or need for therapeutic penetrating keratoplasty in the CXL group. CONCLUSIONS: We were unable to confirm a benefit to adjuvant CXL in the primary treatment of moderate bacterial keratitis. However, CXL may reduce culture positivity and complication rates; therefore, a larger trial to fully evaluate this is warranted. TRIAL REGISTRATION: NCT02570321.

Therapeutic Effect of Corneal Crosslinking on Fungal Keratitis: Efficacy of Corneal Collagen Crosslinking as an Adjuvant Therapy for Fungal Keratitis in a Tertiary Eye Hospital in South India.

PURPOSE: To evaluate the efficacy of CXL in treating fungal keratitis as an adjuvant therapy. METHODS: Detailed clinical examination microbiological investigation was performed. Twenty fungal keratitis patients were recruited and randomized into two groups: group 1 (n= 11, standard antifungal), group 2 (n=9, corneal collagen crosslinking with standard antifungal). Corneal scraping and tear samples collected were subjected to real-time PCR targeting ITS, TLR analysis and cytokine analysis. RESULTS: The mean time for complete resolution of ulcer for group 2 was significantly shorter compared to group 1 and the final mean BCVA was better for group 2. Expression of IL-1ß, IL-8, IFN-γ significantly decreased immediately post CXL in group 2 patients. Significant downregulation of TLR 6, TLR-3, TLR-4 was observed 3-days post CXL compared to group 1 patients. CONCLUSION: Adjuvant effect of CXL was significant in treating fungal keratitis compared to standalone antifungal treatment.

Oxygen Kinetics During Corneal Cross-linking With and Without Supplementary Oxygen.

PURPOSE: To measure and simulate oxygen kinetics during corneal cross-linking at different irradiances with and without supplementary oxygen. DESIGN: Experimental, laboratory study. METHODS: In de-epithelialized porcine eyes, a femtosecond-laser-generated tunnel was used to place a fiber probe in corneal depths of 100, 200, and 300 µm to measure the local oxygen concentration. After riboflavin imbibition, the corneas were irradiated at 3, 9, 18, and 30 mW/cm2 while the oxygen concentration was measured. All experiments were performed under normoxic (21%) and hyperoxic (>95%) conditions. The obtained data were used to identify parameters of a numerical model for oxygen consumption and diffusion. RESULTS: The equilibrium stromal oxygen concentration under atmospheric oxygen at 3 mW/cm2 was 2.3% in 100 µm decreasing to <1% in 300 µm. With 9, 18, and 30 mW/cm2, no oxygen was available in 200 µm, respectively, 100 µm or deeper. Using a hyperoxic environment, the concentration was 50% using 3 mW/cm2 in 100 µm, decreasing to 40% in 300 µm. At 9 mW/cm2, the concentrations were 5%, 3%, and 1% in 100, 200 and 300 µm, respectively. Using 18 and 30 mW/cm2, all oxygen was depleted at 100 µm; however, oxygen half-lives were longer at 18 mW/cm2 than at 30 mW/cm2. The oxygen model was able to reproduce the experiments and indicated an exponential decay with increasing distance to the anterior surface. CONCLUSION: Supplementary oxygen increases the oxygen availability during corneal cross-linking. At higher irradiances, supplementary oxygen is beneficial and eliminates the bottleneck of oxygen allowing a potentially more efficient cross-linking. The calibrated numerical model can quantify the spatial oxygen concentration related to different scenarios such as irradiance or environmental oxygen concentration.

Photorefractive intrastromal corneal crosslinking for treatment of low myopia: clinical outcomes using the transepithelial approach with supplemental oxygen.

PURPOSE: To evaluate the safety and efficacy of photorefractive intrastromal corneal crosslinking (PiXL) for the treatment of low myopia using the epithelium-on approach with supplemental oxygen. SETTING: The Eye Foundation, Coimbatore, India. DESIGN: Prospective interventional case series. METHODS: Myopic nonectatic eyes underwent PiXL using the Mosaic system. Ultraviolet-A (UV-A) irradiation of 365 nm wavelength was delivered in an accelerated (30 mW/cm) pulsed approach to provide a total fluence of 15 J/cm. Supplemental oxygen (concentration greater than 95%) was provided to increase the efficacy of the epithelium-on approach during the UV-A irradiation. Efficacy was determined by improvement in mean refractive spherical equivalent (MRSE), uncorrected distance visual acuity (UDVA), and keratometric flattening. Safety was determined by loss of lines of corrected distance visual acuity, endothelial cell loss, and adverse events. RESULTS: Fifty eyes of 26 patients with a mean age of 22.73 ± 3.74 years were included. A significant improvement in UDVA from baseline (logarithm of the minimum angle of resolution [logMAR] 0.63 ± 0.25) was noted at the 3-month (logMAR 0.08 ± 0.15) and 6-month (logMAR 0.13 ± 0.18) follow-up visits (P < .001). Significant keratometric flattening from baseline was noted at all follow-up visits (P < .001). No significant endothelial cell loss or adverse effects were noted. A significant correlation was noted between the change in MRSE and preoperative corneal biomechanics (deformation amplitude ratio, P = .029). CONCLUSIONS: Transepithelial PiXL with supplemental oxygen might be a safe and effective approach for reduction of myopia. The change in MRSE and keratometric flattening was greater in comparison with earlier protocols, including the epithelium-off approach.

Optimization of Oxygen Dynamics, UV-A Delivery, and Drug Formulation for Accelerated Epi-On Corneal Crosslinking.

Purpose: Corneal collagen crosslinking (CXL) through an intact epithelium (epi-on) at high irradiance could potentially improve patient comfort, visual recovery, and clinical workflow compared to conventional epi-off CXL. However, intact epithelium limits stromal delivery of the oxygen, photosensitizer, and ultraviolet-A (UV-A) radiation needed to drive CXL. This ex vivo study evaluated three different epi-on CXL protocols compared to positive and negative controls, specifically focusing on the impact of supplemental oxygen. Endpoints included stromal oxygen levels, stiffness of crosslinked tissue, and acute flattening of whole eyes.Materials & Methods: Ex vivo porcine eyes were held in a custom environmental chamber. Intrastromal oxygen levels were continuously measured before, during, and after UV illumination by a fiberoptic probe inserted into a laser-cut flap. Accelerated, high irradiance, epi-on CXL protocols using riboflavin formulated with benzalkonium chloride (BAC) were studied, with and without supplemental oxygen. These were compared to an alternate, low irradiance, epi-on protocol using riboflavin formulated with sodium iodide. Both negative (no CXL) and positive (epi-off modified Dresden protocol) controls were performed. Post-CXL elastic modulus was measured using extensiometry and anterior tangential curvature was measured using a Scheimpflug tomographer.Results: Protocols including supplemental oxygen resulted in an approximately 5-fold increase in stromal oxygen levels prior to CXL. During epi-on, high-irradiance UV-A delivery under hyperoxic conditions, an aerobic state was maintained. Conversely, under normoxic conditions, stromal oxygen rapidly depleted to 0-5% for all other protocols. The combination of supplemental oxygen, BAC formulation, and high-irradiance UV-A resulted in the largest biomechanical changes and most pronounced flattening effects of the three epi-on protocols.Conclusions: Ex vivo analysis of stromal oxygen levels, corneal stiffness, and acute anterior curvature change indicates that simultaneous optimization of the oxygen environment, riboflavin formulation, and UV-A protocol can significantly increase the effects of corneal collagen crosslinking.

Cellular and molecular assessment of rose bengal photodynamic antimicrobial therapy on keratocytes, corneal endothelium and limbal stem cell niche.

Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is a novel potential treatment for progressive infectious keratitis. The principle behind this therapy is using Rose Bengal as a photosensitizer that can be activated by green light and results in the production of oxygen free radicals which in turn eradicate the microorganism. Given RB-PDAT's mechanism of action and the potential cytotoxic effects, concerns regarding the safety of this technique have arisen. The purpose of this study was to evaluate the effect of RB-PDAT on keratocytes, while focusing on the safety profile that the photo-chemical reaction has on the limbal stem cell (LSC) niche and endothelial cell layer of the treated cornea. To perform RB-PDAT, Rose Bengal solution (0.1% RB in BSS) was applied to the right cornea of rabbits for 30 min and then irradiated by a custom-made green LED light source (525 nm, 6 mW/cm2) for 15 min (5.4 J/cm2). Three rabbits were sacrificed and enucleated after 24 h for evaluation. TUNEL assay and immunohistochemistry for endothelium and limbal stem cell viability were performed on whole mounts and frozen sections in treated and control eyes. LSC of both eyes were isolated and cultured to perform MTT viability and proliferation, and scratch wound healing assays under time-lapse microscopy. Interestingly, while Rose Bengal dye penetration was superficial, yet associated cellular apoptosis was evidenced in up to 1/3 of the stromal thickness on frozen sections. TUNEL assay on whole mounts showed no endothelial cell death following treatment. Immunohistochemistry on frozen sections of LSC displayed no structural difference between treated and non-treated eyes. There was no difference in LSC proliferation rates and scratch wound healing assay demonstrated adequate cell migration from treated and non-treated eyes. The current study suggests that even though penetration of the RB dye has been shown to be limited, oxidative stress produced by RB-PDAT can reach deeper into the corneal stroma. Nevertheless, our results show that performing RB-PDAT is safe on the corneal endothelium and has no effect on LSC viability or function.

Influence of Biochemical Cues in Human Corneal Stromal Cell Phenotype.

PURPOSE: To identify biochemical cues that could promote a keratocyte-like phenotype in human corneal stromal cells that had become fibroblastic when expanded in serum-supplemented media while also examining the effect on cell proliferation and migration. METHODS: Proliferation was assessed by PrestoBlue™, morphology was monitored by phase contrast microscopy, phenotype was analyzed by real-time polymerase chain reaction (qPCR), immunochemistry and flow cytometry, and migration was studied with a scratch assay. RESULTS: Ascorbic Acid (AA), Retinoic Acid (RA), Insulin-Transferrin-Selenium (ITS), Insulin-like Growth Factor 1 (IGF-1) and 3-isobutyl-1-methylxanthine (IBMX) promoted a dendritic morphology, increased the expression of keratocyte markers, such as keratocan, aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and CD34, and prevented myofibroblast differentiation, while in some cases increasing proliferation. Transforming Growth Factor beta 1 (TGF-ß1) and 3 (TGF-ß3) promoted the differentiation toward myofibroblasts, with increased expression of α-SMA. Fibroblast Growth Factor 2 (FGF-2) supported a fibroblastic phenotype while Platelet-Derived Growth Factor Homodimer B (PDGF-BB) induced a pro-migratory fibroblastic phenotype. A combination of all the pro-keratocyte factors was also compared to the serum-free only, which significantly increased CD34 and keratocan expression. CONCLUSIONS: Partially recovery towards a quiescent keratocyte-like phenotype was achieved by the removal of serum and the addition of AA, IGF-1, RA, ITS and IBMX to a basal medium. These findings can be used to develop cell-based corneal therapies and to study corneal diseases in vitro.

The Effect of Ascorbic Acid (Vitamin C) on Transepithelial Corneal Cross-Linking in Rabbits.

PURPOSE: To evaluate the effects of ascorbic acid (vitamin C), the main antioxidant agent in the cornea on transepithelial corneal cross-linking (CXL) where the main mechanism is oxidation. METHODS: Twenty eyes of 20 rabbits were divided into 3 groups: Group 1 (7 eyes) had transepithelial corneal CXL after being fed with normal diet; Group 2 (7 eyes) had corneal CXL after once-daily subcutaneous injections of 200 mg of ascorbic acid in addition to normal diet; and the control group (6 eyes) was fed with normal diet but did not have corneal CXL performed. Ascorbic acid levels were measured in aqueous humor and plasma, and biomechanical measurements were applied to the cornea. RESULTS: There was a significant difference in ascorbic acid levels of plasma (P = 0.008) and aqueous humor (P = 0.006) between group 1 and 2. The Young's modulus values of group 1 and 2 were similar (P = 0.741) and were significantly higher than the control group (P = 0.02 and P = 0.01). The increase rate in Young's modulus values was 37.3% in group 1 and 43.9% in group 2 compared to control group. The ultimate strain values in group 1 and 2 were similar (P = 0.632) and were significantly higher than control group (P = 0.04, P = 0.03). The ultimate stress values in group 1 and 2 were similar (P = 0.836) and were significantly lower than control group (P = 0.001, P = 0.001). CONCLUSIONS: Systemic vitamin C does not appear to decrease effectiveness of transepithelial corneal CXL. Therefore, there is no reason to stop or reduce vitamin C supplementation before corneal CXL therapy.

Characterization of Rabbit Corneas Subjected to Stromal Stiffening by the Açaí Extract (Euterpe oleracea).

PURPOSE: In this study, we characterized rabbit corneas subjected to corneal cross-linking (CXL) with açaí extract compared with a riboflavin photo-stimulated procedure. MATERIALS AND METHODS: The corneas of the slaughterhouse rabbits were divided into three groups: control, consisting of untreated corneal samples; riboflavin/UVA, where corneas were treated with 0.1% riboflavin photo-stimulated at 365 nm as the standard protocol; and açaí, where the samples were subjected to 4% açaí extract for 0.5-2 h. After the CXL procedure, corneas of the three groups were characterized by analyzing their elastic modulus and thermal denaturation profile. RESULTS: The elastic modulus at 3% strain showed an approximately threefold increase in the riboflavin/UVA group and 10.5 times in the corneas treated with 4% açaí extract for 2 h, compared with the control group (p < 0.01). The denaturation temperature values of the two groups of crosslinked corneas increased significantly (p < 0.05) and were more pronounced in the açaí group. CONCLUSIONS: The açaí extract was effective in promoting CXL in rabbit corneas as characterized by the different techniques.

Microbiologic Examination of Bandage Contact Lenses Used after Corneal Collagen Cross-linking Treatment.

PURPOSE: To investigate the agents of bacterial contamination of contact lenses after corneal collagen cross-linking (CCL), and to present the possible changes of ocular flora after riboflavin/ultraviolet A. METHODS: Seventy-two contact lenses of patients who underwent CCL and 41 contact lenses of patients who underwent photorefractive keratectomy (PRK) as control group were enrolled to the study. After 48 h of incubation, broth culture media was transferred to plates. Samples were accepted as positive if one or more colony-forming units were shown. RESULTS: There were positive cultures in 12 (16.7%) contact lenses in the CCL group and 5 (12.2%) had positive cultures in PRK group. Coagulase-negative staphlycocci (CNS) were the most frequent microorganism. Alpha hemolytic streptococci and Diphteroid spp. were the other isolated microorganisms. CONCLUSIONS: Bacterial colonization can occur during and early after the CCL procedure in epithelial healing. To prevent corneal infections after the treatment, prophylactic antibiotics should be prescribed.

Treatment Effect and Corneal Light Scattering With 2 Corneal Cross-linking Protocols: A Randomized Clinical Trial.

IMPORTANCE: We describe and evaluate a complementary method to indirectly quantify the treatment effect of corneal cross-linking (CXL). Additional methods to indirectly quantify the treatment effect of CXL are needed. OBJECTIVE: To assess the spatial distribution and the time course of the increased corneal densitometry (corneal light backscatter) seen after CXL with riboflavin and UV-A irradiation. DESIGN, SETTING, AND PARTICIPANTS: Open-label randomized clinical trial of 43 patients (60 eyes) who were 18 to 28 years of age and had progressive keratoconus and a plan to be treated with CXL at Umeå University Hospital, Umeå, Sweden. The patients were randomized to receive conventional CXL (n = 30) using the Dresden protocol or CXL with mechanical compression of the cornea using a flat rigid contact lens sutured to the cornea during the treatment (CRXL) (n = 30). All participants were followed up during a 6-month period from October 13, 2009, through May 31, 2012. INTERVENTIONS: Corneal cross-linking according to the Dresden protocol or CRXL. MAIN OUTCOMES AND MEASURES: Change in corneal densitometry after CXL and CRXL for keratoconus. RESULTS: Of the original 60 eyes included, 4 had incomplete data. A densitometry increase was seen after both treatments that was deeper and more pronounced in the CXL group (difference between the groups at 1 month in the center layer, zone 0-2 mm, 5.02 grayscale units [GSU], 95% CI, 2.92-7.12 GSU; P < .001). This increase diminished with time but was still noticeable at 6 months (difference between the groups at 6 months in the center layer, zone 0-2 mm, 3.47 GSU; 95% CI, 1.72-5.23 GSU; P < .001) and was proportional to the reduction in corneal steepness (R = -0.45 and -0.56 for CXL and CRXL, respectively). CONCLUSIONS AND RELEVANCE: The degree of corneal light backscatter relates to the reduction in corneal steepness after cross-linking and may become a relevant complement to other methods in evaluating the cross-linking effect, for example, when comparing different treatment regimens. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02425150.

Efficacy of Primary Collagen Cross-Linking with Photoactivated Chromophore (PACK-CXL) for the Treatment of Staphylococcus aureus-Induced Corneal Ulcers.

PURPOSE: To evaluate the efficacy of corneal collagen cross-linking (CXL) with photoactivated riboflavin (PACK-CXL) as primary therapy for Staphylococcus aureus-induced corneal ulcers in a rabbit model. METHODS: The right eye of 40 rabbits was inoculated with S. aureus to induce formation of central corneal ulcers (day 1). The ulcer was examined on day 5, and rabbits were randomly assigned to 4 groups-group A: no treatment (control); group B: topical antibiotic treatment (cefazolin 50 mg/mL, garamycin 14 mg/mL drops, chloramphenicol 5% ointment every 2 hours); group C: PACK-CXL; group D: PACK-CXL + topical antibiotics. Follow-up by biomicroscopy was performed on day 5 and then every week for 1 month. The main outcome measures included infiltrates or the scar diameter, time to healing, time to full epithelialization, and a change in corneal thickness. RESULTS: After 1 month of treatment, group C ulcers had the smallest mean scar diameter (8.8 mm), followed by groups D (11.2 mm), B (13.0 mm), and A (24.5 mm) (P = 0.011). Group C had the shortest mean healing time (15.5 days), followed by groups D (17.2 days), B (19.7 days), and A (21.8 days). Analysis of relative reduction in the infiltrate size from day 5 yielded better results for groups C (P = 0.039) and D (P = 0.034) than those of group B. CONCLUSIONS: We demonstrate a beneficial effect of PACK-CXL as primary treatment, either as stand-alone or as an adjuvant to antimicrobial therapy.

Development of a convenient ex vivo model for the study of the transcorneal permeation of drugs: histological and permeability evaluation.

In this paper, an ex vivo model for the study of the transcorneal permeation of drugs, based on porcine tissues, was evaluated. The setup is characterized by ease of realization, absence of O2 and CO2 bubbling and low cost; additionally, the large availability of porcine tissue permits a high throughput. Histological images showed the comparability between porcine and human corneas and confirmed the effectiveness of the isolation procedure. A new de-epithelization procedure based on a thermal approach was also set up to simulate cornea permeability in pathological conditions. The procedure did not affect the integrity of the underlying layers and allowed the characterization of the barrier properties of epithelium and stroma. Six compounds with different physicochemical properties were tested: fluorescein, atenolol, propranolol, diclofenac, ganciclovir and lidocaine. The model highlighted the barrier function played by epithelium toward the diffusion of hydrophilic compounds and the permselectivity with regard to more lipophilic molecules. In particular, positively charged compounds showed a significantly higher transcorneal permeability than negatively charged compounds. The comparability of results with literature data supports the goodness and the robustness of the model, especially taking into account the behavior of fluorescein, which is generally considered a marker of tissue integrity.