Lucía Ibares-Frías

Efficacy and safety of short-duration topical treatment with azithromycin oil-based eyedrops in an experimental model of corneal refractive surgery

Purpose To assess the tolerance and side effects of azithromycin eyedrops at the ocular surface after corneal refractive surgery in an experimental animal model. Methods The effect of azithromycin eyedrops was evaluated in hen corneas that underwent laser-assisted in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK) surgery in 1 eye, using the fellow eye (not manipulated) as a control. Animals were treated bid 3 days prior to surgery and 3 days after surgery with T1225 1.5% azithromycin eyedrops or saline eyedrops (balanced salt solution), or were left untreated as a control. Clinical course and cell biology (apoptosis, proliferation, and differentiation) measurements were assessed. Results Infections were present in the following proportions of corneas operated on by LASIK: 0% treated with azithromycin, 60% treated with BSS, and 30% untreated. No corneal abscess or keratitis were present in any PRK or unmanipulated corneas. Conjunctival edema and redness were less prevalent in LASIK-operated eyes treated with azithromycin than in BSS-treated or untreated eyes and were not observed in any PRK or unmanipulated corneas. In PRK-operated eyes treated with azithromycin, a decrease was observed in the apoptosis and an increase in the stromal proliferation. There were no differences in these parameters for LASIK and unmanipulated eyes. Conclusions Topical administration of T1225 oil-based azithromycin eyedrops was well tolerated in both unmanipulated hen corneas and those treated with corneal refractive surgery (PRK and LASIK). T1225 demonstrated a potent antibiotic effect after LASIK treatment.

Scleral changes induced by atropine in chicks as an experimental model of myopia

To determine the effects of intravitreal atropine on scleral growth in the form‐deprived chick as an experimental model of myopia.

Induction of controlled wound healing with PMMA segments in the deep stroma in corneas of hens.

Purpose Polymethylmethacrylate (PMMA) segments are normally used in additive surgery to treat both corneal ectasia post laser-assisted in situ keratomileusis and keratoconus. The aim of this work was to develop an experimental animal model to induce wound healing in the deep stroma in corneas of hens. Methods PMMA segments were implanted in the right eyes of 40 adult hens without suture in the wound incision. Animals were randomized for 5 time points to histopathology study (1, 3, 15, 30, and 90 days) being clinically evaluated during the experiment. Results Thirty-four eyes (85%) had a successful clinical outcome and intraoperative mistakes occurred in 6 eyes (15%), including anterior chamber perforation resulting in abscess (1), excess of lamellar dissection with segment migration (3), and peripheral incisions evolving with neovascularization (2). At 24 hours, all the epithelial injuries were completed in integrity, but a persistent stromal incision, with a fish mouth form, was observed until day 15. Corneal edema disappeared at the fifth day. Haze, keratocyte cell death, keratocyte proliferation, myofibroblast differentiation, and new matrix production were observed in length around the segment. Conclusions Wound healing was induced in the deep corneal stroma by means of PMMA segment implantation in a new animal model developed in hens.

Biomechanical Changes After In Vivo Collagen Cross-Linking With Rose Bengal–Green Light and Riboflavin-UVA

Purpose To compare corneal biomechanical properties after in vivo and ex vivo cross-linking (CXL) using rose bengal-green light (RGX) or riboflavin-UVA (UVX). Methods Corneas of 30 rabbits were treated in vivo by the two CXL modalities monolaterally (Group 1) or bilaterally (Group 2). Rabbits in Group 1 were euthanized 1 month after treatments and in Group 2 two months after treatment. Ex vivo CXL was also performed. Eyes were measured by Scheimpflug air puff corneal deformation imaging (Corvis ST) under constant IOP. Corneal deformation parameters were assessed. Inherent corneal biomechanical properties were estimated using inverse finite element modeling. Results Peak to peak distance decreased 16% 2 months after RGX, and 4% and 20% 1 and 2 months after UVX, respectively. The equivalent Youngs modulus (Eeq) increased relative to the control during the post treatment period for both RGX and UVX. The Eeq increased by factors of 3.4 (RGX) and 1.7 (UVX) 1 month and by factors of 10.7 (RGX) and 7.3 (UVX) 2 months after treatment. However, the Eeq values for ex vivo CXL were much greater than produced in vivo. The ex vivo Eeq was greater than the 1-month in vivo values by factors of 8.1 (RGX) and 9.1 (UVX) and compared with 2 month by factors of 2.5 (RGX) and 2.1 (UVX). Conclusions These results indicate that corneal stiffness increases after CXL, and further increases as a function of time after both RGX and UVX. Also, while biomechanical properties determined after ex vivo CXL are indicative of corneal stiffening, they may not provide entirely accurate information about the responses to CXL in vivo.

Clinical and pathological effects of different acrylic intracorneal ring segments in corneal additive surgery

The objective of this work was to evaluate the potential use of less stiff materials based on acrylic copolymers of methyl methacrylate/2-ethylhexyl acrylate (MMA/EHA) as devices to correct, stabilize and improve the effect of poly(methyl methacrylate) (PMMA) intracorneal ring segments. MMA/EHA and PMMA intracorneal ring segments were surgically implanted in the corneas of Lohmann Classic hens. The effects of the intracorneal ring segments were assessed by optical measurements and corneal tolerance was evaluated through biomicroscopic examination over a 90-day observation period and by conventional histology. The experimental results demonstrated that the intracorneal ring segments made of MMA/EHA copolymers provided a significant change in the corneal curvature and an improved in vivo response compared to those obtained for PMMA rings, which was attributed to the higher flexibility of the copolymeric materials, indicating that these systems might be considered suitable as an alternative to those currently used, for application in clinical practice.

Effects of TGFβ1, PDGF-BB, and bFGF, on human corneal fibroblasts proliferation and differentiation during stromal repair

HighlightsTGF&bgr;1 induces slow and constant proliferation and myofibroblast differentiation, reducing cellular motility during closure.PDGF‐BB induces a quick proliferation and high cellular motility with a low level of myofibroblast differentiation during closure.bFGF accelerates wound closure by increasing cell proliferation inhibiting myofibroblast differentiation. Abstract In an effort to improve the regenerative nature of corneal repair, this study reports the use of an in vitro human corneal fibroblasts (HCFs) wound model after treatment with three of the main growth factors (GFs) involved in corneal healing: transforming growth factor beta 1 (TGF&bgr;1), platelet‐derived growth factor BB‐isoform (PDGF‐BB), and basic fibroblast growth factor (bFGF) in order to delve in cell proliferation and differentiation processes. HCFs were mechanically wounded. The individual effect of TGF&bgr;1, PDGF‐BB, and bFGF on cell proliferation and differentiation during the repair process was studied at different time points until wound closure. Wound dimensions and morphological changes were evaluated by microscopy. Cell proliferation and myofibroblast differentiation were analyzed by immunofluorescence cytochemistry. Changes in cell morphology were apparent at Day 4. PDGF‐BB‐ and bFGF‐treated cells had fibroblast‐like morphology. TGF&bgr;1 stimulated proliferation in the wound edge and surrounding area, induced myofibroblast differentiation and inhibited cellular migration. PDGF‐BB induced rapid wound closure due to proliferation, high motility, and late myofibroblast differentiation. The time course of closure induced by bFGF was similar to that for PDGF‐BB, but was mostly due to proliferation in the wound area, and inhibited myofibroblast differentiation. Each of the GFs induced increases in responses promoting stromal repair differently. This study provides insight regarding how to optimize the outcome of stromal repair following corneal injury.

Clinical, Refractive and Histological Reversibility of Corneal Additive Surgery in Deep Stroma in an Animal Model.

ABSTRACT Purpose: The aim was to evaluate the reversibility of the clinical and histological changes induced in the corneas of an animal model after removing an intracorneal ring segment (ICRS). Methods: Surgery for this study was performed in 38 eyes of an experimental animal model (Gallus domesticus) for ICRS surgery (Ferrara technique). The animals without complications were randomized to two groups; in all of them, 1 segment was implanted in each eye and later removed at different times (1 and 3 months after implantation). In each group, after explantation, corneas were processed at different times for histological analysis with hematoxylin and eosin (H&E) stain and electronic microscopy. The refractive state of the eyes was also measured. Results: In corneas without complications (88.23%), explantation was performed correctly. During the first few days, around the area where the ICRS was implanted we observed deposits of cells and a moderate degree of corneal opacity (haze). These signs decreased progressively without disappearing completely. Histologically, at 7 days, we observed hyperplasia and abnormal arrangement of collagen fibers. Later, these findings also decreased in both groups, albeit at a faster rate in group 1. Minimal changes were observed in electron microscopy up to the end of the study in both groups. Preoperative refractive state was achieved at 1 month after explantation in both groups. Conclusions: ICRS can safely be explanted from the cornea. Refractive reversibility was achieved at 1 month after explantation. However, the clinical and histological findings after ICRS explantation depend on the time from implantation to explantation.

Validation of an experimental animal model for corneal additive surgery

Purpose: To assess the hen cornea as a model for training and future wound healing studies after implantation of intrastromal corneal ring segments (ICRS) by clinical and optical outcomes. Setting: University of Valladolid, Valladolid, Spain. Design: Experimental study. Methods: One 90°, 150-μm thick polymethyl methacrylate Ferrara ICRS segment was manually implanted at 70-80% depth of 192 Gallus domesticus corneas. Clinical follow-up for 6 months included monitoring corneal thickness, epithelial wound closure, edema, haze, and the location and severity of deposits. The refractive state was also measured. After each animal was euthanized, corneas were processed for direct transmittance and histological analysis. Results: Complications were present in 16% of the eyes. Epithelial wound closure was completed at 3 ± 2 days. A slight corneal edema in the channel site was present for the first 15 days. All corneas had deposits by 4 months located along the inner, outer curvatures and under the segments. Corneal haze was present only at the incision site. ICRS induced hyperopic changes in the refractive state without changes in direct transmitance of central cornea. New cells and extracellular matrix were present around the segment where deposits were seen on clinical follow-up. Conclusions: With hen as an animal model, ICRS were implanted in a precise and reproducible way after a learning curve. Similar to humans, the follow-up period during the first 6 months after implantation showed fast wound closure, deposits, and haze at the incision site. ICRS in hens also reduced the refractive power without affecting the central cornea.

Human corneal fibroblast migration and extracellular matrix synthesis during stromal repair: Role played by platelet-derived growth factor-BB, basic fibroblast growth factor, and transforming growth factor-β1: HCFs migration and ECM synthesis during stromal repair: GF effects

The development of treatments that modulate corneal wound healing to avoid fibrosis during tissue repair is important for the restoration of corneal transparency after an injury. To date, few studies have studied the influence of growth factors (GFs) on human corneal fibroblast (HCF) expression of extracellular matrix (ECM) proteins such as collagen types I and III, proteoglycans such as perlecan, or proteins implicated in cellular migration such as α5β1‐integrin and syndecan‐4. Using in vitro HCFs, a mechanical wound model was developed to study the influence of the GFs basic fibroblast GF (bFGF), platelet‐derived GF (PDGF‐BB) and transforming GF‐β1 (TGFβ1) on ECM protein production and cellular migration. Our results show that mechanical wounding provokes the autocrine release of bFGF and TGFβ1 at different time points during the wound closure. The HCF response to PDGF‐BB was a rapid closure due to fast cellular migration associated with a high focal adhesion replacement and a high expression of collagen and proteoglycans, producing nonfibrotic healing. bFGF stimulated nonfibrotic ECM production and limited the migration process. Finally, TGFβ1 induced expression of the fibrotic markers collagen type III and α5β1 integrin, and it inhibited cellular migration due to the formation of focal adhesions with a low turnover rate. The novel in vitro HCF mechanical wound model can be used to understand the role played by GFs in human corneal repair. The model can also be used to test the effects of different treatments aimed at improving the healing process. Copyright

Rose Bengal and Green Light Versus Riboflavin–UVA Cross-Linking: Corneal Wound Repair Response

Purpose To study corneal wound healing after two cross-linking techniques using either rose bengal and green light (RGX) or the conventional treatment using riboflavin and UVA radiation (UVX). Methods Corneas of New Zealand rabbits were monolaterally treated with UVX (21 eyes) or RGX (25 eyes). Treatments involved corneal de-epithelialization (8-mm diameter), soaking with photosensitizer (0.1% riboflavin in 20% dextran for 30 minutes for UVX; 0.1% rose bengal for 2 minutes for RGX), and light irradiation (370 nm, 3 mW/cm2, 30 minutes for UVX; 532 nm, 0.25 W/cm2, 7 minutes for RGX). Contralateral eyes were used as controls. Clinical follow-up included fluorescein staining, haze measurement, and pachymetry. Healing events analyzed after euthanasia at 2, 30, and 60 days included cell death (TUNEL assay), cell proliferation (BrdU [bromodeoxyuridine] immunofluorescence), and differentiation to myofibroblasts (α-SMA [alpha smooth muscle actin] immunohistochemistry). Results Re-epithelialization and pachymetries were similar after RGX and UVX. The haze from day 1 to 15 was greater after UVX. Cell death was deeper after UVX, being localized in the anterior and middle stroma, and was superficial (anterior third) after RGX. Cell proliferation appeared after 2 days and was localized in the middle and posterior stroma in the UVX group but was superficial in the RGX group. After 60 days the number of stromal cells had not returned to the control number in either group. Conclusions The deeper and longer-lasting cell damage caused by UVX compared to RGX may underlie the slower cell repopulation after UVX and other differences in healing. Shallower damage and a shorter treatment time suggest that RGX may be appropriate for stiffening thin corneas.