Suppression of lipopolysaccharide-induced corneal opacity by hepatocyte growth factor

Keratitis induced by bacterial toxins, including lipopolysaccharide (LPS), is a major cause of corneal opacity and vision loss. Our previous study demonstrates hepatocyte growth factor (HGF) promotes epithelial wound healing following mechanical corneal injury. Here, we investigated whether HGF has the capacity to suppress infectious inflammatory corneal opacity using a new model of LPS-induced keratitis. Keratitis, induced by two intrastromal injections of LPS on day 1 and 4 in C57BL/6 mice, resulted in significant corneal opacity for up to day 10. Following keratitis induction, corneas were topically treated with 0.1% HGF or PBS thrice daily for 5 days. HGF-treated mice showed a significantly smaller area of corneal opacity compared to PBS-treated mice, thus improving corneal transparency. Moreover, HGF treatment resulted in suppression of α-SMA expression, compared to PBS treatment. HGF-treated corneas showed normalized corneal structure and reduced expression of pro-inflammatory cytokine, demonstrating that HGF restores corneal architecture and immune quiescence in corneas with LPS-induced keratitis. These findings offer novel insight into the potential application of HGF-based therapies for the prevention and treatment of infection-induced corneal opacity.

Ultrastructural Analysis of Rehydrated Human Donor Corneas After Air-Drying and Dissection by Femtosecond Laser

Purpose: To evaluate the efficiency of femtosecond laser (FSL) incision of rehydrated human donor corneas after air-drying and its effects on corneal structure.Methods: We compared the rehydrated and fresh-preserved corneas by microscopy following Victus-Tecnolas FSL treatment for straight-edge anterior lamellar keratoplasty (ALK). The corneas were dehydrated at room temperature under a laminar-flow hood.Results: To obtain the horizontal cut in rehydrated corneas, we increased the FSL pulse energy to 1.2 μJ from 0.80 μJ applied for the fresh corneas and obtained a clear-cut separation of the lamellar lenticule cap from the corneal bed. Light microscopy showed regular arrangement of stromal collagen lamellae, with spaces in between the fibers in the corneal stroma in the fresh and the rehydrated corneas, but the uppermost epithelial layers in the rehydrated corneas were lost. Transmission electron microscopy (TEM) revealed no signs of thermal or mechanical damage to the corneal structure. The epithelial basal membrane and Bowman's layer maintained their integrity. The epithelial basal layer and cells were separated by large spaces due to junction alteration in the rehydrated corneas. There were gaps between the lamellar layers in the stroma, especially in the rehydrated corneas. Keratocytes displayed normal structure in the fresh corneas but were devoid of microorganules in the rehydrated corneas. Minor irregularities were observed in the vertical incision and the horizontal stroma appeared smooth on scanning electron microscopy.Conclusion: The corneal stroma of rehydrated corneas maintained morphology and integrity, while corneal cellular components were generally altered. When corneas are intended for FSL-assisted ALK, effective stromal bed incision is best achieved at a laser power higher than that currently adopted for fresh corneas.

Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas

Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to −40 °C and 3 °C/min to −120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.

Densitometric assessment of corneal transparency after correction of moderate myopia by femtosecond extraction of the lenticule through a small incision and using laser keratomilesis in situ with femtosecond assistance

Relevance. The study of the mechanisms of insufficiently rapid achievement of high visual acuity in the early postoperative period in the correction of myopia by the SMILE method is relevant. Purpose. To evaluate changes in corneal densitometry parameters after SMILE and FS-LASIK surgery in patients with moderate myopia. Material and methods. A study of 152 patients with moderate myopia was conducted, 68 were operated by SMILE and 84 – FS-LASIK. All procedures were performed using a VisuMax femtosecond laser and a MEL 80 excimer laser (Carl Zeiss Meditec, Germany). Assessment of visual acuity, corneal structure (OCT, Optovue, USA), corneal densitometry (Pentacam Scheimpflug, Germany) were performed before the operation, on the 1st, 5th day, 3, 6, 12 months after the operation. OCT scans were analyzed using the ImageJ program. Results. Оn the 1st day after SMILE, visual acuity (p=0.01) and transparency of the anterior and middle layers of the cornea were reduced than after FS-LASIK in the zone from 0 to 2 mm (p=0.045, p=0.001), from 2 to 6 mm (both p=0.001). These differences became statistically insignificant 5 days after surgery. By three and six months in the FS-ERASER group, the corneal transparency in the middle layers were reduced in the 0–2 mm and 2–6 mm zones (p=0.0001, p=0.001). In both groups, by 12 months, the corneal backscattering reached the values of the preoperative period (p>0,05). Conclusion. Refractive operations SMILE and FS-LASIK are accompanied by a decrease in corneal transparency, which is restored to preoperative values by 12 months. In the early postoperative period, an increase in densitometry indicators and a slower recovery of visual acuity after SMILE surgery may be due to active remodeling of the interface, which includes fragments of collagen fibrils and cellular components inside the intrastromal space. Key words: SMILE, FS-LASIK, densitometry, myopia

Confocal biomicroscopic changes of the corneal layers following MyoRing implantation with corneal collagen crosslinking in keratoconus

Purpose. To study morphological changes in the cornea by confocal microscopy of simultaneous MyoRing implantation with corneal crosslinking in patients with keratoconus. Material and methods. The clinical study included 15 patients (16 eyes) with progressive keratoconus. All patients underwent a combination treatment: implantation of MyoRing intrastromal corneal rings (Dioptex GmBH, Linz, Austria) in combination with corneal crosslinking. The number of cells and quality indicators in each layer of the cornea were compared before and during 36 months after surgery. Results. Qualitative analysis of the cornea showed transient disturbances of the subepithelial nerve plexus, an increase in the reflectivity of stromal keratocytes, and moderate fibroplasia in the middle parts of the stroma in the area of the intrastromal pocket. During the follow-up period of 12 months or more after surgery, no significant changes in the corneal ultrastructure were detected in the dynamics. There was no significant decrease in endothelial cell density in the postoperative period. Conclusion. Analysis of morphological changes confirms the safety of a combination of simultaneous implantation of the MyoRing intrastromal ring and corneal crosslinking in stage II-III keratoconus by confocal microscopy and allows dynamic observation of changes in the corneal structure during intrastromal surgery. Key words: keratoconus, confocal microscopy, intrastromal corneal rings, MyoRing, corneal crosslinking.

Evaluation of in-situ gel-forming eye drop containing bacteriophage against Pseudomonas aeruginosa keratoconjunctivitis in vivo

Introduction: Eradication of Pseudomonas aeruginosa has become increasingly difficult due to its remarkable capacity to resist antibiotics. Bacteriophages have been suggested as an alternative treatment for bacterial infections. Methods: In-situ gel-forming eye drop containing phage against P. aeruginosa keratoconjunctivitis was prepared. The Cystoviridae phage was formulated as in-situ gel-forming formulation which is a solution formulation but turns into gel when it contacts the eye. Therapeutic effectiveness of the in-situ gel forming formulation was evaluated by histological examination on day 12 post-infection. Results: The viscosity of selected formulation increased when it was instilled into the eye. The histological results showed edema, abscesses, and destruction of the stromal structure of cornea in groups where no in-situ gel-forming formulation was used. In the group where in-situ gel forming formulation was used, re-epithelialization and normal corneal structure were observed. Conclusion: In-situ gel-forming ophthalmic formulation containing phage can be effective in the treatment of P. aeruginosa keratoconjunctivitis.

Corneal Structure After Small-Incision Lenticule Extraction in Diabetic Rabbits

Background: To assess the corneal structure after femtosecond laser small-incision lenticule extraction (SMILE) in diabetic rabbits with different blood glucose levels.Methods: Twelve diabetic rabbits were randomly distributed into three groups based on blood glucose levels: group A (G-A), 16-20 mmol/L (n=4); group B (G-B), 4.33-8.61 mmol/L (n=4); and group C (G-C), healthy controls (n=4). After 2 weeks, SMILE was performed. Corneas were evaluated using anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM) preoperatively on day 1 and postoperatively at weeks 1, 4, and 12.Results: Morphological results: Twelve weeks after surgery, a corneal interlamellar stromal scar in three eyes and corneal stroma edema in one eye were found in G-A; no obvious scar edema occurred in G-B and G-C. In G-A, the number of abnormal cells in each corneal layer increased, the cell arrangement was irregular, and nerve fibers decreased significantly; the morphology and arrangement in G-B and G-C were regular. Cell density results: Actual central corneal thickness (CCT) after SMILE was thicker than theoretical CCT in each group (P<0.05). The corneal thickness deviation (△CT) of G-A was thicker than that of G-B and G-C, and the pre-keratocyte density (pre-KD) of G-A was lower than that of G-B and G-C (P<0.05). Endothelial cell density (ECD) at 4 and 12 weeks after surgery only in G-A was lower than that before surgery.Conclusions: Hyperglycemia is an adverse factor affecting corneal structure after SMILE. SMILE is safe and effective for diabetic rabbits with good blood glucose control.