Corneal Healing Process Research Articles

Proteomic Characterization of Corneal Epithelial and Stromal Cell-Derived Extracellular Vesicles.

Communication between the different layers of the cornea (epithelium and stroma) is a complex, yet crucial element in the corneal healing process. Upon corneal injury, it has been reported that the bi-directional cross talk between the epithelium and stroma via the vesicular secretome, namely, extracellular vesicles (EVs), can lead to accelerated wound closure upon injury. However, the distinct protein markers of EVs derived from human corneal epithelial (HCE) cells, keratocytes (HCKs), fibroblasts (HCFs), and myofibroblasts (HCMs) remain poorly understood. All EVs were enriched for CD81 and showed increased expression levels of ITGAV and FN1 in HCM-EVs compared to HCE- and HCF-EVs. All EVs were negative for GM130 and showed minimal differences in biophysical properties (particle concentration, median particle size, and zeta potential). At the proteomic level, we show that HCM-EVs are enriched with proteins associated with fibrosis pathways, such as COL6A1, COL6A2, MMP1, MMP2, TIMP1, and TIMP2, compared to HCE-, HCK-, and HCF-EVs. Interestingly, HCE-EVs express proteins involved with the EIF-2 signaling pathway (stress-induced signals to regulate mRNA translation), such as RPS21, RALB, EIF3H, RALA, and others, compared to HCK-, HCF-, and HCM-EVs. In this study, we isolated EVs from cell-conditioned media from HCE, HCKs, HCFs, and HCMs and characterized their biophysical and protein composition by Western blot, nanoparticle tracking analysis, and proteomics. This study supports the view that EVs from the corneal epithelium and stroma have a distinct molecular composition and may provide novel protein markers to distinguish the difference between HCE-, HCK-, HCF-, and HCM-EVs.

Chlorine-Induced Toxicity on Murine Cornea: Exploring the Potential Therapeutic Role of Antioxidants.

Chlorine (Cl2) exposure poses a significant risk to ocular health, with the cornea being particularly susceptible to its corrosive effects. Antioxidants, known for their ability to neutralize reactive oxygen species (ROS) and alleviate oxidative stress, were explored as potential therapeutic agents to counteract chlorine-induced damage. In vitro experiments using human corneal epithelial cells showed decreased cell viability by chlorine-induced ROS production, which was reversed by antioxidant incubation. The mitochondrial membrane potential decreased due to both low and high doses of Cl2 exposure; however, it was recovered through antioxidants. The wound scratch assay showed that antioxidants mitigated impaired wound healing after Cl2 exposure. In vivo and ex vivo, after Cl2 exposure, increased corneal fluorescein staining indicates damaged corneal epithelial and stromal layers of mice cornea. Likewise, Cl2 exposure in human ex vivo corneas led to corneal injury characterized by epithelial fluorescein staining and epithelial erosion. However, antioxidants protected Cl2-induced damage. These results highlight the effects of Cl2 on corneal cells using in vitro, ex vivo, and in vivo models while also underscoring the potential of antioxidants, such as vitamin A, vitamin C, resveratrol, and melatonin, as protective agents against acute chlorine toxicity-induced corneal injury. Further investigation is needed to confirm the antioxidants' capacity to alleviate oxidative stress and enhance the corneal healing process.

Biomimetic Convex Implant for Corneal Regeneration Through 3D Printing.

Blindness caused by corneal damage affects millions of people worldwide, and this number continues to rise. However, rapid epithelization and a stable epithelium process are the two biggest challenges for traditional corneal materials. These processes are related to corneal curvature, which is an important factor in determination of the corneal healing process and epithelial behavior during corneal damage. In this study, smooth 3D-printed convex corneal implants based on gelatin methacrylate and collagen are generated. As epithelium distribution and adhesion vary in different regions of the natural cornea, this work separates the surfaces into four regions and studies how cells sense topological cues on curvature. It is found that rabbit corneal epithelial cells (RCECs) seeded on steeper slope gradient surfaces on convex structures result in more aligned cell organization and tighter cell-substrate adhesion, which can also be verified through finite element simulation and signaling pathway analysis. In vivo transplantation of convex implants result in a better fit with adjacent tissue and stronger cell adhesion than flat implants, thereby accelerating corneal epithelialization and promoting collagen fibers and neural regeneration within 180 days. Taken together, printed convex corneal implants that facilitate corneal regeneration may offer a translational strategy for the treatment of corneal damage.

Commercial amniotic membrane extract for treatment of corneal ulcers in adult horses.

Amniotic membrane extract enhances the rate of epithelialisation after corneal ulceration in several species but has not been studied in the equine cornea. To evaluate the effect of amniotic membrane extract on re-epithelialisation of equine corneal ulcers compared with ulcers treated with antibiotic, antifungal and mydriatic medical therapy alone, and to evaluate equine corneal healing after experimentally induced superficial ulceration. Masked, randomised, controlled experimental trial. Superficial, 8mm corneal ulcers were created bilaterally in each horse. One eye was treated with amniotic membrane extract and the opposite was control. Both eyes were treated with medical therapy. Treatment eyes received amniotic membrane extract, and control eyes received the amniotic membrane extract vehicle. Ulcers were stained with fluorescein and photographed in 12-hour increments until completely healed. Ulcer surface area was determined by analysing photographs with ImageJ. A mixed linear model was used to compare ulcer surface area and hours until healing between treatment groups. A regression model was also used to calculate corneal re-epithelialisation rate over time. Regardless of therapy, healing occurred in two phases: an initial rapid phase of 0.88mm2 /hr (95% CI: 0.81-0.94mm2 /hr) for approximately 48-54hours followed by a second, slow phase of 0.07mm2 /hr (95% CI: 0.04-0.09mm2 /hr). Most eyes healed within 135.5±48.5hours. Treatment (amniotic membrane extract vs. control) was not significantly associated with size of ulcers over time (P=.984). Discomfort was minimal to absent in all horses. Results achieved experimental studies may differ from outcomes in the clinical setting. There was no significant difference in healing rate with addition of amniotic membrane extract to medical therapy for equine superficial corneal ulcers. A biphasic corneal healing process was observed, with an initial rapid phase followed by a slow phase. Further study will be needed to determine whether amniotic membrane extract will be helpful for infected or malacic equine corneal ulcers.

Treatment of Corneal Alkali Burn with Chestnut Honey, Royal Jelly, and Chestnut Honey-Royal Jelly Mixture.

Objectives:The alkaline burn of the cornea usually results in a decreased vision with opacification. In this study, we investigated the potential role of endemic chestnut honey (CH) and royal jelly on corneal healing after an alkaline burn.Methods:We created an alkaline burn on the center of the corneas of four groups of Wistar rats by applying round filter paper soaked with 1 N NaOH for 30 seconds. The animals were treated with RJ, CH, RJ-CH combination and Na-Hyaluronate (Na-HA) eye drops. We performed a serial evaluation with anterior segment photography on the first, 7th, and 14th days of the experiment. Pathologic examination conducted with hematoxylin and eosin stains and immunostaining for SMA and α4β1 integrin. We evaluated the corneal healing process with a scoring system, which was estimating the degree of corneal edema, the size of the corneal ulcer, and limbal hyperemia.Results:There was no statistically significant difference between groups on the first, 7th, and 14th days concerning the healing scores (p=0.88, p=0.06, p=0.80, respectively). However, there were significantly better scores in the repeated measures of CH (p=0.012) and RJ-CH (p=0.00) groups. The RJ group and Na-HA group did not show a significant difference in repeated measures (p=0.19 and p=0.10, respectively). The α4β1 integrin levels on immunostaining showed a significant difference among groups on the 14th day (p=0.002).Conclusion:We found better corneal healing after treatment with the RJ-CH containing eye drops concerning corneal healing sore and α4β1 integrin staining.

Amniotic Membrane Extract Eye Drop Promotes Limbal Stem Cell Proliferation and Corneal Epithelium Healing.

Objective Human amniotic membrane (HAM) is used as a supporter for limbal stem cell (LSC) expansion and corneal surgery. The aim of study is to use HAM extracts from healthy donors to enhance proliferation of LSCs in vitro and in vivo. Materials and Methods In this interventional experimental study, the effective and cytotoxic doses of the amniotic membrane extract eye drops (AMEED) was assessed by adding different concentrations of AMEED (0-2.0 mg/ml) to LSC cultures for 14 days. Subsequently, the expression levels of ATP-binding cassette sub-family G member 2 (ABCG2, a putative stem cell marker), cytokeratin 3 (K3, corneal maker), K12 and K19 (corneal-conjunctival cell makers) were assessed by real-time polymerase chain reaction (PCR). In the second step, the corneal epithelium of 10 rabbits was mechanically removed, and the right eye of each rabbit was treated with 1 mg/ml AMEED [every 2 hours (group 1) or every 6 hours (group 2)]. The left eyes only received an antibiotic. The corneal healing process, conjunctival infection, degree of eyelid oedema, degree of photophobia, and discharge scores were evaluated during daily assessments. Finally, corneal tissues were biopsied for pathologic evidences. Results In comparison to the positive control [10% foetal bovine serum (FBS)], 0.1-1 mg/ml AMEED induced LSC proliferation, upregulated ABCG2, and downregulated K3. There were no remarkable differences in the expression levels of K12 and K19 (P>0.05). Interestingly, in the rabbits treated with AMEED, the epithelium healing duration decreased from 4 days in the control group to 3 days in the two AMEED groups, with lower mean degrees of eyelid oedema, chemosis, and infection compared to the control group. No pathologic abnormalities were observed in either of the AMEED groups. ConclusionAMEED increases LSCs proliferation ex vivo and accelerates corneal epithelium healing in vivo without any adverse effects. It could be used as a supplement for LSC expansion in cell therapy.

Anti‐glaucoma drugs and their additives, testing the influence of excipients on the corneal healing process in the EX‐Vivo Eye Irritation Test (EVEIT) system

AbstractPurpose This study examined the impact of anti‐glaucoma medications and their additives on the corneal healing process and corneal toxicity.Methods BAK‐free PG analogues (Lumigan UD, Monoprost, Taflotan Sine), soft preserved Travatan BAK free, as well as polyquaternium‐1 (0.001%) and Macrogolglycerolhydroxystearate 40 (5%) were tested regarding corneal irritability and effect on corneal healing against HYLO COMOD (1 mg/mL sodium hyaluronate) as positive and 0.02% benzalkonium chloride (BAK) as negative control. Formulas were applied over three days, six times daily on rabbit corneas cultured on an artificial anterior chamber the Ex Vivo Eye Irritation Test (EVEIT) system. Initially, four corneal abrasions (2.5–4.5 mm2) were induced. All defects were monitored by fluorescein stains and photographs.Results For BAK, the corneal erosion size increased from 14.16 mm² to 88.89 mm². A delay of corneal healing is evident for Monoprost (14.84 mm² to 9.45 mm²) and Macrogolglycerolhydroxystearate 40 (11.58 mm² to 1.56 mm²) in comparison to a complete healing for HYLO COMOD, Lumigan, Taflotan Sine, Travatan and polyquaternium‐1®. For both Monoprost and macrogolglycerolhydroxystearate 40, a bright opaque halo around the remaining erosion area could be noticed. Histology revealed severe alterations of the superficia stroma. Cornea tested without erosions show no corneal toxicity of macrogol 4000.Conclusion Macrogolglycerolhydroxystearate 40, an additive found in Monoprost® shows corneal toxicity solely in case of corneal erosions. This excipient is a known skin irritant. Therefore, the use of Monoprost® in patients with epithelial defects should be questioned. Commercial interest

Comparison of the lubricant eye drops Optive®, Vismed® and Cationorm® on the corneal healing process in an ex‐vivo model of the anterior ocular segment, the Ex Vivo Eye Irritation Test (EVEIT) System.

AbstractPurpose Evaluation of the impact of lubricant eye drops on the corneal healing process and corneal toxicity.Methods Optive and Cationorm were tested regarding corneal irritability and effect on corneal healing against Vismed and 0.01% benzalkonium chloride (BAK) as negative and positive control. Formulas were applied hourly over 3 days on rabbit corneas cultured on artificial anterior chambers (Ex‐Vivo Eye Irritation Test (EVEIT) system). Five corneae were used for each substance except for BAK (n=1). Initially, four corneal abrasions (2 ‐ 5.4 mm²) were induced. All defects were monitored by fluorescein stains and photographes.Results Optive and Vismed showed a complete corneal healing on day 2. In one cornea (Optive) erosion reoccurred on day 3. Erosion sizes of Cationorm treated corneae increased significantly from 12.20 mm² to a subtotal erosion of 51.89 mm² on day 3. Histology revealed epithelial loss and severe alterations of the superficial stroma for Cationorm. Glucose and lactate concentrations were about unchanged after application of Optive and Vismed. In contrast, Cationorm and BAK treated cornea showed a significant increase in lactate concentrations.Conclusion Vismed application led to a rapid corneal healing. Whether the toxicity seen for Optive in one cornea is a valid result should be examined in a long term application study. Cationorm shows considerable corneal toxicity that could be caused by its additive, cetalkonium chloride. Otherwise, Cationorms electrostatic properties lead to a drug film on the area of epithelial loss that could hinders epithelial cell migration and adhesion to heal the lesion. Commercial interest

IL-6 as a corneal wound healing mediator in an in vitro scratch assay

Corneal healing process under inflammatory conditions is not fully understood. We aimed at determining the effect of an inflammatory (presence of IL-6) or anti-inflammatory (presence of IL-10) environment and a mixture of both in the expression of IL-6 signaling pathway mediators, and on corneal wound healing in an in vitro scratch assay. For that purpose, human corneal epithelial cells were cultured until confluence. The effect of IL-6 (10 ng/ml), IL-10 (20 ng/ml) or IL-6 + IL-10 exposure on the expression of IL-6R, gp130, and STAT3 was determined by Western blotting and quantitative PCR, at different time points. The monolayer was mechanically wounded using a sterile 10 μl pipette tip. Wound healing rate in the presence or absence of these cytokines was measured immediately after cytokine exposure and after 4, 8, and 24 h. The effect of mitomycin C on wound healing rate, in control and IL-6-stimulated cells, was also evaluated. Detection of proliferative cells was performed with an EdU imaging kit. For the visualization of migrating cells, cold methanol-fixed cells were incubated with an α-actinin antibody. For the statistical analysis a two-factor design of experiment method was applied. Levene test was used to contrast equality of variances. If variances were equal, ANOVA was performed to test the equality of means. If variances were not equal, a Mood's median test was performed. We observed that IL-6 and IL-10 stimulation, and their combination, increased gp130 production at different time points. STAT3 production was increased in IL-6-stimulated cells, at 72 h. An increase in pSTAT3 production was found in IL-6- and IL-10-stimulated cells, that was sustained in time in IL-6 + IL-10 co-stimulated cultures. Scraped areas had an initial width of 570.57 ± 75.82 μm. In IL-6-exposed cells wound healing closure was faster than in control cells or IL-10-exposed cells. After 8 h, wound width in IL-10-exposed cells, was also significantly smaller than that of control cells. Cells exposed to IL-6 + IL-10 had the slowest wound healing rate, similar to control cells. Wounds were closed after 24 h regardless the experimental condition. Mitomycin C exposure increased the wound closure rate in every experimental condition. No significant differences in the percentage of proliferative cells at the edge of the scratch and in distant areas of the monolayer were found. At the edge of the scratch, some actin filaments of non-proliferative cells were directed through the cell-free area, independently of the stimulating condition. In conclusion, the presence of IL-10 and, most importantly, of IL-6, increased the wound healing rate in an in vitro corneal wound healing model. The combination of both cytokines did not have a synergistic action in wound healing. In our model, wound closure was the result of the combination of cell proliferation and cell migration.

Topical application of autologous adipose‐derived mesenchymal stem cells (AdMSCs) for persistent sterile corneal epithelial defect

AbstractPurpose To report a patient with post‐traumatic persistent sterile corneal epithelial defect treated with topical application of autologous AdMSCs.Methods A 35‐yr‐old man was referred to our clinic for treatment of post‐traumatic persistent corneal epithelial defect previously treated as infection. 1 yr before CxL was performed for keratoconus stabilization. Examination showed a central corneal epithelial defect with stromal opacification and thinning. Corneal sensitivity was decreased, while corneal scraping cultures were negative for bacterial or fungal infection. Despite conventional treatment (patching, artificial tears, soft contact lens & autologous serum) no corneal epithelial healing progress occurred 50 days after injury. A PKP was scheduled due to risk of corneal perforation. While waiting for graft, we proposed to the patient the alternative treatment of autologous AdMSCs. IRB was obtained and approved from the Hospital.Results Corneal epithelial healing process was started 10 days after topical application of MSCs, isolated through liposuction. 1 month later, complete corneal epithelial healing was observed. 1 yr later, corneal epithelium remained intact with mild anterior stromal opacification. The patient’s current UCVA lies between 6‐7/10.Conclusion Topical application of autologous adipose‐derived MSCs seems to promote corneal epithelial healing in a patient with persistent sterile corneal epithelial defect refractory to other treatments. Differentiation or trans‐differentiation of MSCs into corneal epithelial/stromal cells and/or production of trophic factors could be possible mechanisms behind the corneal healing process.

Corneal NF-κB activity is necessary for the retention of transparency in the cornea of UV-B-exposed transgenic reporter mice

To determine the dynamics of Nuclear Factor-κB (NF-κB) in murine corneal pathology and the role of NF-κB in maintaining corneal clarity after ultraviolet B radiation insult, transgenic mice containing NF-κB- luciferase reporter were exposed to LPS (bacterial lipopolysaccharide), TNF-α (Tumor Necrosis Factor-alpha) or 4 kJ m −2 UV-B radiation. NF-κB decoy oligonucleotides were also administered in some of the UV-B experiments. Following various exposure times, the mice were sacrificed and whole eyes or corneal tissues were obtained. Whole eyes were examined for scattering using a point-source optical imaging technique. Tissue homogenates were examined for luciferase activity using a luminometer. TNF-α and LPS-injected NF-κB-luciferase transgenic mice demonstrated 3–10-fold increases in cornea NF-κB with peak activities at 4 and 6 hr post-injection, respectively. Mice exposed to 4 kJ m −2 UV-B exhibited a 3-fold increase in NF-κB activity 4 hr post-exposure. The administration of NF-κB-decoy oligonucleotides to mice had the effect of reducing UV-B-induced NF-κB activity in the cornea and significantly increasing the amount of light scattering in UV-B exposed corneas 7 days post-UV-B exposure when compared to sham injected mice. These results indicate that NF-κB is activated in cornea in pathologies that involves increased plasma levels of LPS and TNF-α, as well as direct UV-B exposure, and suggest that NF-κB activation play an essential part in the corneal healing process.

Excimer laser photorefractive keratectomy resulting more myopia postoperatively than preoperatively

SUMMARY Purpose: To present three patients who experienced excessive myopic regression which resulted in more myopia after excimer laser surgery. Methods: All patients, white males, 31 to 35 years old, were treated with the VISX 20/20 excimer laser. Results: Patient 1 had a manifest refraction of -5.00 diopters before PRK and 9 months later it was -4.25 +0.50 x 65o. Six months after retreatment it was -6.00 diopters and the patient had a corneal haze grade 3. Patient 2 had a manifest refraction of -12.25 +2.25 x 10o before surgery and -14.25 +1.00x 105o after wards with a corneal haze grade 3. Patient 3 had a manifest refraction of -5.75 +0.25 x 25o and four months after surgery it was -6.25 with a corneal haze grade 2. Conclusion: The corneal healing process after excimer is notwell understood and patients should be informed about the remote possibility that their myopia may increase after PRK and that they may develop a significanthaze.

Electron microscopy of corneal wound healing

Perforating incisions about 1·5 mm in length were made in the center of rabbit corneas. The eyes were enucleated after various periods, and the corneal wound was studied by light and electron microscopy. The cuts showed excellent apposition anteriorly, and were closed posteriorly by a fibrin clot. The epithelium had started proliferation by 3·5 hr, reaching its peak in 2 days, and a large epithelial plug was formed. The keratocytes in a 300 μm zone adjacent to the cut degenerated and disappeared. Intact peripheral keratocytes migrated toward this acellular zone and underwent mitosis. They reached the cut edge after 2 days, and showed characteristics of fibroblasts. After 3 and 4 days the epithelial plug regressed, and fibroblasts entered the anterior cut in large numbers, forming collagen fibers. However, fibroblasts did not enter the posterior part of the cut. Intact endothelial cells remained very near the cut edge of Descemet's membrane, and began proliferating by 24 hr. Regenerating endothelial cells extended on the posterior surface of the fibrin clot toward the center, covering the wound completely in 4–8 days. These cells were separated from the stroma by the fibrin mass. In late stages, they formed a mass of endothelial cells which filled the posterior portion of the incision. A normal endothelium and a new Descemet's membrane formed on the posterior surface of the mass between the cut edges of the old Descemet's membrane. Finally, the cellular elements in both anterior and posterior portions of the cuts were reduced in number until the border of the scar could not be detected. It is concluded, therefore, that the corneal healing process is different in the anterior and posterior portions of the incision, and that both epithelial cells and fibroblasts participate in healing of the anterior portions, while only endothelial cells play a role in the most posterior portion of the corneal wound.