Corneal Wound Healing Process Research Articles

Establish an In Vitro Cell Model to Explore the Impacts of UVA on Human Corneal Endothelial Wound Healing

ABSTRACT Purpose To provide scientific data for clinical practice in making strategies for accelerating corneal endothelial wound healing, we investigated the impact of UVA on the corneal endothelial wound healing process and the underlying mechanism using an in vitro cell model. Materials and methods An in vitro cell model for corneal endothelial wound healing was established by scratching the in vitro cultured human corneal endothelial cell (HCEnC) confluent layer. Then, we investigated the impacts of UVA irradiation and Ascorbic acid-2-phosphate (Asc-2p) on the wound healing process of the in vitro HCEnC model by examining wound-healing index, F-actin+ rate, Ki-67+ rate, and ROS production. Results After scratching, the Ki-67+ and F-actin+ HCEnCs occupied the scratching gap. Furthermore, the F-actin+ rates were significantly higher than Ki-67+ rates in the wound closure area. After irradiated with UVA, the wound-healing indexes, Ki-67+ rates and F-actin+ rates of the wound-healing model significantly reduced, whereas the ROS production significantly increased in a dose-dependent manner. Pretreatment with Asc-2p significantly reduced the ROS production as well as increased the wound-healing indexes, Ki-67+rates and F-actin+ rates of the UVA irradiated wound-healing model. Conclusion The migration of HCEnC plays a major role in the wound healing process of the established cell model, which is like the wound healing process in vivo. UVA decreases the wound closure of the in vitro HCEnC model dose-dependently, while antioxidant Asc-2p can attenuate the damage to UVA to HCEnCs probably via reducing ROS to improve their migration.

Kinetics of Tear Fluid Proteins after Endothelial Keratoplasty and Predictive Factors for Recovery from Corneal Haze.

Endothelial keratoplasty (EK) is less invasive with faster recovery as compared to conventional penetrating keratoplasty, however, it relies on the clarity of the host corneal stroma. Corneal transplantation involves the induction of immune tolerance for allogeneic tissues as well as the corneal wound healing process, in which coordinated interactions between cytokines and growth factors are critical. In this study, we profiled the expression of 51 soluble factors in the tear fluid over the course of EK and have provided evidence of dynamic changes in cytokine expression in the ipsilateral and contralateral eyes. Cluster analyses classified the cytokine expression kinetics into five groups. Group 1 proteins included TGF-b1, IL-1b, and innate proinflammatory cytokines, which bilaterally increased after surgery, despite the use of topical corticosteroid in the transplanted eyes. Local corticosteroids suppressed cytokines involved in adaptive immunity in the transplanted eyes but not in the contralateral eyes. We found tear protein expression at baseline and one week post-surgery to be a potential predictive biomarker of delayed recovery after EK in terms of the corneal haze and visual acuity. Furthermore, Group 1 tear proteins were most associated with persistent corneal haze pre-surgery as well as visual acuity at one month-post transplant.

Cellular Factor XIII, a Transglutaminase in Human Corneal Keratocytes.

Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.

Dynamic changes of the extracellular matrix during corneal wound healing

The extracellular matrix (ECM) confers transparency to the cornea because of the precise organization of collagen fibrils and a wide variety of proteoglycans. We monitored the corneal wound healing process after alkali burns in rabbits. We analyzed the location and expression of collagens and proteoglycans, the clinical impact, and the recovery of optical transparency.After the animals received both general and ocular topical anesthesia, the central cornea of the left eye was burned by placing an 8-mm diameter filter paper soaked in 0.5 N NaOH for 60 s. The eyes were evaluated under a surgical microscope at 1, 3, and 6 months after burning. At each time point, the clinical conditions of the burned and control corneas were observed. The arrangement of collagen fibers in the corneal stroma was visualized by Picrosirius-red staining, Gomori's silver impregnation and transmission electronic microscopy. Corneal light transmittance was also measured. Myofibroblasts presence was analyzed by immunohistochemistry. mRNA expression levels of collagen types I and III, lumican, decorin, keratocan and alpha-smooth muscle actin were determined by quantitative real-time polymerase chain reaction.One month after alkali burn, the ECM was disorganized and filled with lacunae containing different types of cells and collagen type III fibers in the wound area. Corneal opacities were present with attendant loss of light transmittance. Collagen and proteoglycan mRNA expression levels were up-regulated. After three months, wound healing progress was indicated by reduced corneal opacity, increased light transmittance, reorganization of collagen fibers and only collagen type I expression levels were at control levels. After six months, the wound area ECM morphology was similar to controls, but transmittance values remained low, denoting incomplete restoration of the stromal architecture.This multidisciplinary study of the stromal wound healing process revealed changes in corneal transmittance, collagen organization, myofibroblasts presence and ECM composition at 1, 3, and 6 months after alkali burning. Documenting wound resolution during the six-month period provided reliable information that can be used to test new therapies.

Utility of nanomedicine targeting scar-forming myofibroblasts to attenuate corneal scarring and haze.

Corneal scarring refers to the loss of normal corneal tissue, replaced by fibrotic tissue (during wound repair) thereby affecting corneal transparency and vision quality. The corneal wound healing process involves a complex series of physiological events resulting in the transformation of transparent keratocytes into opaque myofibroblasts; the prominent cause of irregular extracellular matrix synthesis leading to the development of corneal opacity/hazy vision. Globally, corneal scarring/haze is one of the most prevalent causes of blindness. Ocular trauma (physical and chemical) and microbial infections induce corneal tissue damage. Although great progress has been made in the clinical management of ocular diseases, the global rates of corneal blindness remain high, nonetheless. The topical conventional modalities treating corneal wounds/injuries have inherent limitations/side effects such as low bioavailability of a therapeutic agent, upregulation of the intraocular pressure and the toxicity/allergy of the drug. These limitations/side effects rather than treating the wound, often negatively affect the healing process, especially, when applied frequently for longer periods. Recently, there has been an increasing evidence provided by the preclinical studies that nanotechnology-based drug-delivery systems can improve drug bioavailability, through controlled drug release and targeted delivery. After reviewing the epidemiology, risk factors of corneal scarring/haze and the conventional ocular medicines, we review here the different nanodrug-delivery systems and potential drug candidates including nanoherbal formulations investigated for their efficacy to heal the damaged cornea. Finally, we discuss the challenges of using these nanomedicinal platforms.

Changes in Corneal Wound Healing and Graft Biomechanics After Primary Penetrating Keratoplasty Versus Repeat Penetrating Keratoplasty.

To evaluate and compare the influence of the wound healing process at the graft edge on corneal biomechanics after primary penetrating keratoplasty (PK) and repeat PK. This retrospective comparative study included 95 eyes: 35 eyes of 35 patients had PK, 40 eyes of 40 patients underwent repeat PK, and 20 eyes of 20 normal subjects served as the control group. The ocular response analyzer was used to measure and compare corneal hysteresis and corneal resistance factor among the study groups at least 6 months after all sutures were removed. In vivo confocal microscopy was used to study the corneal wound healing process at the graft edge in PK and repeat PK groups and to correlate these findings with corneal biomechanics. Corneal hysteresis and corneal resistance factor were significantly lower in the PK group (6.71 ± 1.3 and 5.99 ± 1.2 mm Hg, respectively) as compared with the repeat PK group (9.4 ± 1.03 and 8.77 ± 1.1 mm Hg, respectively) (P < 0.0001). The repeat PK group demonstrated biomechanical parameters comparable with normal subjects (9.88 ± 1.29 and 9.24 ± 1.13 mm Hg, respectively) (P = 0.12). Confocal microscopy revealed more reflective and activated keratocytes at the graft edge in the repeat PK group as compared with the primary PK group. The healing process at the graft edge is the key to optimal corneal integrity after PK. Repeat PK stimulated more activated keratocytes than primary PK and hence a stronger healing response, providing superior corneal biomechanics comparable with normal subjects.

Induction of Fibroblast Senescence During Mouse Corneal Wound Healing.

To investigate the presence and role of fibroblast senescence in the dynamic process of corneal wound healing involving stromal cell apoptosis, proliferation, and differentiation. An in vivo corneal wound healing model was performed using epithelial debridement in C57BL/6 mice. The corneas were stained using TUNEL, Ki67, and α-smooth muscle actin (α-SMA) as markers of apoptosis, proliferation, and myofibroblastic differentiation, respectively. Cellular senescence was confirmed by senescence-associated β-galactosidase (SA-β-gal) staining and P16Ink4a expression. Mitogenic response and gene expression were compared among normal fibroblasts, H2O2-induced senescent fibroblasts, and TGF-β-induced myofibroblasts in vitro. The senescence was further detected in mouse models of corneal scarring, alkali burn, and penetrating keratoplasty (PKP). The apoptosis and proliferation of corneal stromal cells were found to peak at 4 and 24 hours after epithelial debridement. Positive staining of SA-β-gal was observed clearly in the anterior stromal cells at 3 to 5 days. The senescent cells displayed P16Ink4a+ vimentin+ α-SMA+, representing the major origin of activated corneal resident fibroblasts. Compared with normal fibroblasts and TGF-β-induced myofibroblasts, H2O2-induced senescent fibroblasts showed a nonfibrogenic phenotype, including a reduced response to growth factor basic fibroblast growth factor (bFGF) or platelet-derived growth factor-BB (PDGF-BB), increased matrix metalloproteinase (MMP)1/3/13 expression, and decreased fibronectin and collagen I expression. Moreover, cellular senescence was commonly found in the mouse corneal scarring, alkali burn, and PKP models. Corneal epithelial debridement induced the senescence of corneal fibroblasts after apoptosis and proliferation. The senescent cells displayed a nonfibrogenic phenotype and may be involved in the self-limitation of corneal fibrosis.

Role of Bone Morphogenetic Protein 7 (BMP7) in the Modulation of Corneal Stromal and Epithelial Cell Functions.

In the cornea, healing of the wounded avascular surface is an intricate process comprising the involvement of epithelial, stromal and neuronal cell interactions. These interactions result to the release of various growth factors that play prominent roles during corneal wound healing response. Bone morphogenetic proteins (BMPs) are unique multi-functional potent growth factors of the transforming growth factor-beta (TGF-β) superfamily. Treatment of corneal epithelial cells with substance P and nerve growth factor resulted to an increase in the expression of BMP7 mRNA. Since BMP7 is known to modulate the process of corneal wound healing, in this present study, we investigated the influence of exogenous rhBMP7 on human corneal epithelial cell and stromal cell (SFs) function. To obtain a high-fidelity expression profiling of activated biomarkers and pathways, transcriptome-wide gene-level expression profiling of epithelial cells in the presence of BMP7 was performed. Gene ontology analysis shows BMP7 stimulation activated TGF-β signaling and cell cycle pathways, whereas biological processes related to cell cycle, microtubule and intermediate filament cytoskeleton organization were significantly impacted in corneal epithelial cells. Scratch wound healing assay showed increased motility and migration of BMP7 treated epithelial cells. BMP7 stimulation studies show activation of MAPK cascade proteins in epithelial cells and SFs. Similarly, a difference in the expression of claudin, Zink finger E-box-binding homeobox 1 was observed along with phosphorylation levels of cofilin in epithelial cells. Stimulation of SFs with BMP7 activated them with increased expression of α-smooth muscle actin. In addition, an elevated phosphorylation of epidermal growth factor receptor following BMP7 stimulation was also observed both in corneal epithelial cells and SFs. Based on our transcriptome analysis data on epithelial cells and the results obtained in SFs, we conclude that BMP7 contributes to epithelial-to-mesenchymal transition-like responses and plays a role equivalent to TGF-β in the course of corneal wound healing.

Impact of Hyaluronic Acid-Containing Artificial Tear Products on Reepithelialization in an In Vivo Corneal Wound Model.

Purpose: To evaluate the effect of 6 commercially available hyaluronic acid (HA)-containing topical artificial tear products on corneal reepithelialization following injury, in an in vivo mouse model.Methods: Ninety-six C57Bl/6 mice (16 per treatment group; male to female ratio, 1:1 per group) were anesthetized. Epithelial debridement was performed on 1 cornea per animal, and the debrided eye was imaged. A 30 μL masked test solution containing 1 of 6 artificial tear products was instilled, immediately on debridement, and subsequently, every 2 h, for a total of 4 administrations. At 24 h post debridement, corneas were stained with fluorescein and imaged to calculate corneal healing rate (number of fluorescein-negative corneas).Results: All 6 artificial tear products used in this study permitted the initial process of corneal wound healing. However, the corneal reepithelialization rate after 24 h was higher with Hydroxypropyl guar (HPG)/HA (53.33%) compared with other HA-containing artificial tear products [HA1 (12.5%), HA2 (26.67%), HA3 (31.25%), HA4 (6.25%), and HA5 (43.75%)]. The average area and percentage area of reepithelialization after 24 h were also higher with HPG/HA compared with other treatment groups.Conclusions: Percentage of eyes with complete corneal reepithelialization 24 h post debridement was highest with HPG/HA compared with other HA-containing artificial tear products tested. The results of this study provide additional evidence on the potential benefits of HPG/HA in the management of dry eye and its role in the rapid restoration of a healthy ocular epithelium. However, further studies are required to confirm the effects on human corneal wounds.

Topical Administration of Spironolactone-Loaded Nanomicelles Prevents Glucocorticoid-Induced Delayed Corneal Wound Healing in Rabbits.

The objective was to investigate whether mineralocorticoid receptor antagonism using a novel topical micellar formulation of spironolactone could prevent glucocorticoid-induced delayed corneal wound healing in New Zealand white rabbits. Spironolactone micelles (0.1%, w/v) with a mean number weighted diameter of 20 nm were prepared using a pegylated copolymer (mPEG-dihexPLA) and showed a preliminary stability of at least 12 months at 5 °C. Preclinical studies in New Zealand white rabbits demonstrated that the 0.1% spironolactone micellar formulation was well-tolerated since no reaction was observed in the cornea following multiple daily instillation over 5 days. As expected, the preclinical studies also confirmed that dexamethasone significantly delayed epithelial wound healing as compared to untreated control (percentage re-epithelialization after day 4: 84.6 ± 13.9% versus 99.5 ± 1.0% for the control, p < 0.05). However, the addition of the 0.1% spironolactone micellar formulation significantly improved the extent of re-epithelialization, countering the dexamethasone induced delayed wound healing with a percentage re-epithelialization that was statistically equivalent to the control (96.9 ± 7.3% versus 99.5 ± 1.0%, p > 0.05). The biodistribution study provided insight into the ocular metabolism of spironolactone and hence the relative contributions of the parent molecule and its two principal metabolites, 7α-thiomethylspironolactone and canrenone, to the observed pharmacological effects. Comparison of the efficacies of spironolactone and potassium canrenoate (a water-soluble precursor of canrenone) in overcoming the dexamethasone-induced delayed wound healing confirmed that the former had greater efficacy. The results pointed to the greater potency of 7α-thiomethylspironolactone over canrenone as a mineralocorticoid receptor antagonist, which explained its superior ability in countering the glucocorticoid-induced overactivation that was responsible for the delayed wound healing. In conclusion, the preliminary results supported the above-mentioned hypothesis suggesting that coadministration of mineralocorticoid receptor antagonists to patients under glucocorticoid therapy might prevent the deleterious effects of glucocorticoids on complex corneal wound healing processes.

Over-expression of ΔNp63α facilitates rat corneal wound healing in vivo.

To investigate the roles of ΔNp63α during corneal wound healing and the genes regulated by ΔNp63α in limbal epithelial cells. Adenovirus or shRNA targeting ΔNp63α were pre-injected into the anterior chamber of rat eyeballs and the central corneal epithelium was then wounded with NaOH. The effects of ΔNp63α expression during wound healing were observed by propidium iodide staining. In addition, limbal epithelial cells were cultured and ectopically expressed ΔNp63α by transfecting Ad-ΔNp63α. Total RNA was extracted from transfected epithelial cells and subjected to a gene expression microarray assay. The results showed that over-expression of ΔNp63α accelerated the process of corneal wound healing while knockdown of ΔNp63α impaired the process. ΔNp63α positively up-regulated several cell growth promoter genes and could be referred as a positive regulator of limbal epithelial cell proliferation. It might also inhibit cell differentiation and cell death by differential target gene regulation.

Visual outcomes and management after corneal refractive surgery: A review

Corneal refractive surgery procedures are widely performed to permanently correct refractive errors. Overall, refractive surgeries are safe, predictable and present high rates of satisfaction. Nevertheless, the induced epithelial, stromal and nerve damage alters corneal integrity and function, triggering a regenerative response. Complications that arise from corneal wound healing process might directly impact on visual outcomes of keratorefractive procedures. Most of these complications can be prevented or effectively treated with minimal consequences and minor impact on optical quality. Nevertheless, it is crucial to accurately and timely identify these corneal regeneration-related complications for successful counseling and management. Optometrists, as primary eye care providers, play an essential role in detecting anatomic and functional alterations in vision. It is therefore of great interest for optometrists to be familiar with the principal postoperative complications derived from alterations in regenerative process after corneal laser refractive surgeries. This review aims to provide a basis for optometrists to better understand, identify and manage the main wound healing-related complications after refractive surgery.

Descemet\u2019s Membrane Supports Corneal Endothelial Cell Regeneration in Rabbits

Descemet’s membrane (DM) helps maintain phenotype and function of corneal endothelial cells under physiological conditions, while little is known about the function of DM in corneal endothelial wound healing process. In the current study, we performed in vivo rabbit corneal endothelial cell (CEC) injury via CEC scraping, in which DM remained intact after CECs removal, or via DM stripping, in which DM was removed together with CECs. We found rabbit corneas in the CEC scraping group healed with transparency restoration, while there was posterior fibrosis tissue formation in the corneas after DM stripping on day 14. Following CEC scraping on day 3, cells that had migrated toward the central cornea underwent a transient fibrotic endothelial-mesenchymal transition (EMT) which was reversed back to an endothelial phenotype on day 14. However, in the corneas injured via DM stripping, most of the cells in the posterior fibrosis tissue did not originate from the corneal endothelium, and they maintained fibroblastic phenotype on day 14. We concluded that corneal endothelial wound healing in rabbits has different outcomes depending upon the presence or absence of Descemet’s membrane. Descemet’s membrane supports corneal endothelial cell regeneration in rabbits after endothelial injury.

Thymic stromal lymphopoietin is expressed in human corneal stromal cells and secreted upon protease-activated receptor 1 activation.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7) like cytokine that triggers inflammatory responses through activating dendritic cell. It is demonstrated to be involved in a number of disorders, including cancer, gastrointestinal diseases, and autoimmune diseases. In addition, TSLP is also found to be able to induce itch sensation by directly activating the itch sensory neurons. The expression of human TSLP is detected in various tissues such as heart, liver, testis, and prostate. In this study, we report that TSLP was expressed in the human corneal stromal cells, the highly specialized cells that are critical for the function of the cornea. Furthermore, we found that activation of the protease-activated receptor 1 (PAR1) induced secretion of TSLP by the corneal stromal cells. Beside the canonical activator thrombin, PAR1 can also be activated by a few matrix metalloproteases (MMPs) such as MMP1 and MMP13. Since MMPs including MMP1 and MMP13 are upregulated in many corneal diseases as well as the corneal wound healing process, we proposed that TSLP might function as the link between increased protease activity and inflammatory responses or itch sensation in the corneas. © 2017 IUBMB Life, 69(8):606-610, 2017.

Plasminogen activator activity in tears of pregnant women.

PurposePlasminogen activator activity (PAA) in tears of pregnant women was investigated at various gestation times to assess the availability of plasminogen activator for aiding potential corneal wound healing processes during pregnancy.MethodsPAA was measured by a spectrophotometric method. The analysis used 91 tear samples from pregnant and non-pregnant women, supplemented with 10 additional tear PAA measurements from non-pregnant women obtained in a previous study.ResultsTear levels of PAA in pregnant women formed a bimodal distribution. Either the tear PAA level was zero or non-zero during pregnancy. When non-zero, the tear PAA level was dissociated from gestation time and not different than non-pregnant and post-pregnant levels. The frequency of occurrence of zero level tear PAA increased with gestation: 16%, 17% and 46% had zero tear PAA in samples taken from women in the first, second and third trimester, respectively.ConclusionsOverall, of the tear samples taken from women during pregnancy, a total of 26% were at zero tear PAA. The remaining tear samples had non-zero tear PAA values throughout gestation equivalent to non-pregnant tear PAA values, suggesting local control of the source of PAA in tears. Given the importance of the plasminogen activator system in tears to wound healing in the cornea, and the high occurrence of zero tear PAA in our sample of pregnant women, elective corneal surgery would be contraindicated. If corneal surgery is nevertheless necessary, the tear PAA level would be worth checking and patients with low level should be closely observed during the postoperative period.

Silk-Derived Protein Enhances Corneal Epithelial Migration, Adhesion, and Proliferation.

PurposeThe corneal surface is vulnerable to a myriad of traumatic insults including mechanical, chemical, and thermal injuries. The resulting trauma may render the naturally occurring regenerative properties of the cornea incapable of restoring a healthy epithelial surface, and may result in the loss of corneal transparency and vision. Healing of the corneal epithelium requires a complex cascade of biological processes that work to restore the tissue after injury. New therapeutic agents that act on the multiple steps of the corneal wound-healing process would offer a potential for improving patient outcomes. Here, a novel silk fibroin–derived protein (SDP) was studied for potential impacts on wound healing through studying an in vitro model.MethodsSolubilized SDP, produced from the Bombyx mori silkworm cocoon, was added to human corneal limbal-epithelial (hCLE) cultures to evaluate the material's effects on epithelial cell migration, proliferation, and adhesion through the use of various scratch wound assays and flow chamber studies.ResultsResults indicated that the addition of SDP to culture increased hCLE migration rate by over 50%, and produced an approximate 60% increase in cell proliferation. This resulted in a nearly 30% enhancement of in vitro scratch wound closure time. In addition, cultures treated with SDP experienced increased cell-matrix focal adhesion formation by over 95% when compared to controls.ConclusionsThe addition of SDP to culture media significantly enhanced hCLE cell sheet migration, proliferation, and attachment when compared to untreated controls, and indicates SDP's potential utility as an ophthalmic therapeutic agent.

Change of Optical Intensity during Healing Process of Corneal Wound on Anterior Segment Optical Coherence Tomography.

The purpose of this study is to investigate the process of corneal wound healing after penetrating injury with the change in optical intensity on anterior segment optical coherence tomography (AS-OCT) and to investigate factors associated with severity of corneal scar. Forty-seven eyes from 47 patients with repaired corneal laceration were included. AS-OCT was performed on 1day, 1week, 1, 3 and 6 months after primary repair. Internal aberrations of wound edges were observed on AS-OCT images. Parameters including height of steps, width of gaps, maximal corneal thickness, area and optical intensity of corneal wound/scar were measured. The relationship between the parameters at day 1 and the optical intensity at 6 months were analyzed. The results showed that optical intensity of corneal wound/scar increased from 124.1 ± 18.8 on day 1 postoperatively to 129.3 ± 18.7, 134.2 ± 23.4, 139.7 ± 26.5, 148.2 ± 26.4 at 1 week, 1 month, 3 months and 6 months postoperatively. Height of steps at 1 day after surgery was the only factor identified as correlated with optical intensity of corneal scar at 6 months (beta = 0.34, p = 0.024). The increase of optical intensity represents the process of fibrosis of corneal wound healing. Higher step after suturing is associated with more severity of corneal scar at last.

Effect of fibrin glue on corneal lamellar healing and how it correlates to biomechanical properties: biomechanical wavefront analysis and confocal study.

BackgroundTo evaluate, using a rabbit model, the influence of the wound healing process at the flap edge on corneal biomechanics after sutured, glued, and non-augmented microkeratome flaps.MethodsUnilateral 160 μm thick laser in situ keratomileusis (LASIK) flaps using a mechanical microkeratome were performed on the corneas of the left eyes of 36 rabbits. Animals were then divided into 3 groups of 12 rabbits each: A: the flaps were glued with human fibrin tissue adhesive (Tisseel); B: the flaps were sutured; and C: the flaps were allowed to heal without the use of sutures or glue (non-augmented). The contralateral eyes served as controls. Reichert ocular response analyzer (ORA) was used to measure corneal hysteresis (CH), corneal resistance factor (CRF), Goldmann-correlated intraocular pressure (IOPg) and cornea-compensated IOP (IOPcc) at 6 weeks and 3 months postoperatively. In vivo confocal microscopy (IVCM) was also used to study the corneal wound healing process in all groups.ResultsBoth mean CH and mean CRF were significantly higher in sutured and glued groups compared with the non-augmented group at 6 weeks and 3 months postoperatively (P < 0.0001). No statistically significant difference in corneal biomechanics was found between controls and groups A and B at any time points. Activated keratocytes were detected at the wound edge and peripheral flap interface in sutured and glued groups.ConclusionThe healing process at the wound edge is critical for optimal corneal integrity. Fibrin glue may serve as a safe and effective substitute to sutures in enhancing the corneal flap edge healing response and in increasing its mechanical strength.

Somatostatin supports corneal wound healing in vivo

PurposeTo evaluate the influence of somatostatin (SST) and its analog octreotid (Oct) on corneal wound healing processes. MethodsThe wound healing rate in C57BL/6 mice eyes under SST and Oct treatment was analyzed using an alkali-induced corneal wounding model. Effects of SST and Oct on cell proliferation, migration and quantified protein expression of vascular endothelial growth factor (VEGF) on human corneal epithelial cells (HCE, cell line) were evaluated by means of electric cell-substrate impedance sensing, scratch migration assays and ELISA. ERK1/2 and p38 phosphorylation was investigated by semi-quantitative western blot analysis. ResultsTen nanograms per microliters of SST significantly accelerated the wound closure rate of corneal defects in vivo. SST and Oct had no influence on HCE cell proliferation and migration and did not activate ERK1/2 or p38 signaling in HCE cells. However, there was increased VEGF protein expression in cytosolic proteins and medium supernatants of HCE upon Oct stimulation for 24h. One and 10ng/ml Oct led to a 2.5-fold and 100ng/ml Oct to a 4-fold upregulation of VEGF protein expression. ConclusionThe data implicate that SST promotes corneal wound healing in a mouse model. However, using a HCE cell line in vitro, the wound healing mechanism does not seem to be supported by proliferation and migration processes or by activation of ERK1/2 and p38 signaling pathways. Other possible mechanisms could be the activation of other pathways and the induction of growth factors such as VEGF that modulate the observed corneal wound healing process.

Substance P Enhances Keratocyte Migration and Neutrophil Recruitment through Interleukin-8

Keratocytes, the resident cells of the corneal stroma, are responsible for maintaining turnover of this tissue by synthesizing extracellular matrix components. When the cornea is injured, the keratocytes migrate to the wounded site and participate in the stromal wound healing. The neuropeptide substance P (SP), which is also known to be produced by non-neuronal cells, has previously been implicated in epithelial wound healing after corneal injury. Corneal scarring, which occurs in the stroma when the process of wound healing has malfunctioned, is one of the major causes of preventable blindness. This study aimed to elucidate the potential role of SP in keratocyte migration and therefore in stromal wound healing. We report that the expression and secretion of SP in human keratocytes are increased in response to injury in vitro. Moreover, SP enhances the migration of keratocytes by inducing the actin cytoskeleton reorganization and focal adhesion formation through the activation of the phosphatidylinositide 3-kinase and Ras-related C3 botulinum toxin substrate 1/Ras homolog gene family, member A pathway. Furthermore, SP stimulation leads to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8 (IL-8), which also contributes significantly to SP-enhanced keratocyte migration and is able to attract neutrophils. In addition, the preferred SP receptor, the neurokinin-1 receptor, is necessary to induce keratocyte migration and IL-8 secretion. In conclusion, we describe new mechanisms by which SP enhances migration of keratocytes and recruits neutrophils, two necessary steps in the corneal wound-healing process, which are also likely to occur in other tissue injuries.