Corneal Stromal Wound Healing Research Articles

Patterned collagen films loaded with miR-133b@MBG-NH2 for potential applications in corneal stromal injury repair

Corneal stromal injury is a common surgical disease. With the development of tissue engineering materials, many artificial corneal scaffolds have been developed to replace allograft corneal transplantation and solve the problem of corneal donor shortage. However, few researchers have paid attention to corneal stromal wound healing. Herein, a nanocomposite of amino modified mesoporous bioactive glass (MBG-NH2) and microRNA-133b (miR-133b) was introduced into the patterned collagen films to achieve corneal stromal injury repair. MBG-NH2 nanoparticles as a nano delivery carrier could efficiently load miR-133b and achieve the slow release of miR-133b. The physicochemical properties of collagen films were characterized and found the microgrooved collagen films loaded with miR-133b@MBG-NH2 nanoparticles possessed similar swelling properties, optical clarity, and biodegradability to the natural cornea. In vitro cell experiments were also conducted and proved that the patterned collagen films with miR-133b@MBG-NH2 possessed good biocompatibility, and miR-133b@MBG-NH2 nanoparticles could be significantly uptake by rabbit corneal stromal cells (RCSCs) and have a significant impact on the orientation, proliferation, migration, and gene expression of RCSCs. More importantly, the patterned collagen films with miR-133b@MBG-NH2 could effectively promote the migration of RCSCs and accelerate wound healing process, and down-regulate the expression levels of α-SMA, COL-I, and CTGF genes associated with myofibroblast differentiation of corneal stromal cells, which has a potential application prospect in the repair of corneal stromal injury.

Metal Oxide Engineered Nanomaterials Modulate Rabbit Corneal Fibroblast to Myofibroblast Transformation.

PurposeCorneal keratocyte-fibroblast-myofibroblast (KFM) transformation plays a critical role in corneal stromal wound healing. However, the impact of engineered nanomaterials (ENMs), found in an increasing number of commercial products, on this process is poorly studied. This study investigates the effects of metal oxide ENMs on KFM transformation in vitro and in vivo.MethodsCell viability of rabbit corneal fibroblasts (RCFs) was tested following treatment with 11 metal oxide ENMs at concentrations of 0.5 to 250 µg/ml for 24 hours. Messenger RNA (mRNA) and protein expression of αSMA, a marker of myofibroblast transformation, were measured using RCFs after exposure to 11 metal oxide ENMs at a concentration that did not affect cell viability, in media containing either 0 or 10 ng/ml of TGF-β1. Additionally, the effect of topical Fe2O3 nanoparticles (NPs) (50 ng/ml) on corneal stromal wound healing following phototherapeutic keratectomy (PTK) was determined.ResultsV2O5, Fe2O3, CuO, and ZnO ENMs were found to significantly reduce cell viability as compared to vehicle control and the other seven metal oxide ENMs tested. V2O5 nanoflakes significantly reduced mRNA and protein αSMA concentrations in the presence of TGF-β1. Fe2O3 NPs significantly increased αSMA mRNA expression in the presence of TGF-β1 but did not alter αSMA protein expression. Topically applied Fe2O3 NPs in an in vivo rabbit corneal stromal wound healing model did not delay healing.ConclusionsFe2O3 NPs promote corneal myofibroblast induction in vitro but do not impair corneal stromal wound healing in vivo.Translational RelevanceThese experimental results can apply to human nanomedical research.

L-carnitine suppresses transient receptor potential vanilloid type 1 activity and myofibroblast transdifferentiation in human corneal keratocytes

Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells.

Cell-Free Biological Approach for Corneal Stromal Wound Healing.

Corneal opacification is the fourth most common cause of blindness globally behind cataracts, glaucoma, and age-related macular degeneration. The standard treatment of serious corneal scarring is corneal transplantation. Though it is effective for restoring vision, the treatment outcome is not optimal, due to limitations such as long-term graft survival, lifelong use of immunosuppressants, and a loss of corneal strength. Regulation of corneal stromal wound healing, along with inhibition or downregulation of corneal scarring is a promising approach to prevent corneal opacification. Pharmacological approaches have been suggested, however these are fraught with side effects. Tissue healing is an intricate process that involves cell death, proliferation, differentiation, and remodeling of the extracellular matrix. Current research on stromal wound healing is focused on corneal characteristics such as the immune response, angiogenesis, and cell signaling. Indeed, promising new technologies with the potential to modulate wound healing are under development. In this review, we provide an overview of cell-free strategies and some approaches under development that have the potential to control stromal fibrosis and scarring, especially in the context of early intervention.

Biological Variance in Corneal Stromal Wound Healing: A Hurdle to Translation and Tool for Discovery

Biological Variance in Corneal Stromal Wound Healing: A Hurdle to Translation and Tool for Discovery

Wound Healing, Inflammation, and Corneal Ultrastructure After SMILE and Femtosecond Laser–Assisted LASIK: A Human Ex Vivo Study

To assess the wound healing, inflammation, and tissue ultrastructure in the human corneal stroma after small incision lenticule extraction (SMILE) and femtosecond laser-assisted LASIK (FS-LASIK). Sixteen corneoscleral discs of 16 human donors unsuitable for corneal transplantation were obtained from an eye bank. Eight eyes underwent SMILE with -5.00 diopters (D) of myopic correction; in 3 of them the lenticule was not extracted. Further 5 donor corneas were subjected to FS-LASIK with -5.00 D ablation, and 3 eyes served as the control group without surgical intervention. Postoperatively, specimens were incubated in organ culture medium for 72 hours before being subjected to immunofluorescence staining for CD11b, Ki67, fibronectin, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay, and high-magnification scanning electron microscopy. Keratocyte apoptosis, keratocyte proliferation, and infiltration of immune cells were generally mild and comparable between FS-LASIK and SMILE (irrespective of surgical lenticule extraction). By staining for fibronectin, we observed a trend toward milder fibrotic response in the corneal stroma after SMILE than after FS-LASIK. On the contrary, scanning electron microscopy analysis revealed a smoother, more regular ultrastructural appearance of the residual corneal bed after FS-LASIK. Corneal stromal wound healing after SMILE and FS-LASIK was virtually identical with respect to keratocyte proliferation and apoptosis in the human donor eye model. Although reactive fibrosis adjacent to the laser application site appeared less marked after SMILE, the stromal bed after LASIK exhibited a smoother surface texture. [J Refract Surg. 2018;34(6):393-399.].

Regulatory Mechanism of Collagen Degradation by Keratocytes and Corneal Inflammation: The Role of Urokinase-Type Plasminogen Activator.

Keratocytes, corneal resident cells in the corneal stroma, exist between collagen lamellae and maintain the corneal stromal structure. When the corneal stroma is damaged, keratocytes are transformed to myofibroblasts to aid corneal wound healing by phagocytizing debris. Keratocytes and extracellular collagen influence each other because keratocytes cultured in a 3D collagen gel undergo morphological changes and keratocytes produce metalloproteases that degrade extracellular collagen. IL-1 and plasminogen are critical mediators for collagen degradation. The plasminogen system contributes to tissue repair by activating matrix metalloproteinases (MMPs), releasing growth factors from the extracellular matrix and extracellular matrix degradation. Urokinase-type plasminogen activator (uPA) is thought to be involved in corneal disorders and regulates corneal wound healing. uPA is a serine protease synthesized by various cells such as corneal epithelial cells, corneal fibroblasts, vascular endothelial cells, smooth muscle cells, monocytes, macrophages, and malignant tumor cells of different origins. Here, we review the role of uPA in corneal stromal wound healing. uPA is expressed in leukocytes and corneal fibroblasts in the corneas of patients with corneal ulcerations suggesting it is a key regulator of corneal stromal wound healing. uPA is directly involved in plasmin-mediated collagen degradation induced by IL-1. Moreover, uPA is critically involved in promoting leukocyte infiltration in corneal inflammation by activating MMP-9. This activation is presumably directly and indirectly mediated by the plasminogen/plasmin cascade. Moreover, uPA mediates the release of inflammatory cytokines from corneal fibroblasts to promote leukocyte infiltration.

Modulation of Smad signaling by non-TGFβ components in myofibroblast generation during wound healing in corneal stroma

Corneal scarring/fibrosis disturbs normal transparency and curvature of the tissue and thus impairs vision. The lesion is characterized by appearance of myofibroblasts, the key player of the fibrogenic reaction, and excess accumulation of extracellular matrix. Inflammatory/fibrogenic growth factors or cytokines expressed in inflammatory cells that infiltrate into injured tissues play a pivotal role in fibrotic tissue formation. In this article the pathogenesis of fibrosis/scarring in the corneal stroma is reviewed focusing on the roles of myofibroblast, the key player in corneal stromal wound healing and fibrosis, and cytoplasmic signals activated by the fibrogenic cytokine, transforming growth factor β (TGFβ). Although it is established that TGFβ/Smad signal is essential to the process of keratocyte-myofibroblast transformation in a healing corneal stroma post-injury. This article emphasizes the involvement of non-TGFβ molecular mechanisms in modulating Smad signal. We focus on the roles of matricellular proteins, i.e., osteopontin and tenascin C, and as cellular components, the roles of transient receptor potential (TRP) cation channel receptors are discussed. Our intent is to draw attention to the possibility of signal transduction cascade modulation (e.g., Smad signal and mitogen-activated protein kinases, by gene transfer and other related technology) as being beneficial in a clinical setting to reduce or even prevent corneal stromal tissue fibrosis/scarring and inflammation.

Hevin Plays a Pivotal Role in Corneal Wound Healing

BackgroundHevin is a matricellular protein involved in tissue repair and remodeling via interaction with the surrounding extracellular matrix (ECM) proteins. In this study, we examined the functional role of hevin using a corneal stromal wound healing model achieved by an excimer laser-induced irregular phototherapeutic keratectomy (IrrPTK) in hevin-null (hevin-/-) mice. We also investigated the effects of exogenous supplementation of recombinant human hevin (rhHevin) to rescue the stromal cellular components damaged by the excimer laser.Methodology/Principal FindingsWild type (WT) and hevin -/- mice were divided into three groups at 4 time points- 1, 2, 3 and 4 weeks. Group I served as naïve without any treatment. Group II received epithelial debridement and underwent IrrPTK using excimer laser. Group III received topical application of rhHevin after IrrPTK surgery for 3 days. Eyes were analyzed for corneal haze and matrix remodeling components using slit lamp biomicroscopy, in vivo confocal microscopy, light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) and western blotting (WB). IHC showed upregulation of hevin in IrrPTK-injured WT mice. Hevin -/- mice developed corneal haze as early as 1-2 weeks post IrrPTK-treatment compared to the WT group, which peaked at 3-4 weeks. They also exhibited accumulation of inflammatory cells, fibrotic components of ECM proteins and vascularized corneas as seen by IHC and WB. LM and TEM showed activated keratocytes (myofibroblasts), inflammatory debris and vascular tissues in the stroma. Exogenous application of rhHevin for 3 days reinstated inflammatory index of the corneal stroma similar to WT mice. Conclusions/SignificanceHevin is transiently expressed in the IrrPTK-injured corneas and loss of hevin predisposes them to aberrant wound healing. Hevin -/- mice develop early corneal haze characterized by severe chronic inflammation and stromal fibrosis that can be rescued with exogenous administration of rhHevin. Thus, hevin plays a pivotal role in the corneal wound healing.

FGF-2– and TGF-β1–Induced Downregulation of Lumican and Keratocan in Activated Corneal Keratocytes by JNK Signaling Pathway

Downregulation of lumican and keratocan expression is an undesirable phenotypic change that occurs during corneal wound healing. The present study was intended to determine whether the activation of Jun N-terminal kinase (JNK)-signaling pathway is involved in their downregulation in TGF-β1- and FGF-2-activated keratocytes. Keratocytes, isolated from rabbit corneal stroma, and cultured in a serum-free medium, pretreated or not treated with JNK inhibitor (SP600125), were activated with FGF-2/heparin sulfate (HS) or TGF-β1 in the presence or absence of SP600125. In another set of experiments, keratocytes were transfected with JNK1/2 Dicer-substrate RNA (DsiRNA) and then activated with TGF-β1 or FGF-2/HS. Specific phenotypic changes were analyzed immunocytochemically and correlated with Western blot analyses. The relative levels of specific mRNAs were estimated by quantitative RT-PCR using specific reagents. The FGF-2/HS- or TGF-β-induced activation of corneal stromal keratocytes to fibroblast- or myofibroblast-phenotype, respectively, resulted in marked decreases in cell surface-associated and secreted keratan sulfate proteoglycans (KSPGs). Both keratocan and lumican proteins and their mRNAs were downregulated in the activated keratocytes. However, JNK inhibition during the activation of keratocytes, pretreated with the JNK inhibitor, suppressed the reduction in the cell-surface associated and secreted KSPGs (lumican and keratocan), and their mRNA transcripts. Downregulation of total KSPGs and their mRNAs was also inhibited by decreasing JNK1 and JNK2 levels via JNK1/2 DsiRNA transfection of keratocytes before their activation. Extrapolating from the present study, FGF-2- and TGF-β1-activation of JNK signaling pathway may be partly responsible for the downregulation of keratocan and lumican expression in activated corneal keratocytes observed during corneal stromal wound healing.

Synergistic Effect of Platelet-Activating Factor and Tumor Necrosis Factor-α on Corneal Myofibroblast Apoptosis

Elimination of myofibroblasts after repair of corneal injury is essential for the maintenance of corneal transparency. In the current study, the role of platelet-activating factor (PAF) in combination with tumor necrosis factor (TNF)-alpha in corneal myofibroblast apoptosis was explored. Porcine corneal myofibroblasts (PCMs) were obtained from subcultured fibroblasts plated at a low density (5 cells/mm2). Mouse anti-alpha-smooth muscle actin antibody was used to identify the cell phenotype. Immunofluorescence was performed to localize PAF and TNF-alpha receptors in those cells. The reactivity of the antibodies was characterized by Western blot analysis. To induce myofibroblast apoptosis, PCMs were treated for 24 to 72 hours with methylcarbamyl-PAF (cPAF, 300 nM), a nonhydrolyzable PAF analogue, TNF-alpha (20 ng/mL), and TNF-alpha+cPAF, with or without LAU-0901 (150 nM), a novel PAF antagonist. Apoptosis was assayed by Hoechst 33258 and TUNEL staining and DNA laddering. 6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Images were recorded by fluorescence microscope. Immunofluorescence with a PAF-receptor (N terminus) polyclonal antibody showed that the receptor was expressed in both plasma and nuclear membranes of myofibroblasts. TNF-alpha receptor II (TNF-RII) was localized in the cytoplasm, whereas TNF-receptor I (TNF-RI) was found in both cytoplasm and plasma membrane. Treatment with TNF-alpha for 24, 48, and 72 hours induced apoptosis in 18%, 24%, and 32%, respectively, of the myofibroblasts. Western blot analysis showed expression of single bands corresponding to the molecular weights of the receptors. Treatment with cPAF induced apoptosis in 10%, 18%, and 26% of the cells, respectively. However, treatment with both cytokines induced apoptosis in 42%, 78%, and 86%, respectively, of the cells, demonstrating a synergistic action between PAF and TNF-alpha. Blocking the PAF receptor with LAU-0901 inhibited the synergistic effect induced by PAF. Corneal myofibroblasts express a PAF receptor in the nuclear membrane, and they also express TNF-RI and RII. The synergistic effect on myofibroblast apoptosis by PAF and TNF-alpha suggests that during corneal stromal wound healing, PAF acting in conjunction with other cytokines could play an important role in eliminating these cells.

RANK, RANKL, OPG, and M-CSF expression in stromal cells during corneal wound healing.

To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ. Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry. A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury. Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.

Mitomycin C alters corneal stromal wound healing and corneal haze in rabbits after argon-fluoride excimer laser photorefractive keratectomy.

To investigate the effects of mitomycin C (MMC) on rabbit cornea wound healing after photorefractive keratectomy (PRK). Rabbit corneas were stained with dichlorotriazinyl aminofluorescein immediately after PRK. MMC was applied to the right eye and phosphate-buffered salt solution (PBS) to the left. Corneal epithelial wound healing rate and corneal haze were examined. Ultrasound pachymetry was performed. Stromal collagen regeneration was evaluated by fluorescent microscopy. We used terminal deoxyribonucleotidyl transferase-mediated D-uridine 5'-triphosphated-digoxigenin nick-end labeling (TUNEL) assay and transmission electron microscopy (TEM) to evaluate keratocyte apoptosis. In eyes treated with MMC, there was no delay to the healing rate of corneal epithelial wound, and less haze 4 weeks after PRK. Ultrasound pachymetry showed thinner corneal thickness in MMC-treated eyes at week 4. Corneal stromal thickness regression was less in MMC-treated eyes observed by fluorescent microscope at week 4. Keratocyte apoptosis was noted in both MMC- and PBS-treated eyes by TUNEL assay and TEM observation. This study discovered the phenomenon that MMC prolongs keratocyte apoptosis. Applying MMC after PRK is an effective method to decrease haze formation and corneal stromal thickness regression in rabbit corneas. The effect may be related to MMC prolonging keratocyte apoptosis.

Apoptosis in keratocytes caused by mitomycin C.

The purpose of this study was to quantify the effect of mitomycin C on rabbit keratocytes, with a view to determining its potential in modulating corneal stromal wound healing. In addition, the pathway by which this regulation occurs was investigated. Keratocytes were isolated from New Zealand White rabbits and cultured. Hoechst staining and flow cytometric analyses with annexin V were used to identify the nature of the keratocyte response to mitomycin C. The response of cultured keratocytes to 0.005%, 0.01%, 0.02%, 0.04%, and 0.06% mitomycin C was evaluated with the lactate dehydrogenase (LDH) assay. In addition, after exposure of keratocytes to 0.01% mitomycin C, the LDH assay was performed at different times of 6, 12, and 24 hours. Keratocytes were preincubated with various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK), caspase-8 inhibitor (Z-IETD-FMK), and caspase-9 inhibitor (Z-LEHD-FMK) and treated with 0.01% mitomycin C. The LDH assay was performed after 12 hours. Cytochrome c immunostain was performed after exposure to 0.01% mitomycin C. Hoechst staining revealed shrinkage of the cytoplasm, formation of apoptotic bodies, and nuclear fragmentation. Apoptotic changes in cells were detected by flow cytometry. LDH activities increased significantly at concentrations of 0.005% mitomycin C or greater and were time dependent until 24 hours. Treatment with a CPP32-like protease inhibitor caused a decrease in LDH activity, although the results were not statistically significant. Specific inhibitors of caspase-8 and -9 significantly reduced the LDH activity induced by mitomycin C. Cytochrome c immunostaining of keratocytes pretreated with mitomycin C showed strongly positive findings. Mitomycin C induced apoptosis, not necrosis, in cultured corneal keratocytes through the caspase pathway-specifically, caspase-8 and -9-related to the mitochondrial pathway.

Corneal wound healing after excimer laser keratectomy

Excimer laser keratectomy is widely used to correct refractive errors. Several complications of excimer laser keratectomy are reported including corneal infection, regression, corneal haze formation, glare and halo. Most of the complications are closely related to the corneal stromal wound healing process. In order to perform the excimer laser keratectomy with minimum complications, we should understand the mechanism of the corneal stroma wound healing process. In addition, such knowledge will help us to regulate the corneal stromal wound healing process in the future. In the present article, we discuss the molecular mechanism of the corneal stromal wound healing process after excimer laser keratectomy and its regulation by anti-inflammatory agents.

Corneal wound healing after excimer laser keratectomy

Excimer laser keratectomy is widely used to correct refractive errors. Several complications of excimer laser keratectomy are reported including corneal infection, regression, corneal haze formation, glare and halo. Most of the complications are closely related to the corneal stromal wound healing process. In order to perform the excimer laser keratectomy with minimum complications, we should understand the mechanism of the corneal stroma wound healing process. In addition, such knowledge will help us to regulate the corneal stromal wound healing process in the future. In the present article, we discuss the molecular mechanism of the corneal stromal wound healing process after excimer laser keratectomy and its regulation by anti-inflammatory agents.

Toxicity of topical anesthetic agents to human keratocytes in vivo

Purpose To test the potential toxicity on human keratocytes of topical anesthetic agents used after photorefractive keratectomy (PRK) to reduce or eliminate pain. Setting Department of Ophthalmology, Doheny Eye Institute, University of Southern California, Los Angeles, California, USA. Methods Cultured human keratocytes were incubated with commercially available tetracaine and proparacaine at reduced concentrations of 0.001%, 0.01%, 0.1%, and 0.25%. Evaluations were performed by phase-contrast microscopy and tetrazolium salt colorimetric assay every 2 hours for 12 hours after adding 1 of the anesthetic agents to the media. Results After time of incubation and concentration were adjusted, both drugs reduced overall cell viability; however, tetracaine produced a larger decrease in cell viability than proparacaine ( P = .008). For both drugs, significant differences were found among concentrations for and across time ( P < .001 and P = .004, respectively). Conclusion Both tetracaine and proparacaine had toxic effects on stromal keratocytes related not only to drug concentrations but also to time exposure. These findings underscore the widespread concern that anesthetic drugs may affect corneal stromal wound healing after PRK.

Corneal stromal wound healing in refractive surgery: the role of myofibroblasts.

While laser and incisional refractive surgery offer the promise to correct visual refractive errors permanently and predictably, variability and complications continue to hinder wide-spread acceptance. To explain variations, recent studies have focused on the role of corneal wound healing in modulating refractive outcomes. As our understanding of the corneal response to refractive surgery broadens, it has become apparent that the response of one cell, the corneal stromal keratocyte, plays a pivotal role in defining the results of refractive surgery. Studies reviewed herein demonstrate that injury-induced activation and transformation of keratocytes to myofibroblasts control the deposition and organization of extracellular matrix in corneal wounds. Myofibroblasts establish an interconnected meshwork of cells and extracellular matrix that deposits new matrix and contracts wounds using a novel and unexpected "shoe-string-like" mechanism. Transformation of keratocytes to myofibroblasts is induced in culture by transforming growth factor beta (TGFbeta) and blocked in vivo by antibodies to TGFbeta. Overall, myofibroblast appearance in corneal wounds is associated with wound contraction and regression following incisional keratotomy and the development of "haze" or increased scattered light following laser photorefractive keratectomy (PRK). By contrast, absence of myofibroblasts is associated with continued widening of wound gape and progressive corneal flattening after incisional procedures. Based on these studies, we have arrived at the inescapable conclusion that a better understanding of the cellular and molecular biology of this one cell is required if refractive surgery is ever to achieve predictable and safe refractive results.

Three-dimensional organization of collagen fibrils during corneal stromal wound healing after excimer laser keratectomy

Purpose: To investigate the structural changes in corneal stromal collagen fibrils after excimer laser keratectomy in relation to the degree of corneal haze.Setting: University of Tokyo Hospital, Tokyo, Japan.Methods: Corneal haze was quantitatively measured by analyzing the light scattering in Scheimpflug images of the corneas of white rabbits after excimer laser keratectomy. Collagen fibril structure was examined using scanning electron microscopy after chemical digestion with sodium hydroxide solution; the same specimens were examined by transmission electron microcopy after re-embedding.Results: Corneal haze reached a peak 4 weeks after excimer laser keratectomy and then gradually decreased, The collagen fibrils of the normal cornea were regularly arranged parallel to the surface of the cornea, with small interfibrillar distances. After excimer laser keratectomy, the arrangement was highly disordered, with increased interfibrillar distances. These structural changes were most prominent 4 weeks after excimer laser keratectomy.Conclusion: The structural changes in the collagen fibrils of the corneal stroma, especially the increase in interfibrillar distances and the disordered arrangement, were associated with corneal haze after excimer laser keratectomy.

In vitro effects of mitomycin-C on human keratocytes.

The purpose of the present study is to quantify the in vitro antiproliferative and cytotoxic effects of mitomycin-C on human keratocytes for their potential to modulate corneal stromal wound healing. Cultured human keratocytes were exposed to various concentrations of mitomycin-C for periods of 5 minutes and 1 hour. Keratocyte proliferation and viability were assessed by phase-contrast microscopy, 3H-thymidine uptake, and electronic cell counting. Cytotoxic changes and inhibition of keratocyte proliferation exhibited after exposure to mitomycin-C were both dose- and time-dependent. The lowest concentrations to significantly (> 50%) inhibit keratocyte proliferation after 5-minute exposures were 0.05 mg/ml (P < .005) and after 1-hour exposures were 0.005 mg/ml (P < .001). At 5 minutes, ID50 was 0.038 mg/ml and LD50 was much higher than the greatest concentration tested (0.5 mg/ml). Mitomycin-C's median inhibitory dose (ID50) and median lethal dose (LD50) after 1 hour of exposure differed by a magnitude of 50 (0.0048 vs. 0.28 mg/ml). Mitomycin-C has antiproliferative effects at concentrations below those cytotoxic to human keratocytes. If used after photorefractive keratectomy, the drug should be administered at antiproliferative rather than cytotoxic concentrations.