Wound healing in the cornea: a review of refractive surgery complications and new prospects for therapy

PURPOSE: The corneal wound healing response is of particular relevance for refractive surgical procedures since it is a major determinant of efficacy and safety. The purpose of this review is to provide an overview of the healing response in refractive surgery procedures. METHODS: Literature review. RESULTS: LASIK and PRK are the most common refractive procedures; however, alternative techniques, including LASEK, PRK with mitomycin C, and Epi-LASIK, have been developed in an attempt to overcome common complications. Clinical outcomes and a number of common complications are directly related to the healing process and the unpredictable nature of the associated corneal cellular response. These complications include overcorrection, undercorrection, regression, corneal stroma opacification, and many other side effects that have their roots in the biologic response to surgery. The corneal epithelium, stroma, nerves, inflammatory cells, and lacrimal glands are the main tissues and organs involved in the wound healing response to corneal surgical procedures. Complex cellular interactions mediated by cytokines and growth factors occur among the cells of the cornea, resulting in a highly variable biologic response. Among the best characterized processes are keratocyte apoptosis, keratocyte necrosis, keratocyte proliferation, migration of inflammatory cells, and myofibroblast generation. These cellular interactions are involved in extracellular matrix reorganization, stromal remodeling, wound contraction, and several other responses to surgical injury. CONCLUSIONS: A better understanding of the complete cascade of events involved in the corneal wound healing process and anomalies that lead to complications is critical to improve the efficacy and safety of refractive surgical procedures. Recent advances in understanding the biologic and molecular processes that contribute to the healing response bring hope that safe and effective pharmacologic modulators of the corneal wound healing response may soon be developed.     

角膜伤口愈合机制对准分子激光角膜屈光手术的启示

角膜伤口愈合反应是由细胞因子、生长因子、趋化因子介导的复杂级联反应。角膜上皮细胞和基底膜在这一过程中起了重要的作用,而这二者在准分子激光角膜屈光手术中常常被损伤。因此,深刻理解角膜伤口愈合过程以及并发症产生的原因,对提高准分子激光角膜屈光手术的有效性和安全性具有重要意义。就角膜伤口愈合过程及其机制进行综述。     

角膜基质修复的机制和调控因素研究进展

角膜基质是维持角膜透明度的重要结构。外伤、感染、手术等可造成角膜基质损伤,引起修复的过程包括基质细胞表型改变、细胞外基质重塑、免疫细胞迁移。当基质严重受损,肌成纤维细胞增多和细胞外基质沉积发生基质纤维化反应,形成角膜瘢痕,是全球致盲的主要原因。目前治疗方式主要是角膜移植手术,因角膜供体资源短缺、手术技巧要求和术后移植排斥风险等治疗效果不佳。近年来,各种分子、细胞和组织对角膜基质损伤修复的调控机制取得一定研究进展。本文就角膜基质损伤修复的机制和角膜损伤原因、角膜结构、分子因素对角膜基质损伤修复的调控进行综述,为探索促进角膜基质修复和再生的途径提供新思路,希望帮助临床预防角膜瘢痕的发生。     

Genomics of corneal wound healing: a review of the literature

Corneal wound healing is a complex process: its mechanisms and the underlying genetic control are not fully understood. It involves the integrated actions of multiple growth factors, cytokines and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells and lacrimal gland cells. Following an epithelial insult, multiple cytokines are released triggering a cascade of events that leads to repair the epithelial defect and remodelling of the stroma to minimize the loss of transparency and function. In this review, we examine the literature surrounding the genomics of corneal wound healing with respect to the following topics: epithelial and stromal wound healing (including inhibition); corneal neovascularisation; the role of corneal nerves in wound healing; the endothelium; the role of aquaporins and aptamers. We also examine the effect of ectasia on corneal wound healing with regard to keratoconus and following corneal surgery. A better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design treatments that selectively modulate key phases of the healing process resulting in scars that more closely resemble normal corneal architecture.     

Corneal stromal wound healing: Major regulators and therapeutic targets

Corneal stromal wound healing is a complex event that occurs to restore the transparency of an injured cornea. It involves immediate apoptosis of keratocytes followed by their activation, proliferation, migration, and trans-differentiation to myofibroblasts. Myofibroblasts contract to close the wound and secrete extracellular matrix and proteinases to remodel it. Released proteinases may degenerate the basement membrane allowing an influx of cytokines from overlying epithelium. Immune cells infiltrate the wound to clear cellular debris and prevent infections. Gradually basement membrane regenerates, myofibroblasts and immune cells disappear, abnormal matrix is resorbed, and transparency of the cornea is restored. Often this cascade deregulates and corneal opacity results. Factors that prevent corneal opacity after an injury have always intrigued the researchers. They hold clinical relevance as they can guide the outcomes of corneal surgeries. Studies in the past have shed light on the role of various factors in stromal healing. TGFβ (transforming growth factor-beta) signaling is the central player guiding stromal responses. Other major regulators include myofibroblasts, basement membrane, collagen fibrils, small leucine-rich proteoglycans, biophysical cues, proteins derived from extracellular matrix, and membrane channels. The knowledge about their roles helped to develop novel therapies to prevent corneal opacity. This article reviews the role of major regulators that determine the outcome of stromal healing. It also discusses emerging therapies that modulate the role of these regulators to prevent stromal opacity.     

The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells

The corneal wound healing cascade is complex and involves stromal-epithelial and stromal-epithelial-immune interactions mediated by cytokines. Interleukin-1 appears to be a master modulator of many of the events involved in this cascade. Keratocyte apoptosis is the earliest stromal event noted following epithelial injury and remains a likely target for modulation of the overall wound healing response. Other processes such as epithelial mitosis and migration, stromal cell necrosis, keratocyte proliferation, myofibroblast generation, collagen deposition, and inflammatory cell infiltration contribute to the wound healing cascade and are also likely modulated by cytokines derived from corneal cells, the lacrimal gland, and possibly immune cells. Many questions remain regarding the origin and fate of different cell types that contribute to stromal wound healing. Over a period of months to years the cornea returns to a state similar to that found in the unwounded normal cornea.     

Corneal wound healing in tenascin knockout mouse

PURPOSE: Tenascin (TN) is a large hexameric extracellular matrix glycoprotein that is expressed in developing organs and tumors. It has also been reported that TN is expressed in the embryonic cornea and during corneal wound healing. However, the role of TN in the cornea is not fully known. In this study, the role of TN in corneal wound healing was examined using the TN knockout (KO) mouse. METHODS: Two different injuries (a linear perforation wound and two 10-0 nylon suture wounds) were made separately on the corneas of both TNKO and congenic wild-type mice. The corneal wound healing was compared histologically, and the expression of TN and fibronectin (FN) on the injured cornea was examined immunohistochemically and by immunoblot analysis. RESULTS: Based on histologic analysis, there was no significant difference in the wound healing process between wild-type and TNKO mice in the linear incision experiment. However, the corneal stromata of TNKO mice were compressed prominently and devoid of migrating keratocytes in suture injury, which induced a more significant amount of TN than perforation wounds. Although FN expression on the sutured corneas of TNKO mice was upregulated during suture injury, the amount of FN protein was smaller than that of wild-type mice at the same time points after injury. CONCLUSIONS: In suture wounds, TN appears to enhance the amount of FN expression, and a lack of TN may impair stromal cell migration. TN plays a significant role in corneal wound healing, especially for wounds with mechanical stress.     

羊膜移植对家兔PRK术后伤口愈合作用的研究

目的：观察羊膜对家兔PRK术后角膜伤口愈合反应的影响。方法：对30只家兔常规施行双眼PRK手术（－8．0D切削程序），即行羊膜移植，观察术后早期角膜切削区炎性细胞的浸润和角膜上皮愈合情况，晚期角膜成纤维细胞增生和上皮增生情况，观察haze并评分；早期测量角膜SOD活力和MDA含量，对结果进行统计学分析。结果：早期移植相炎性细胞的数目比对照组少，上皮愈合时间较短，晚期上皮增生较轻，晚期移植组成纤维细胞数目较少；移植组haze评分较低；早期移植组自由基反应产物MDA的含量较低，而SOD含量较高，差异均具有显著性。结论：羊膜能促进术后上皮愈合，减轻炎性反应和自由基反应，减少成纤维细胞增生，减轻haze的形成。     

准分子激光角膜切削术与准分子激光原位角膜磨镶术角膜创口愈合的对比实验研究

比较准分子激光角膜切削术(photorefractive keratectomy,PRK)与准分子激光原位角膜磨镶术(1aser in situ keratomileusis,LASIK)后激光对角膜组织的切削效应及角膜的愈合情况,从组织学角度探讨角膜雾状混浊(Haze)及屈光度数回退的成因。方法24只新西兰白兔按预矫屈光度数随机分为-4.00 D组和-8.00 D组,每只兔右眼行PRK,左眼行LASIK。术后10d,1、3及6个月观察Haze情况并验光,每组随机处死3只兔取角膜行光镜、电镜及免疫组化检查,检测胶原Ⅲ、胶原Ⅳ、纤维连结蛋白(fibronectine,FN)及转化生长因子-β(transforming growth factor-β1,TGF-β2)的含量。结果行PRK术的右眼术后出现不同程度的Haze及屈光度数回退,其程度与预矫正屈光度数成正比。行LASIK术的左眼术后除少数角膜瓣周围半环形混浊外,手术区域角膜透明,屈光度数回退较右眼轻。-4.00 D组右眼与左眼术后屈光度数均稳定,-8.00 D组右眼较左眼屈光度数回退明显。右眼术后角膜愈合反应重,恢复慢。6个月时角膜基质仍处于修复阶段。左眼术后除形成角膜上皮栓及对应处基质轻度增生外,手术区域角膜瓣与基质床间界面清晰,无明显增生,角膜基质愈合反应轻、恢复快。术后所有兔眼角膜均有TGF-β1表达及活化,持续时间与角膜愈合时间一致。Haze及屈光度数回退组织学改变为：角膜上皮细胞增生,基底膜不成熟,前基质角膜细胞活化、增殖,新生胶原Ⅲ合成、排列紊乱,细胞外基质FN在角膜上皮下沉积。结论LASIK矫正近视尤其是高度近视优于PRK;角膜伤口愈合特别是基质愈合的反应程度,是。Haze及屈光度数回退的关键;TGF-β1是角膜愈合过程中重要调节因子,可通过介导角膜上皮一基质相互作用,调节胶原Ⅲ及FN的含量,参与瘢痕形成。     

Extracellular matrix and wound healing

Over the years, most researchers have approached corneal epithelial and stromal wound healing as separate events. However, it is becoming increasingly clear that even the simplest epithelial debridement wound results in keratocyte death and a subsequent stromal response to regenerate the affected area. Thus, the interaction between stromal and epithelial healing must be considered to fully understand corneal wound healing. Although wound healing has been an active area of research for many years, the advent of refractive surgery has stimulated research into the regulation of wound repair and provided important insights into the molecular components involved in repair. Epithelial and stromal wound healing are influenced by extracellular matrix components. The purpose of the current article is to review progress in the year 2000 toward understanding mechanisms involved in corneal wound healing and how extracellular matrix affects the healing processes.     

Thymosin-beta4 modulates corneal matrix metalloproteinase levels and polymorphonuclear cell infiltration after alkali injury

PURPOSE: Corneal alkali injury is highly caustic, and present clinical therapies are limited. The purpose of this study was to investigate the ability of thymosin-beta4 (Taubeta4) to promote healing in an alkali injury model and the mechanisms involved in that process. METHODS: Corneas of BALB/c mice were injured with NaOH, irrigated copiously with PBS, and treated topically with either Tbeta4 or PBS twice daily. At various time points after injury (PI), corneas from the Tbeta4- versus the PBS-treated group were examined for polymorphonuclear leukocyte (PMN) infiltration, chemokine, and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) expression. RESULTS: Tbeta4-treated corneas demonstrated improved corneal clarity at day 7 PI. Whereas Tbeta4 decreased corneal MMP-2 and -9 and MT6-MMP levels after alkali injury, no change in TIMP-1 and -2 expression was detected. Tbeta4 treatment also decreased corneal KC (CXCL1) and macrophage inflammatory protein (MIP)-2 chemokine expression and PMN infiltration. Immunohistochemistry studies demonstrated MMP-9 expression at the leading edge of the epithelial wound, in the the limbus (containing stem cells), and in stromal PMNs. CONCLUSIONS: Tbeta4 treatment decreases corneal inflammation and modulates the MMP/TIMP balance and thereby promotes corneal wound repair and clarity after alkali injury. These results suggest that Tbeta4 may be useful clinically to treat severe inflammation-mediated corneal injuries.     

Proteoglycan alterations in the rabbit corneal stroma after a lamellar incision

PURPOSE: To investigate proteoglycans under minimally damaged epithelium after a lamellar microkeratome incision. SETTING: Collaborating university departments. METHODS: Anterior lamellar caps were excised from rabbit corneas and then resutured in place. Healing tissue was examined by electron microscopy with proteoglycan staining. RESULTS: In the weeks after surgery, regions of disorganized stromal matrix were populated by sulfated proteoglycan filaments that were much larger (up to 300 nm long) than those in quiescent stroma. CONCLUSIONS: Large, sulfated proteoglycans existed in rabbit corneas healing from lamellar incisions. These molecules appear to be a normal feature of corneal wound healing; because of their water-binding capacity, they might aid tissue restructuring.     

Stromal-epithelial interactions in the cornea

Stromal-epithelial interactions are key determinants of corneal function. Bi-directional communications occur in a highly coordinated manner between these corneal tissues during normal development, homeostasis, and wound healing. The best characterized stromal to epithelial interactions in the cornea are mediated by the classical paracrine mediators hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF). HGF and KGF are produced by the keratocytes to regulate proliferation, motility, differentiation, and possibly other functions, of epithelial cells. Other cytokines produced by keratocytes may also contribute to these interactions. Epithelial to stromal interactions are mediated by cytokines, such as interleukin-1 (IL-1) and soluble Fas ligand, that are released by corneal epithelial cells in response to injury. Other, yet to be identified, cytokine systems may be released from the unwounded corneal epithelium to regulate keratocyte viability and function. IL-1 appears to be a master regulator of corneal wound healing that modulates functions such as matrix metalloproteinase production, HGF and KGF production, and apoptosis of keratocyte cells following injury. The Fas/Fas ligand system has been shown to contribute to the immune privileged status of the cornea. However, this cytokine-receptor system probably also modulates corneal cell apoptosis following infection by viruses such as herpes simplex and wounding. Pharmacologic control of stromal-epithelial interactions appears to offer the potential to regulate corneal wound healing and, possibly, treat corneal diseases in which these interactions have a central role.     

Comparison of stromal remodeling and keratocyte response after corneal incision and photorefractive keratectomy]

PURPOSE: We investigated the keratocyte response and stromal remodeling after corneal incision and photorefractive keratectomy, respectively to learn the difference between the two surgeries histophysiologically and immunohistochemically. METHODS: We performed corneal incision or photorefractive keratectomy in rabbits or rats, and then we chronologically observed the histological changes and the changes in localization of extracellular matrix proteins. RESULTS: In both types of surgery, the keratocyte population in the damaged stroma became sparse, and the cells were undergoing apoptosis immediately after the procedures. After that, activated keratocytes migrated into the acellular zone, and the cells formed multiple layers at the resurfaced subepithelial regions. Deposition of amorphous substances was observed between migrated keratocytes, and stromal remodeling began. Three months after the surgery, corneal structure had recovered to near normal condition in the corneal incisions. In photorefractive keratectomy, however, strong immunoreactivity of extracellular matrix proteins was observed in the subepithelial regions. CONCLUSIONS: These results suggested that stromal wound healing processes were similar in both corneal incision and photorefractive keratectomy. Corneal incision may induce transient keratocyte response during the stromal remodeling, but photorefractive keratectomy may induce sustained keratocyte response.     

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.     

Effect of topical anti-inflammatory drugs on corneal and limbal wound healing

PURPOSE: To examine the effect of topical treatment with either steroidal or nonsteroidal anti-inflammatory drugs (NSAIDs) on the wound healing of corneal and limbal incisions using histologic criteria. SETTING: Loyola University Medical Center, Maywood, Illinois, USA. METHODS: Eighteen eyes of 9 cats were used for the study. All right eyes received a temporal limbal incision and the left eyes, a clear corneal incision. All eyes were treated with ofloxacin 0.3% postoperatively for 3 days. The animals were equally divided among 3 groups. Group 1 received topical steroid drops (prednisolone acetate 1%) for 7 days maximum; Group 2 received topical NSAID drops (ketorolac tromethamine 0.5%) for 7 days maximum; Group 3 received no additional treatment. One animal from each group was killed and the eye enucleated 3, 7, or 28 days postoperatively. The eyes were processed for staining with hematoxylin-eosin and smooth muscle actin, and corneal sections were evaluated in a blind fashion by an ophthalmic pathologist. RESULTS: Three and 7 days after surgery (1) the limbal incision exhibited more prominent wound healing than the similarly treated clear corneal incision, and (2) the steroid-treated corneas had less wound healing than untreated or NSAID-treated corneas. At 28 days, the wounds in all eyes were almost completely healed. CONCLUSIONS: Limbal incisions heal faster than clear corneal incisions. Steroids, but not NSAIDs, inhibit wound healing. Cataract surgery using limbal incisions and postoperative topical NSAIDs may result in faster wound healing and provide a reduced risk of related postoperative complications.     

In vivo confocal microscopy for evaluation of wound healing following corneal refractive surgery

Understanding of corneal wound healing plays an important role, not only in management of corneal infections, but especially in refractive surgery. A better control of wound healing mechanisms might improve the results of such resculpturing techniques and help to avoid complications arising from these procedures. While studies have been focused in different aspects of corneal wound healing, our knowledge has increased greatly during the last years. Many problems associated with corneal healing also contribute to clinical pathology following corneal surgery. Understanding of such conditions has been augmented by the continuously developing corneal imaging techniques. We have used in vivo confocal microscopy (IVCM) for assessing corneas subjected to refractive surgery as well as corneas with common complications resulting from such procedures. IVCM has become a powerful tool for examining corneal cells, nerves, inflammations and infections. It allows information to be acquired repeatedly and at subbiomicroscopic levels that earlier had been obtainable only by invasive microscopic methods. Pre-examining corneas preoperatively by IVCM in order to reveal diseases or conditions in which elective refractive surgical procedures should not be undertaken or to select the ideal operation technique may help to avoid complications in the future. Measurement of the thickness of corneal sublayers or estimation of the thickness of a laser in situ keratomileusis flap or wound bed are other applications in which confocal microscopy may be valuable. In this article we attempt to describe the in vivo confocal findings of common refractive procedures and their complications, and discuss their biology in light of the existing knowledge on wound healing phenomena.     

Wakayama Symposium: New Therapies for Modulation of Epithelialization in Corneal Wound Healing

Many factors are involved in the corneal wound healing mechanism, including adhesion, migration, and proliferation of corneal epithelial cells. Abnormal corneal wound healing leads to corneal edema, neovascularization, scar formation, and poor vision. Three agents, 17β-estradiol, nicergoline, and β-glucan, have demonstrated positive effects on the wound healing response in laboratory experiments and may be of help in controlling wound healing in corneas that have suffered epithelial damage or have undergone refractive surgery.     

翼状胬肉术后不同处理方法对创面上皮修复的影响

目的：比较包扎术眼及局部滴用重组人表皮生长因子对翼状胬肉术后角膜上皮修复的影响。 方法：单侧原发性翼状胬肉38例38眼,胬肉侵入角膜在2~5mm范围内。均行翼状胬肉单纯切除术,术后将病例随机分为两组,A组19例术后每日结膜囊内涂氧氟沙星眼膏,绷带包眼至角膜上皮完全愈合;B组19例术后当日给予结膜囊内涂氧氟沙星眼膏包眼,自术后第2d起每日给予氧氟沙星眼水4次+重组人表皮生长因子眼水4次滴眼。对两组病例的角膜情况进行观察,统计角膜上皮完全愈合的时间,进行比较分析。 结果：角膜上皮平均愈合时间A组为4.1±1.4d,B组为65±2.9d,两组间比较有显著统计学差异（P<0.01）。 结论：翼状胬肉术后包眼较局部滴用表皮生长因子眼水更能加快角膜上皮愈合。     

Partial dislocation of laser in situ keratomileusis flap by air bag injury

PURPOSE: A patient developed significant corneal complications from air bag deployment, 17 months after laser in situ keratomileusis (LASIK). METHODS: Case report, slit-lamp microscopy, and review of the medical literature. RESULTS: A 37-year-old woman underwent bilateral LASIK with resultant 20/20 uncorrected visual acuity. Seventeen months later, she sustained facial and ocular injuries from air bag deployment during a motor vehicle accident. Examination revealed bilateral corneal abrasions, partial dislocation of the right corneal LASIK flap, and a hyphema in the right eye. The LASIK flap was realigned, but recovery was complicated by a slowly healing epithelial defect and flap edema. One month following the injury, epithelial ingrowth beneath the LASIK flap was noted. Surgical elevation of the flap and removal of the epithelial ingrowth was performed. Eight months later, epithelial ingrowth was absent and the visual acuity was 20/40. Residual irregular astigmatism necessitated rigid gas permeable contact lens fitting to achieve 20/20 visual acuity. CONCLUSIONS: Air bags may cause significant ocular trauma. The wound healing response of LASIK allows corneal flap separation from its stromal bed for an indeterminate time after surgery. Discussion of the possible risk of corneal trauma as part of informed consent prior to LASIK may be appropriate.