Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

Role of TGF-β in tissue eosinophilia associated with vernal keratoconjunctivitis

To determine the role of TGF-β₁ in the tissue eosinophilia associated with vernal keratoconjunctivitis (VKC), we investigated the immunohistochemical expression of TGF-β₁ and TGF-β₁-related proteins in giant papillae obtained from VKC patients. We also investigated the effect of TGF-β₁ on production of eotaxin by cultured conjunctival and corneal fibroblasts using ELISA. Finally, the effects of glucocorticoids, cyclosporine, and tacrolimus on eotaxin production by corneal fibroblasts were assessed. Our investigations revealed that eosinophils expressing TGF-β₁ and TGF-β₁-related proteins (such as phosphorylated Smad2, integrin αvβ₆, α-smooth muscle actin, type I procollagen, and tenascin-C) were expressed in the giant papillae. TGF-β₁ and IL-4/IL-13 caused a synergistic increase of eotaxin production in cultured conjunctival and corneal fibroblasts. This effect of TGF-β₁ and IL-4/IL-13 was inhibited by glucocorticoids, but neither by cyclosporine nor by tacrolimus. In conclusion, TGF-β₁ has an important role in the tissue eosinophilia associated with VKC.     

Interleukin-1 and Transforming Growth Factor Beta: Commonly Opposing,but Sometimes Supporting,Master Regulators of the Corneal Wound Healing Response to Injury

PurposeInterleukin (IL)-1α/IL-1β and transforming growth factor (TGF)β1/TGFβ2 have both been promoted as “master regulators” of the corneal wound healing response due to the large number of processes each regulates after injury or infection. The purpose of this review is to highlight the interactions between these systems in regulating corneal wound healing.MethodsWe conducted a systematic review of the literature.ResultsBoth regulator pairs bind to receptors expressed on keratocytes, corneal fibroblasts, and myofibroblasts, as well as bone marrow-derived cells that include fibrocytes. IL-1α and IL-1β modulate healing functions, such as keratocyte apoptosis, chemokine production by corneal fibroblasts, hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) production by keratocytes and corneal fibroblasts, expression of metalloproteinases and collagenases by corneal fibroblasts, and myofibroblast apoptosis. TGFβ1 and TGFβ2 stimulate the development of myofibroblasts from keratocyte and fibrocyte progenitor cells, and adequate stromal levels are requisite for the persistence of myofibroblasts. Conversely, TGFβ3, although it functions via the same TGF beta I and II receptors, may, at least in some circumstances, play a more antifibrotic role—although it also upregulates the expression of many profibrotic genes.ConclusionsThe overall effects of these two growth factor-cytokine-receptor systems in controlling the corneal wound healing response must be coordinated during the wound healing response to injury or infection. The activities of both systems must be downregulated in coordinated fashion to terminate the response to injury and eliminate fibrosis.Translational RelevanceA better standing of the IL-1 and TGFβ systems will likely lead to better approaches to control the excessive healing response to infections and injuries leading to scarring corneal fibrosis.     

Localization of thrombospondin-1 and myofibroblasts during corneal wound repair

Thrombospondin-1 (TSP-1) is a multifunctional matrix protein that has recently been examined in various wound processes, primarily for its ability to activate the latent complex of transforming growth factor-beta (TGF-β). TGF-β has been shown to play a major role in stimulating mesenchymal cells to synthesize extracellular matrix. After injury, corneal keratocytes become activated and transform into fibroblasts and myofibroblasts. Our hypothesis is that TSP-1 regulates the transformation of keratocytes into myofibroblasts (MF) via TGF-β. In the current study, we examined the expression of TSP-1 and α-smooth muscle actin (SMA), a marker of MF, during rat corneal wound healing. Three-mm keratectomy or debridement wounds were made in the central rat cornea and allowed to heal from 8 hours to 8 weeks in vivo. Unwounded rat corneas served as controls. Expression of TSP-1, SMA and Ki67, a marker of proliferating cells, were examined by indirect-immunofluorescence microscopy. In unwounded corneas, TSP-1 expression was observed primarily in the endothelium. No expression was seen in the stroma, and only low levels were detected in the epithelium. Ki67 was localized in the epithelial basal cells and no SMA was present in the central cornea of unwounded eyes. After keratectomy wounds, TSP-1 expression was seen 24 h after wounding in the stroma immediately subjacent to the wound-healing epithelium. The expression of TSP-1 increased daily and peaked 7–8 days after wounding. SMA expression, however, was not observed until 3–4 days after wounding. Interestingly, SMA-positive cells were almost exclusively seen in the stromal zone expressing TSP-1. Peak expression of SMA-positive cells was observed 7–8 days after wounding. Ki67-expressing cells were seen both in the area expressing TSP-1 and the adjacent area. In the debridement wounds, no SMA expressing cells were observed at any time point. TSP-1 was localized in the basement membrane zone from 2 to 5 days after wounding, and the localization did not appear to penetrate into the stroma. These data are in agreement with our hypothesis that TSP-1 localization in the stromal matrix is involved in the transformation of keratocytes into myofibroblasts.     

Corneal cells: chatty in development,homeostasis, wound healing,and disease

PURPOSE: To provide an overview of cell-cell interactions in the cornea that have a critical role in corneal development, homeostasis, wound healing, and disease. DESIGN: Review. METHODS: Review of the literature. RESULTS; Cell-cell interactions make critical contributions to development, homeostasis, and wound healing in the cornea. Many of these interactions are mediated by cytokines, growth factors, and chemokines. The best characterized are stromal-epithelial interactions between epithelial cells and stromal cells such as keratocytes, keratoblasts, and myofibroblasts. However, interactions also occur between corneal nerves and epithelial cells and between corneal cells (epithelial cells and stromal cells) and corneal immune cells. Although investigations are limited, it is likely that there are interactions between corneal endothelial cells and keratocytes in the posterior stroma. CONCLUSIONS: Cellular communications in the cornea are critical during development, homeostasis, and wound healing. Disorders of cellular communication likely contribute to many corneal diseases.     

Progress in corneal wound healing

Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β (TGF-β) system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells.     

TGF-β在角膜瘢痕形成及无瘢痕愈合中的作用

转化生长因子β（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒ-β,ＴＧＦ-β）是一种多功能的细胞因子。体外和多种动物模型中研究已证实在角膜ＴＧＦ-β是主要参与角膜损伤后启动瘢痕化的重要生长因子。近年来针对ＴＧＦ-β抗瘢痕化研究已取得一些进展。本文综述了ＴＧＦ-β分子结构、生物学功能,角膜损伤修复中瘢痕形成过程中基质细胞在ＴＧＦ-β调控下的变化及ＴＧＦ-β治疗角膜瘢痕的进展。     

Vitronectin or fibronectin is required for corneal fibroblast-seeded collagen gel contraction

PURPOSE: The wound healing process in the corneal stroma involves the activation of corneal keratocytes and the expression of associated phenotypes (fibroblasts and myofibroblasts). One of these phenotypes, the myofibroblasts, synthesizes alpha-smooth muscle actin in order to affect wound closure by contracting the surrounding matrix. Excessive contraction results in the formation of unresolvable scars that are undesirable in the corneal stroma. The authors tested the effect of vitronectin and fibronectin on the contraction process associated with corneal wound healing. METHODS: Collagen gels were prepared and were exposed to different treatments of fetal calf serum (FCS). The FCS used was either depleted of fibronectin and vitronectin or contained a known concentration of fibronectin, vitronectin, or both at 50 microg/ml. Contraction was measured using image analysis and cross sections of contracted gels were examined for alpha-smooth muscle actin expression using laser confocal microscopy. RESULTS: Fibroblasts seeded in collagen gels paralleled the morphologic characteristics and cell distribution of keratocytes in unwounded cornea. Matrix contraction was dependent on the presence of fibronectin and/or vitronectin where myofibroblasts were present. The cell-mediated contraction process was maximal at 0.5 x 10(5) fibroblasts/ml. CONCLUSIONS: These studies showed that vitronectin or fibronectin is required for the myofibroblast-associated contraction to occur in this in vitro model of stromal wound healing. This model system shows a distinct potential for further studies relating to the corneal wound healing process.     

兔眼SBK、LASIK、PRK术后角膜转化生长因子β、α-平滑肌肌动蛋白的表达及创口愈合机制的对比研究

背景 目前对飞秒激光前弹力层下角膜磨镶术(SBK)术后创面愈合的研究较多,关于新型角膜板层刀SBK方面的研究尚少.观察术后早期成纤维细胞的活化程度,可以了解术后不同时间的角膜细胞增生情况. 目的 检测转化生长因子(TGF-β)和α-平滑肌肌动蛋白(α-SMA)的表达,比较SBK、准分子激光屈光性角膜切削术(PRK)和准分子激光角膜原位磨镶术(LASIK)手术后早期角膜细胞增生活化情况以及角膜创面的愈合特点,探讨不同手术方式术后组织愈合机制及生物力学差异.方法 新西兰白兔27只,用随机数字表法分为SBK组、LASIK组和PRK组,每组9只,均取右眼为手术眼,左眼为正常对照.于术后7d、1个月、3个月取兔眼角膜,光学显微镜下观察,并行免疫组织化学法检测角膜组织中TGF-β及α-SMA的表达,计数成肌纤维细胞活化数量.结果 SBK组术眼角膜瓣创口边缘上皮细胞增生明显,成肌纤维细胞数量增多,术后7d创口周围TGF-β及α-SMA呈阳性表达,1个月达高峰,3个月减弱.与LASIK组相比,SBK组和PRK组TGF-β的表达差异均有统计学意义(SBK组:t=2.226、2.158、2.330,P＜0.05;PRK组:t=4.745、6.524、6.293,P＜0.05);成纤维细胞数量的差异亦均有统计学意义(SBK组:t=4.439、5.692、4.175,P＜0.05;PRK组:t=6.330、6.723、5.267,P＜0.05).SBK组各时间点TGF-β的表达量均低于PRK组,差异均有统计学意义(t =4.691、5.527、4.399,P＜0.05);α-SMA的表达差异亦均有统计学意义(t=9.637、10.282、8.197,P＜0.05);成纤维细胞数量除3个月时差异无统计学意义外,其余时间点SBK组活化均较PRK组少,差异均有统计学意义( t=5.188、4.529,P＜0.05).结论 与传统LASIK相比,SBK手术的创面活化的成肌纤维细胞、TGF-β和α-SMA表达增多,其愈合反应更强,具有更好的术后生物力学优势,但同PRK相比仍有差距.     

Expression and function of toll-like receptor-3 and -9 in human corneal myofibroblasts

PURPOSE: To investigate the expression and function of toll-like receptor (TLR)-3 and -9 in corneal myofibroblasts. METHODS: Two types of human keratocytes were used, which were freshly isolated keratocytes from donor corneas and cultured keratocytes. Expression of the mRNAs for various molecular markers was analyzed in these cells by RT-PCR, and TLR-2, -3, -4, and -9 mRNAs were also analyzed by RT-PCR. Expression of TLR-3 and -9 at the protein level was assessed by flow cytometry. In addition, an antibody array and ELISA were used to detect chemokines and cytokines in the supernatant of cultured keratocytes, with or without stimulation by poly inosine-polycytidylic acid (poly (I:C)) or CpG-DNA. Furthermore, a phagocytosis assay was performed to evaluate whether signaling via TLR-3 and -9 enhances phagocytosis. RESULTS: Keratocytes cultured for three passages underwent differentiation into corneal myofibroblasts. TLR-3 and -9 were detected in corneal myofibroblasts at the mRNA and protein levels, but not in freshly isolated keratocytes. Stimulation of corneal myofibroblasts with poly (I:C) or CpG-DNA enhanced the production of IL-6, IL-8, GRO, ENA-78, and RANTES compared with that by untreated cells. Phagocytic activity of myofibroblasts was upregulated by signaling via TLR-3 and -9. CONCLUSIONS: This is the first report on the in vitro expression and function of TLR-3 and -9 in corneal myofibroblasts. The findings suggest that the keratocyte phenotype determines the expression of TLR-3 and -9 and that corneal myofibroblasts may have an important role in bacterial and viral clearance.     

Nerve growth factor effect on human primary fibroblastic-keratocytes: possible mechanism during corneal healing

In response to corneal injury, cytokines and growth factors play a crucial role by influencing epithelial-stromal interaction during the healing and reparative processes which may resolve in tissue remodeling and fibrosis. While transforming growth factor-beta1 (TGF-beta1) is considered the main profibrogenic modulator of these process, recently the nerve growth factor (NGF) appears as a pleiotropic modulator of wound-healing and inflammatory responses. Interestingly in the cornea, where NGF, trkA(NGFR) and p75(NTR) are expressed by epithelial cells and keratocytes, the NGF eye-drop induces the healing of neurotrophic or autoimmune corneal ulcers. During corneal healing, quiescent keratocytes are replaced by active fibroblast-like keratocytes/myofibroblasts. While the NGF effect on epithelial cells has been investigated, no data are reported for NGF effects on fibroblastic-keratocytes, during corneal healing. NGF, trkA(NGFR) and p75(NTR) were found expressed by fibroblastic-keratocytes. NGF was able to induce fibroblastic-keratocyte differentiation into myofibroblasts, migration, Metalloproteinase-9 expression/activity and contraction of a 3D collagen gel, without affecting their proliferation and collagen production. These data also show a two-directional control of fibroblastic-keratocytes by NGF and TGF-beta1. To sum up, the findings of this study indicate that NGF can modulate some functional activities of fibroblastic-keratocytes, thus substantiating the healing effects of NGF on corneal wound-healing.     

Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing

PURPOSE: To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo. METHODS: The expression of VSX1 was evaluated through semiquantitative RT-PCR, immunofluorescence and in situ hybridization both in corneas (either freshly obtained or wounded) and in collagenase/hyaluronidase-isolated keratocytes grown in the absence or presence of serum to promote keratocyte-to-myofibroblast differentiation. RESULTS: Quiescent or resting keratocytes normally residing in the corneal stroma or cultured in vitro in the absence of serum did not express VSX1. In wounded corneas or when cultured in the presence of serum to mimic wound-healing responses, keratocytes underwent fibroblastic transformation (with appearance of alpha-SMA and disappearance of CD-34 and keratocan signals) and started expressing VSX1. CONCLUSIONS: The results show that VSX1 is expressed in vitro and in vivo during human corneal wound healing, a process in which differentiation of corneal keratocytes into myofibroblasts occurs. These data may help to elucidate the role of VSX1 in cornea physiology suggesting a potential involvement in cornea-related diseases such as keratoconus.     

TGF-β在角膜损伤修复中的时间和空间分布

角膜损伤后的纤维化修复是角膜瘢痕形成的主要原因.转化生长因子-β(transforming growth factor-beta,TGF-β)在角膜的稳态平衡中起着至关重要的作用,是角膜损伤修复的重要参与者.同时,角膜上皮基底膜是角膜创伤修复过程中角膜上皮与基质相互作用的重要屏障.角膜损伤修复的不同阶段,各亚型TGF-β在角膜各种细胞及各个不同部位存在着分布差异,角膜上皮基底膜是否完整是影响该过程的重要因素.TGF-β不同亚型在时间和空间上的分布差异及变化与角膜的创伤修复过程中细胞的迁移、增殖、表型变化及细胞外基质沉着都紧密相关,是瘢痕愈合及无瘢痕愈合的细胞分子生物学基础.本文就TGF-β的生物学功能及其亚型在角膜损伤修复中的时间和空间分布情况作一综述.     

Regulatory role of miR-146a in corneal epithelial wound healing via its inflammatory targets in human diabetic cornea

PurposeMiR-146a upregulated in limbus vs. central cornea and in diabetic vs. non-diabetic limbus has emerged as an important immune and inflammatory signaling mediator in corneal epithelial wound healing. Our aim was to investigate the potential inflammation-related miR-146a target genes and their roles in normal and impaired diabetic corneal epithelial wound healing.MethodsOur previous data from RNA-seq combined with quantitative proteomics of limbal epithelial cells (LECs) transfected with miR-146a mimic vs. mimic control were analyzed. Western blot and immunostaining were used to confirm the expression of miR-146a inflammatory target proteins in LECs and organ-cultured corneas. Luminex assay was performed on conditioned media at 6- and 20-h post-wounding in miR-146a mimic/inhibitor transfected normal and diabetic cultured LECs.ResultsOverexpression of miR-146a decreased the expression of pro-inflammatory TRAF6 and IRAK1 and downstream target NF-κB after challenge with lipopolysaccharide (LPS) or wounding. Additionally, miR-146a overexpression suppressed the production of downstream inflammatory mediators including secreted cytokines IL-1α, IL-1β, IL-6 and IL-8, and chemokines CXCL1, CXCL2 and CXCL5. These cytokines and chemokines were upregulated in normal but not in diabetic LEC during wounding. Furthermore, we achieved normalized levels of altered secreted cytokines and chemokines in diabetic wounded LEC via specific inhibition of miR-146a.ConclusionOur study documented significant impact of miR-146a on the expression of inflammatory mediators at the mRNA and protein levels during acute inflammatory responses and wound healing, providing insights into the regulatory role of miR-146a in corneal epithelial homeostasis in normal and diabetic conditions.     

Keratocyte activity during wound healing of alkali-burned cornea

The proliferation and collagen synthesis of keratocytes during corneal wound healing after alkali-burn were investigated using 3H-thymidine or 3H-proline autoradiography. The effect of epidermal growth factor (EGF) on keratocytes was also examined. On day 1 after burn, the wounded stroma lacked keratocytes, and keratocytes at the periphery of the wound started to proliferate from the endothelial side. On day 7 and day 14, the population of keratocytes returned to normal. Collagen synthesis activity of the keratocytes was observed from day 7 through day 56, with the highest activity on around day 21. After day 14, keratocytes at the site of epithelial reopening or ulceration started to proliferate again. In the early stage of wound healing more keratocytes incorporated H-thymidine in eyes treated with EGF than those treated without EGF, suggesting that EGF may stimulate the proliferation of keratocytes in the early stage of wound healing.     

Aquaporin-1-facilitated keratocyte migration in cell culture and in vivo corneal wound healing models

Aquaporin-1 (AQP1) water channels are expressed in corneal keratocytes, which become activated and migrate following corneal wounding. The purpose of this study was to investigate the role of AQP1 in keratocyte migration. Keratocyte primary cell cultures from wildtype and AQP1-null mice were compared, as well as keratocyte cultures from pig cornea in which AQP1 expression was modulated by RNAi knockdown and adenovirus-mediated overexpression. AQP1 expression was found in a plasma membrane pattern in corneal stromal and cultured keratocytes. Osmotic water permeability, as measured by calcein fluorescence quenching, was AQP1-dependent in cultured keratocytes, as was keratocyte migration following a scratch wound. Keratocyte migration in vivo was compared in wildtype and AQP1 knockout mice by histology and immunofluorescence of corneal sections at different times after partial-thickness corneal stromal debridement. AQP1 expression in keratocytes was increased by 24 h after corneal debridement. Wound healing and keratocyte appearance near the wound margin were significantly reduced in AQP1 knockout mice, and the number of neutrophils was increased. These results implicate AQP1 water permeability as a new determinant of keratocyte migration in cornea.     

Expression of basic fibroblast growth factor in rabbit corneal alkali wounds in the presence and absence of granulocytes

PURPOSE: To study the expression of basic fibroblast growth factor (bFGF) in the early phases of corneal wound healing in the presence or absence of granulocytes. METHODS: A central penetrating corneal alkali wound was inflicted to one eye in each of 14 rabbits under general anaesthesia. Subsequently, seven of the rabbits were given fucoidin i.v. for 36 hours in order to block the selectins on the vascular endothelium, thus preventing blood granulocytes from entering the tissues. Then, corneas were prepared, stained for bFGF and evaluated by light microscopy. RESULTS: Whereas normal corneal epithelium expressed bFGF weakly, conjunctival epithelium did so strongly, particularly the goblet cells. The corneal endothelium showed medium staining, while keratocytes and vascular endothelial cells did not consistently express bFGF. After 36 hours of wound healing, a marked up-regulation of bFGF expression was observed in the corneal epithelial and endothelial cells, as well as in the keratocytes, that were migrating into the wound. No other changes were noted. None of these features were modulated when granulocyte emigration was prevented by fucoidin administration. CONCLUSIONS: The difference in bFGF expression between the corneal and conjunctival epithelium suggests a role for this growth factor in the barrier function at the limbus. Moreover, the specific presence of bFGF in cells migrating into the wound indicates the participation of bFGF in corneal wound healing. Expression of bFGF was independent of granulocytes.     

ICAM-1 mediates surface contact between neutrophils and keratocytes following corneal epithelial abrasion in the mouse

Corneal epithelial abrasion elicits an inflammatory response involving neutrophil (PMN) recruitment from the limbal vessels into the corneal stroma. These migrating PMNs make surface contact with collagen and stromal keratocytes. Using mice deficient in PMN integrin CD18, we previously showed that PMN contact with stromal keratocytes is CD18-dependent, while contact with collagen is CD18-independent. In the present study, we wished to extend these observations and determine if ICAM-1, a known ligand for CD18, mediates PMN contact with keratocytes during corneal wound healing. Uninjured and injured right corneas from C57Bl/6 wild type (WT) mice and ICAM-1^−/− mice were processed for transmission electron microscopy and imaged for morphometric analysis. PMN migration, stromal thickness, and ICAM-1 staining were evaluated using light microscopy. Twelve hours after epithelial abrasion, PMN surface contact with paralimbal keratocytes in ICAM-1^−/− corneas was reduced to  ˜ 50% of that observed in WT corneas; PMN surface contact with collagen was not affected. Stromal thickness (edema), keratocyte network surface area and keratocyte shape were similar in ICAM-1^−/− and WT corneas. WT keratocyte ICAM-1 expression was detected at baseline and ICAM-1 staining intensity increased following injury. Since ICAM-1 is readily detected on mouse keratocytes and PMN-keratocyte surface contact in ICAM-1^−/− mice is markedly reduced, the data suggest PMN adhesive interactions with keratocyte-stromal networks is in part regulated by keratocyte ICAM-1 expression.     

The Changes of TGF-α, TGF-β_1 and Basic FGF Messenger RNA Expression in Rabbit Cornea after Photorefractive Keratectomy

Objective : To study the mechanism of haze formation and investigate the expression changes of transforming growth factor-α(TGF-α), transforming growth factor-β1 (TGF-β1) and basic fibroblast growth factor (bFCF) mRNA in corneal epithelium and stroma after photorefractive keratectomy (PRK).Methods: Sixteen white rabbits were randomly divided into 4 groups, and PRK was performed on each eye of 12 rabbits. The haze formation was examined under a slit-lamp microscope at the 1st, 2nd and 3rd month after PRK, and the expressions of TGF-α , TGF-β1 and bFGF mRNA were detected with in situ hybridization.Results : The corneal haze formed at the 1" month after PRK. The most prominent haze formation was observed at the 2nd month, and declined gradually at the 3rd month after ablation. TGF-a mRNA expression was presented on the normal corneal epithelium and not on the corneal stroma. TGF-β1 and bGFG mRNA were expressed by both corneal epithelium and stroma. The capacities for cornea tissue expression of thre