Role of fibronectin in the healing of superficial keratectomies in vitro

A fibronectin (Fn)-fibrinogen (Fg) surface matrix is not essential for epithelial cell migration in a corneal epithelial scrape wound model, in which the basement membrane is preserved. We have therefore tested whether such a provisional scaffolding becomes more critical in a superficial keratectomy model, when the basement membrane is surgically removed. Exogeneous Fn at 0.3 mg/ml was added to the medium of organ cultures of rabbit superficial keratectomies. At 48 hr after wounding, the healing rate was 1.12 +/- 0.03 mm2/hr in control corneas and 1.11 +/- 0.03 mm2/hr in those cultured with Fn. At 64 hr after wounding, the healing rates were also not significantly different (P greater than 0.5). Immunofluorescence studies showed that Fn was not detectable on the surface of control corneas but could be depicted under the migrating epithelium. In corneas cultured with Fn, it diffused throughout the entire stroma but did not deposit as a surface matrix. We therefore attempted to obtain formation of a provisional Fn-Fg surface matrix before establishment of the in vitro organ culture by leaving the superficial keratectomies in vivo for 8 hr, 24 hr or 64 hr. At 64 hr after wounding, their healing rate was 0.80 +/- 0.04 mm2/hr, 0.86 +/- 0.04 mm2/hr and 0.85 +/- 0.06 mm2/hr, respectively, which were not significantly different from that of contralateral ex vivo-wounded cultured corneas (0.83 +/- 0.04 mm2/hr, P greater than 0.5). Immunofluorescence studies revealed a Fn matrix on the bare surface of in vivo-wounded specimen, which was not detectable on ex vivo-wounded cultured corneas; there was also no diffuse stromal Fn.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ectopic epithelial tissue within the stroma on keratocyte apoptosis,mitosis, and myofibroblast transformation

The purpose of this study was to examine the effects of the epithelium on processes involved in stromal wound healing. Lamellar epithelial-stromal flaps were produced in rabbit corneas with a microkeratome. Peripheral corneal epithelial tissue, central corneal epithelial tissue, or no epithelial tissue (control) was introduced beneath the flap. Corneas were removed at time points from 4 hr to 1 month after surgery. Tissue sections were analyzed with immunocytochemistry for Keratin 3 (K3) to detect epithelial antigen, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay to detect apoptosis, immunocytochemistry for Ki67 to detect cell proliferation, and immunocytochemistry for alpha-smooth muscle actin (SMA) to detect myofibroblasts. K3 was detected at the level of the interface from 4 hr to 1 month after surgery in corneas in which epithelial tissue was introduced, but not control corneas, with the exception of one that developed epithelial in growth. Keratocyte apoptosis was significantly higher at 4 hr after flap formation in both groups in which corneal epithelial tissue was introduced beneath the flap compared with controls. Keratocyte proliferation was significantly greater at 72 hr in corneas in which epithelial tissue was introduced beneath the flap compared to the controls. Corneas in which epithelial tissue was introduced into the interface, but not control corneas, had stromal cells expressing alpha-SMA in the stroma anterior and posterior to the interface at 1 week and 1 month after surgery. This was also noted in the control cornea in which there was epithelial ingrowth. Signals derived from the corneal epithelium promote keratocyte apoptosis. Keratocyte proliferation is higher in corneas that have lamellar surgery when epithelial tissue is introduced into the interface. Epithelium-derived signals also participate in the generation and/or maintenance of myofibroblasts in the corneal stroma.     

Expression of fibronectin isoforms bearing the alternatively spliced EIIIA, EIIIB, and V segments in corneal alkali burn and keratectomy wound models in the rat

PURPOSE: To better understand the healing process in the wounded cornea, fibronectin (FN) isoforms bearing the alternatively spliced EIIIA, EIIIB, and V segments (EIIIA+, EIIIB+, and V+ FNs) were evaluated in alkali burn and keratectomy wound models in the rat. METHODS: Alkali burn or keratectomy wounds (both 2 mm) were created, and corneas were harvested at various time points and analyzed by indirect immunofluorescence using antibodies specific for the EIIIA, EIIIB, and V segments as well as for the total pool of FN (total FN). RESULTS: There was minimal staining for any variety of FN in the epithelium or basement membrane zone (BMZ) in normal cornea, but each antibody produced granular staining in the stroma. Bright staining for V+ and total FNs was evident at the denuded stromal surface 1 day following keratectomy. In contrast, staining for EIIIA+ and EIIIB+ FNs was negligible at 24 hours but appeared on the wound surface under the migrating unstained epithelium by the second day. BMZ staining for FN then gradually subsided, such that there was little or no staining by 6 weeks. In contrast, alkali burn wounds exhibited very little BMZ staining throughout the time course. Although there was preferential staining of the anterior aspect of Descemet membrane by anti-EIIIA and anti-EIIIB antibodies under normal conditions, the staining intensity of the anterior and posterior aspects became similar following corneal wounding. CONCLUSION: Deposition of EIIIA+ and EIIIB+ FNs in the BMZ of the keratectomy wound occurs more slowly than deposition of V+ and total FNs. EIIIA+ FN is expressed in a distribution that overlaps with that previously described for the alpha 9 integrin subunit following corneal debridement, suggesting that EIIIA-alpha 9 interactions could occur during corneal wound healing. In contrast, the relative lack of FN deposition in alkali burn wounds suggests that proteolytic degradation of FN may occur; and this, along with impairment of new FN synthesis because of cellular damage, could play a role in the high prevalence of recurrent epithelial erosions in alkali-wounded corneas.     

Transplant of corneal epithelium to rabbit corneal wounds in vivo

Sheets of corneal epithelium removed from 9-mm buttons of adult rabbit corneas using Dispase II were placed on abraded (basement membrane intact) or keratectomized corneas of anesthetized rabbits. Both types of wounds extended from limbus to limbus. The host animals were maintained under deep anesthesia for 3 hr, during which time culture medium was dripped onto the surface of the transplant. A soft contact lens then was placed over the cornea and the eye bandaged shut. Short-term experiments indicated that after 24 hr the transplanted epithelium was adherent to both abraded and keratectomized corneas (n = 4). Hemidesmosomes had formed between basal cells of donor epithelium and denuded host basement membrane, and cytoplasmic blebs had extended from donor epithelium into host keratectomized stroma. Seven transplants to abraded corneas and 17 transplants to keratectomized corneas were followed for longer time periods. Six of the seven transplants to abraded corneas were maintained until termination of the experiment (four at 4 weeks, one at 2 weeks, one at 1 week). Three of the 17 transplants to keratectomized corneas were maintained until termination (one at 4 weeks, one at 2 weeks, and one at 6 days). The remaining 14 sloughed between days 2 and 6. These data indicate that it is feasible to transplant corneal epithelial sheets and that they can be maintained most successfully if the host basement membrane is present.     

The effect of ophthalmic preservatives on the healing rate of the rabbit corneal epithelium after keratectomy

Most ophthalmic preparations contain preservatives. Some are toxic to healthy corneal epithelium or retard healing of corneas from which part or all of the epithelium has been removed leaving an intact basement membrane. In this paper, the effect of commonly used concentrations of benzalkonium chloride (BAC), thimerosal, and ethylenediaminetetracetic acid (EDTA) upon the healing of rabbit corneas after partial lamellar keratectomy is investigated. This model has not been used previously for a study of preservative toxicity. Thimerosal (0.004%), BAC (0.01%), or EDTA (0.1%) applied four times per day had no significant effect on the corneal healing or epithelial migration rates. A slight retardation occurred when BAC (0.01%) and EDTA (0.1%) were used together, while healing failed to occur as long as benzalkonium chloride (0.02%) was administered.     

Human diabetic corneas preserve wound healing,basement membrane,integrin and MMP-10 differences from normal corneas in organ culture

The authors have previously documented decreased epithelial basement membrane (BM) components and alpha3beta1 epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar-collagen gel at the air-liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 alpha5 chain, nidogen-1/entactin, integrin alpha3beta1, and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of alpha3beta1 integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized alpha3beta1 integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas.     

Intracellular potentials of isolated rabbit and human corneal epithelium

Cell membrane potentials (p.d.) of corneal epithelium were studied with 3 m-KCl-filled microelectrodes using the glass fibre method of filling. In the isolated rabbit cornea a staircase-like potential profile with five to seven single steps was obtained and a mean intracellular p.d. of 68·5±0·6 mV was recorded in basal epithelial cells. This p.d. was stimulated to 78·2±0·7 mV by 3·7 mm ascorbic acid and inhibited by KCl and NaCN. Epithelial p.d. in isolated human corneas of enucleated eyes (with normal corneal function) was 57·4± 0·4 mV. Cadaver corneas removed 2–5 hr after death or additionally stored for 12–18 hr in a moist chamber at 4°C exhibited, at least for 2 hr of incubation in modified Ringer solution, an adequate epithelial cell potential.     

An organ culture system for study of adherence of Pseudomonas aeruginosa to normal and wounded corneas

An organ culture system has been developed to study the adherence of Pseudomonas aeruginosa to unwounded corneas and to corneas healing after a 3 mm central epithelial debridement. The Pseudomonas strain was isolated from a human corneal ulcer; suspensions containing 1 X 10(8) colony-forming units/ml (CFU/ml) of bacteria were incubated with the corneas for the last 30 min of the 18 hr culture period. The distribution pattern and number of adherent bacteria on the ocular surface were determined by morphometric analysis of scanning electron micrographs. Few bacteria (25 +/- 15/mm2) adhered to the apical cells of unwounded corneas. There was a definite region-specific distribution of adherent bacteria on healing corneas. There was a definite region-specific distribution of adherent bacteria on healing corneas. Most bacteria were found on the denuded basal lamina in front of the leading edge of the migrating epithelium (360,700 +/- 49,000/mm2). Appreciable but lower numbers adhered to the apical membrane of leading-edge cells (37,700 +/- 6,100/mm2) and to the central portion of the denuded basal lamina (28,800 +/- 10,700/mm2). No bacteria were found adherent to the apical cells of the stratified epithelium behind the leading edge of the epithelium migrating to cover the wound. A similar region-specific distribution of adherent bacteria was found when corneas were inverted in the bacterial suspension and when corneas were incubated in the bacterial suspension for 15 rather than 30 min. Corneas preincubated with the lectin, succinyl-concanavalin A, showed significantly decreased bacterial adherence, indicating a possible role for mannose moieties of wound surface glycoconjugates in bacterial adherence.     

The contribution of blood flow by the anterior ciliary arteries to the anterior segment in the primate eye

The rate and mode of corneal wound healing in severely diabetic rats were studied by light microscopy and scanning electron microscopy. Diabetes mellitus was induced in 52 rats by alloxan injection, and 52 nondiabetic rats were used as controls. After 3 weeks, a nonpenetrating razor-blade wound was made in the central cornea of both eyes in 48 diabetic and 48 normal rats. The incision passed through the epithelium and into the stroma. The effects of diabetes on the unwounded cornea were observed by comparison with corneas from eight unwounded rats (four diabetic and four normal). Whole corneas from wounded diabetic and normal rats were studied at 0, 1, 3, 6, 12 and 24 hr and at 2–7 days after wounding. The rate and mode of healing were not found to differ between diabetics and normals. The surfaces of corneal wounds in both groups appeared to be completely healed and indistinguishable from the surrounding unwounded epithelium after 24 hr. The epithelial cells involved in the initial healing process were derived primarily from the layer of wing cells which progressed across the wound close to the connective-tissue base. Only in the final stages of healing, after the wound had been filled by the deeper epithelial cells, did superficial epithelial cells migrate. There appeared to be more exfoliating superficial epithelial cells over the entire cornea in diabetic rats than in normals. Because the healing of central corneal incisions occurs initially and primarily by sliding of the deeper epithelial cells, and because the diabetic condition appears to be associated with increased exfoliation of surface cells, the healing of central incisions may be less affected by diabetes than the healing of defects of the whole corneal surface, where the superficial epithelial cells have been reported to be the main migratory cells in the initial healing process and where healing in diabetics is delayed.     

The epidermal growth factor receptor (EGFR): role in corneal wound healing and homeostasis

To evaluate the role of the epidermal growth factor receptor (EGFR) in corneal epithelial wound healing, the effect of an EGFR inhibitor on epithelial cell proliferation and cell stratification during wound healing was investigated. From 3 days prior to wounding until wound healing was complete, rats were systemically treated with either an EGFR tyrosine kinase inhibitor (ZD1839) at 40 mg kg(-1) day(-1)or 80 mg kg(-1) day(-1), or with vehicle only (control). A single corneal wound was made in the center of 66 rat corneas, using a 6.0 mm glass tube wrapped in tissue paper soaked in n-heptanol. Subsequently, each wound was photographed and measured by a computer-assisted digitizer every 12 hr. To determine the number of cells in S phase, entire corneas were labelled with (3)H-thymidine and subjected to autoradiography at 0, 12, 24 and 48 hr after wounding. Epithelial thickness was also measured at these time points by microscopy. Epithelial wound healing was significantly and dose-dependently delayed following administration of ZD1839. At 24 hr after wounding, the number of S-phase cells in the limbal corneal epithelium was significantly lower in both the treated groups compared with the control group (P < 0.05). In the cornea before wounding (0 hr) and at 48 hr post-wounding, epithelial thickness was also significantly less in treated rats compared with controls (P < 0.05). These results indicate that EGFR inhibition affects epithelial cell proliferation and stratification during corneal epithelial wound healing and may play a role in maintaining normal corneal epithelial thickness.     

Effects of soft contacts of differing thickness on corneal wound healing in rabbits

This study was undertaken to determine the effects of thin (60 microns) and thick (240 microns) soft contact lenses of equal water content (70%) and power on nonlesioned and lesioned rabbit corneas. In one group of animals, corneas were not lesioned. Thin lenses were placed on left corneas and thick lenses on right corneas. In a second group, lesions were made in both corneas. Left corneas were covered with thin lenses and right corneas with thick lenses. Post-treatment times were 8 hr and 24 hr. At sacrifice, one-half of the cornea was fixed in 4% buffered glutaraldehyde for SEM study. The other half was cut into segments, fixed in a buffered glutaraldehyde-ruthenium red (RR) solution post-osmicated in osmium containing RR and prepared for TEM. At both 8 hr and 24 hr SEM showed cell migration in lesioned corneas covered with thin lenses but not in lesioned corneas covered with thick lenses. At 8 hr, TEM of nonlesioned and lesioned corneas showed no changes in the thickness of the corneal epithelium or the RR staining of the surface. At 24 hr, in nonlesioned corneas covered with thick lenses, the RR staining of microvilli and the height of the corneal epithelium were less than in nonlesioned corneas covered with thin lenses. In lesioned corneas covered with thick lenses, the thickness of the cornea was markedly reduced, the RR staining of microvilli was less and basal cells were more compressed than in lesioned corneas covered with thin lenses. The results of this study indicate that the thickness of a soft contact lens is important in treating corneal trauma.     

脱细胞猪角膜基质的生物相容性与组织工程化兔角膜上皮移植的研究

目的 探讨脱细胞猪角膜基质的生物相容性,评价组织工程化角膜上皮组织作供体的可行性,观察支架材料的细胞化情况和种子细胞的存活情况.方法 实验研究.采用完全随机化设计的方法,用Dispase-Triton-X-100处理猪角膜基质,脱去角膜细胞;以角膜基质囊袋内植入的方法,观察异种角膜基质植入后的生物相容性,A组:脱细胞猪角膜基质,B组:新鲜猪角膜基质,C组:空白对照组.以组织工程化雄性角膜上皮组织为供体,同种雌性为受体,作板层角膜移植,观察角膜的混浊、水肿、新生血管等情况;组织病理学和免疫组化方法检测支架材料的细胞化情况,Y染色体性别决定基因(SRY)-聚合酶链反应(PCR)方法追踪种子细胞的存活情况.结果 猪角膜基质植入兔角膜囊袋后,角膜逐渐恢复透明,排斥反应指数＜6,组织病理学观察角膜结构完整,胶原纤维平行排列,少许细胞长入脱细胞猪角膜基质边缘,各组免疫组化检测未见CIM+、CD8+T淋巴细胞浸润.组织工程化角膜上皮作异体板层角膜移植后,3～4 d上皮光滑,10～20 d变为透明;15 d时角膜上皮、基质、内皮完整,上皮细胞约4或5层结构,少许基质细胞长入支架,1个月时可见角膜上皮细胞约7或8层细胞,基质纤维排列规则,多量细胞长入脱细胞角膜基质.上皮细胞表达CK3,支架内新生细胞表达波形蛋白.SRY-PCR结果显示种子细胞可以在受体内长期存活.结论 脱细胞猪角膜基质生物相容性良好,组织工程化角膜上皮可作为板层角膜移植的供体,脱细胞猪角膜基质细胞化良好,种子细胞可以在受体内长期存活.     

Alteration of epithelial paracellular permeability during corneal epithelial wound healing.

We studied the paracellular permeability to mannitol of corneas with epithelium of corneal, limbal, or conjunctival origin. Corneas with epithelial defects reepithelialized by corneal or limbal epithelium were nonvascularized; the corneal permeability was initially increased and returned to normal 3 days later. When epithelial defects extended beyond the limbus, they were healed by conjunctival epithelium. If corneas remained avascular or minimally vascularized, the conjunctiva-derived epithelium underwent a transdifferentiation process into a cornealike morphology in which the corneal permeability was initially increased upon complete reepithelialization, and gradually decreased to a level similar to that of normal cornea, 4 weeks after healing. However, when corneas became vascularized, the conjunctiva-derived epithelium retained its original phenotype, and corneal permeability remained increased throughout the 8-month period of study. The deranged barrier functions noted in the above vascularized cornea were demonstrated further by horseradish peroxidase tracer, which was found in the intercellular spaces of conjunctiva-derived epithelium of vascularized corneas but not in the avascular corneas with epithelia of corneal or limbal origin, or transdifferentiated conjunctival epithelium. To study further the effect of subsequent ocular surface trauma, conjunctival biopsy was performed on transdifferentiated avascular corneas 3 months after initial wounding. The biopsy resulted in extensive vascularization in three of eight previously nonvascularized corneas. Two weeks later, the corneal permeability was increased to a level similar to that of conjunctiva. These results indicate that corneal epithelial paracellular permeability correlates well with the status of the epithelial phenotype.     

Expression of K12 keratin in alkali-burned rabbit corneas.

The healing of alkali-injured corneas is characterized by the persistence of polymorphonuclear leukocytes (PMN) in tissues and recurrent corneal epithelial defects. It has been suggested that the proteolytic enzymes secreted by PMN may account in part for the recurrent epithelial defects in the alkali-burned corneas. Cytoplasmic keratins, which form intracellular intermediate filaments, participate in the formation of hemidesmosomes and play a key role in the focal adhesion of epithelial cells to the basement membranes. The K3/K12 keratin pair is a major constituent of differentiated and stratified corneal epithelium. We have recently cloned the cDNA encoding the rabbit K12 keratin. In the present study we examined the expression of K12 keratin during the healing of alkali-burned rabbit corneas by slot-blot and in situ hybridization. Our results indicate that in normal cornea K12 keratin is equally expressed in all cell layers of stratified corneal epithelium and suprabasal layers of limbal epithelium, but not in bulbar conjunctival and other epithelia, i.e., lens, iris, and retinal pigment epithelium. The basal cells of the detached regenerating epithelium of the injured cornea express a very low level of K12 keratin. These observations are consistent with the notion that defective expression of K3/K12 keratins may play a role in the abnormal attachment of the regenerating epithelium to the basement membrane.     

Effects of substance P and IGF-1 in corneal epithelial barrier function and wound healing in a rat model of neurotrophic keratopathy

PURPOSE: To establish a rat model of neurotrophic keratopathy and to examine the effects of the combination of substance P (SP) and insulin-like growth factor (IGF)-1 on corneal epithelial barrier function and wound healing in this model. METHODS: Corneal denervation was achieved by thermocoagulation of the ophthalmic branch of the trigeminal nerve. A modified Schirmer test was performed without topical anesthesia. Corneal epithelial barrier function was assessed by measurement of fluorescein permeability with an anterior fluorophotometer. Epithelial wound healing was evaluated by measurement of the area of the defect at various times after removal of the entire epithelium. Eye drops containing both 1 mM SP and IGF-1 (1 micro g/mL) were administered six times daily. RESULTS: The Schirmer test result in eyes subjected to trigeminal denervation was lower than that in control eyes. The fluorescein permeability of the corneal epithelium of denervated eyes was increased relative to that of control eyes. Furthermore, trigeminal denervation induced a delay in corneal epithelial wound healing. Application of eye drops containing SP and IGF-1 to denervated corneas restored the fluorescein permeability of the corneal epithelium to control levels and abolished the delay in epithelial wound healing. CONCLUSIONS: A rat model of neurotrophic keratopathy, characterized by reduced tear secretion, loss of corneal sensation, impaired epithelial barrier function, and delayed epithelial wound healing, was established by trigeminal denervation. Treatment with both SP and IGF-1 improved corneal epithelial barrier function and stimulated corneal epithelial wound healing in this model.     

Neutral glycolipids of migrating and nonmigrating rabbit corneal epithelium in organ and cell culture

It is generally believed that plasma membrane glycoconjugates influence corneal epithelial cell migration after wounding. Previous studies have focused on the role of glycoproteins in this event. The present study was designed to determine whether migration-specific glycolipids are synthesized by epithelium of healing rabbit corneas. Migrating and nonmigrating rabbit corneal epithelia were incubated with [3H]-galactose in an organ culture system for 48 hr. At the end of the labeling period, a neutral glycosphingolipid (NGSL) fraction was isolated from each radiolabeled epithelium and was analyzed by thin-layer chromatography. Three radiolabeled NGSL components, M1, M2 and M3 (M1-M3), were present in significantly higher amounts in the extracts of migrating as compared to nonmigrating epithelium. Chromatographic mobility of M3 was similar to that of a standard glucosylceramide; M1 and M2 migrated more slowly than M3. For characterization of the migration-related NGSL, a large amount of the starting material is required. Experiments, therefore, were conducted using cell cultures of rabbit corneal epithelium. Confluent (nonmigrating) cell cultures of rabbit corneal epithelium were found to synthesize either minimal or undetectable amounts of NGSL M1-M3. In contrast, we found that the NGSL M1-M3 are synthesized as major components by sparse (migrating) corneal epithelial cell cultures. Components M1-M3 were synthesized as major components by sparse cultures even in the absence of cell mitosis. This suggests that the increased synthesis of components M1-M3 by sparse cell cultures may be related to cell migration rather than cell mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Change in epithelial keratin expression during healing of rabbit corneal wounds

Corneal epithelial wound healing following full-thickness trephination and transcorneal freeze injury was studied by electron microscopy and immunofluorescent microscopy using monoclonal antibodies AE1, AE2, and AE3 to human epithelial keratin. Wounds were evaluated at various time intervals between 4 hr and 2 mo after injury. By scanning and transmission electron microscopy, epithelial migration was evident 4 hr after injury and was characterized by thinning of the epithelium and extension of filopodial processes. AE1 monoclonal antibody, which stains specifically the superficial cells of normal corneal epithelium, reacted to cells at the leading edge of the migrating epithelium. By 24 hr, all cells migrating over the wound displayed positive fluorescence with AE1 while the epithelium over the undamaged cornea exhibited normal fluorescence limited to the superficial epithelial cells. In full-thickness corneal wounds, reepithelialization was complete by 1-2 wk; however, all epithelial cells covering the wound remained positive for the AE1 antikeratin antibody. By 2 mo, the AE1 fluorescence returned to normal. In transcorneal freeze injuries, reepithelialization was complete by 4 to 7 days after injury, with all cells overlying the wound reacting with the AE1 antibody. By 2 wk after freeze injury, all epithelial cells appeared to express a normal AE1 staining pattern. No change was noted in the fluorescent distribution of either AE2 antibody, which did not react with the corneal epithelium, or AE3, which reacts with all corneal epithelial cells. These results suggest that healing of corneal epithelial wounds involves changes in keratin expression of the corneal epithelium.     

实时荧光聚合酶链反应定量检测LASIK术后兔角膜纤维连接蛋白1的表达

目的　定量检测纤维连接蛋白 1(fibronectin 1,FN1)在LASIK术后不同时段角膜中的表达水平 ,探讨FN1与LASIK术后早期伤口愈合的关系。方法　 2 0只新西兰白兔 ,左眼按近视 4D行LASIK术 ,右眼作对照组。分别于术后 4、4 8h ,1、2周取角膜 ,荧光半定量PCR方法检测中央手术区及周边非手术区角膜FN1mRNA的表达。结果　LASIK术后 4h左眼手术区FN1的mRNA表达水平比右眼相应对照区明显升高 ,4 8h降低至对照组水平 ,其后随时间的延长无明显改变。结论　实时荧光定量PCR定量检测FN1在角膜中的表达结果提示 ,FN1参与LASIK术后角膜瓣与基质床的粘附过程     

Corneal endothelial healing rate and the effect of topical retinoic acid

These studies were undertaken to evaluate wound healing rates of the corneal endothelium in vivo. After insertion of a 26-gauge needle into the anterior chamber of the rabbit eye through the limbus, a 5-0 nylon monofilament was introduced through the needle, and endothelial wounds were made by scratching the cells with the filament. The wounds were photographed with a wide-field specular microscope at various intervals. Montages of the wounds were made, and the areas of the wounds were determined by planimetry. Wound closure occurred rapidly in a linear manner during the first 6 hr after wounding, after which the rate of cell migration decreased. Healing rates (micron2/hr) during the first 6 hr were calculated by linear regression analysis. There was a direct linear correlation between the healing rate and initial wound area. The slope of this line for nine normal (untreated) corneas was 0.093 hr-1. Nine corneas were treated with 0.1% retinoic acid in petrolatum ointment, while eight control corneas received vehicle alone. The slope of healing rate versus initial wound area for treated corneas (0.11 hr-1) was significantly greater than control (0.097 hr-1). This was interpreted as a stimulation of corneal endothelial migration during healing by retinoic acid. As a result of this study, a method for analysis of corneal endothelial healing rate has been developed which can be used for comparison of healing rates among treatments when initial wound area cannot be standardized.     

Impaired epithelial wound healing and EGFR signaling pathways in the corneas of diabetic rats

PURPOSE. The purpose of the study was to investigate the effects of hyperglycemia on EGFR (epidermal growth factor receptor)-mediated wound response and signal transduction in the corneal epithelium of rats with type I diabetes mellitus (DM). METHODS. Corneal epithelia were removed from streptozotocin (STZ)- and weight-matched normal rats. Wound healing was monitored by fluorescein staining at 24 or 48 hours after epithelial debridement. Phosphorylation of EGFR, AKT, ERK, and BAD was determined by Western blot analysis. The distribution of phospho-AKT and proliferating cell nuclear antigen (PCNA) in rat corneas was examined by immunohistochemistry. Cell death was evaluated by TUNEL staining. RESULTS. A significant delay in corneal epithelial wound healing was observed 48 hours after wounding in the diabetic rats compared with the weight-matched control rats. In the DM rat corneas, epithelial cells demonstrated diminished responses to wounding, as assessed by the phosphorylation of EGFR and its downstream signaling molecules, AKT and ERK. Furthermore, although the distribution pattern of phospho-AKT suggested a role for AKT in epithelial migration and proliferation in the normoglycemic rat corneas, it was abrogated in the healing epithelia of the DM rats. Consistent with impaired AKT activity, the number of PCNA-stained cells was also greatly reduced in the healing corneas of the diabetic rats. Finally, decreases in pBAD (Ser(136) and Ser(112)) and increases in TUNEL-positive cells were observed in both the uninjured and healing corneal epithelia of the DM rats, but not of the control rats. CONCLUSIONS. In the corneas of SZT rats, EGFR-PI3K-AKT and ERK, as well as their downstream BAD signaling pathways in migratory epithelium, were altered, resulting in increased apoptosis, decreased cell proliferation, and delayed wound closure.