The effect of sub-conjunctival platelet-rich plasma in combination with topical acetylcysteine on corneal alkali burn ulcer in rabbits

An experimental study examined the effect of sub-conjunctival platelet-rich plasma (sPRP) in combination with topical acetylcysteine on corneal alkali burn ulcers in rabbits. A total of 20 rabbits were used in this study. After collecting intracardiac blood samples from ten rabbits, platelet-rich plasma was obtained by centrifugation. Alkali wounds were inflicted on the central corneas of rabbits by applying a round filter paper, 6.0 mm in diameter, soaked in 1 M NaOH for 60 s. Only one eye in each rabbit was used. A total of 20 rabbits were allocated into four groups of five animals each. Group 1 served as the control group. Group 2 received 3% N-acetylcysteine (NAC) topically three times daily for 2 weeks. The third group received only sPRP whereas group 4 received sPRP with topical 3% NAC three times daily for 2 weeks. Clinical outcome was monitored by evaluation of epithelial defects, corneal opacity, duration of blepharospasm, corneal vascularisation, duration of ocular discharge and wound area diameter measurement. After 3 weeks eyes were enucleated and corneas were excised for histopathological analysis. Samples were assessed by evaluating the number of epithelial rows, stromal vascularisation and inflammation and stromal collagen arrangement. Comparison between groups showed that group 3 had significantly shorter duration of blepharospasm than the control group. Additionally, group 3 had smaller mean defect area and greater wound healing. Histopathlogical investigation revealed significantly less inflammation and vascularisation in the corneas in group 3; this group also had the best stromal collagen arrangement. In conclusion sPRP seems to improve corneal epithelial burn healing. However, acetylcysteine and sPRP combination may have a retarded healing effect as compared with platelet-rich plasma alone.     

Expression of Smad7 in mouse eyes accelerates healing of corneal tissue after exposure to alkali

Damage to the cornea from chemical burns is a serious clinical problem that often leads to permanent visual impairment. Because transforming growth factor (TGF)-beta has been implicated in the response to corneal injury, we evaluated the effects of altered TGF-beta signaling in a corneal alkali burn model using mice treated topically with an adenovirus (Ad) expressing inhibitory Smad7 and mice with a targeted deletion of the TGF-beta/activin signaling mediator Smad3. Expression of exogenous Smad7 in burned corneal tissue resulted in reduced activation of Smad signaling and nuclear factor-kappaB signaling via RelA/p65. Resurfacing of the burned cornea by conjunctival epithelium and its differentiation to cornea-like epithelium were both accelerated in Smad7-Ad-treated corneas with suppressed stromal ulceration, opacification, and neovascularization 20 days after injury. Introduction of the Smad7 gene suppressed invasion of monocytes/macrophages and expression of monocyte/macrophage chemotactic protein-1, TGF-beta1, TGF-beta2, vascular endothelial growth factor, matrix metalloproteinase-9, and tissue inhibitors of metalloproteinase-2 and abolished the generation of myofibroblasts. Although acceleration of healing of the burned cornea was also observed in mice lacking Smad3, the effects on epithelial and stromal healing were less pronounced than those in corneas treated with Smad7. Together these data suggest that overexpression of Smad7 may have effects beyond those of simply blocking Smad3/TGF-beta signaling and may represent an effective new strategy for treatment of ocular burns.     

The lectin KM+ induces corneal epithelial wound healing in rabbits

Neutrophil influx is essential for corneal regeneration ( Gan et al. 1999 ). KM+, a lectin from Artocarpus integrifolia , induces neutrophil migration ( Santos-de-Oliveira et al. 1994 ). This study aims at investigating a possible effect of KM+ on corneal regeneration in rabbits. A 6.0-mm diameter area of debridement was created on the cornea of both eyes by mechanical scraping. The experimental eyes received drops of KM+ (2.5 μg/ml) every 2 h. The control eyes received buffer. The epithelial wounded areas of the lectin-treated and untreated eyes were stained with fluorescein, photographed and measured. The animals were killed 12 h (group 1, n  = 5), 24 h (group 2, n  = 10) and 48 h (group 3, n  = 5) after the scraping. The corneas were analysed histologically (haematoxylin and eosin and immunostaining for proliferation cell nuclear antigen, p63, vascular endothelial growth factor, c-Met and laminin). No significant differences were found at the epithelial gap between treated and control eyes in the group 1. However, the number of neutrophils in the wounded area was significantly higher in treated eyes in this group. Three control and seven treated eyes were healed completely and only rare neutrophils persisted in the corneal stroma in group 2. No morphological distinction was observed between treated and control eyes in group 3. In treated corneas of group 2, there was an increase in immunostaining of factors involved in corneal healing compared to controls. Thus, topical application of KM+ may facilitate corneal epithelial wound healing in rabbits by means of a mechanism that involves increased influx of neutrophils into the wounded area induced by the lectin.     

Comparative evaluation in the use of topical corticosteroid in the management of corneal alkali burn ulcers in rabbits

This study was performed to evaluate the addition of steroidal or non-steroidal, anti-inflammatory agents or topical medroxyprogesterone acetate to tetracycline and vitamin C in the management of ocular alkali burns. Alkali wounds were created on the central corneas of rabbits by applying a round filter paper, 12?mm in diameter, soaked in 1?M NaOH for 30?s. Only one eye in each rabbit was treated. A total of 25 rabbits were divided into five groups with five animals each. Five rabbits without treatment after the alkali burn were designated as the control group. In the remaining four groups, topical tetracycline and systemic vitamin C treatment were included. In group A, 1% topical medroxyprogesterone acetate, in group B, 1% topical prednisolone acetate and in group C, topical diclofenac were used. In group D, corneal ulcers were treated with only tetracycline and vitamin C. Clinical outcome was monitored daily by corneal opacity, duration of blepharospasm, corneal vascularization, and duration of ocular discharge. After 3?weeks, corneas were excised for histopathological analysis. Samples were monitored by evaluating corneal thickness (μm), numbers of epithelial rows, keratocyte density, stromal vascularization, stromal inflammation, and stromal collagen arrangement. Comparison between groups showed that groups A, B, and C had significantly lower discharge days, and groups B and C had significantly shorter duration of blepharospasm than the control group. In the microscopic evaluation of the corneas, groups B and C had a significantly lower degree of corneal vascularization, and group B had significantly lower degree of corneal inflammation than the control group. In conclusion, the topical application of 1% prednisolone combined with vitamin C and tetracycline may be therapeutically valuable in the early treatment (first 3?weeks) of corneal alkali burns.     

Triptolide Suppresses Alkali Burn‐Induced Corneal Angiogenesis Along with a Downregulation of VEGFA and VEGFC Expression

Triptolide (TPL) is an active compound extracted from a Chinese herbal medicine tripterygium wilfordii Hook. f. (Celastraceae), which has been used as an anti‐inflammatory drug for years. It also inhibits the growth and proliferation of different types of cancer cells. The inhibitory effect of TPL on angiogenesis after chemical‐induced corneal inflammation was studied in vivo. The effects of TPL on the proliferation, apoptosis, migration, and tube formation of rat aortic endothelial cells (RAECs) were studied in vitro. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. Migration was analyzed using the scratch wound healing assay and transwell assay. Tube formation assay was used to examine angiogenesis. Real‐time PCR and Western blot were used to determine the expression of vascular endothelial growth factor A (VEGFA) and VEGFC. To study the in vivo effects of TPL, the mouse model of alkali burn‐induced corneal angiogenesis was used. The angiogenesis was analyzed by determining the density of the newly generated blood vessels in corneas. We found that TPL induced apoptosis and inhibited the proliferation of RAECs in a dose‐dependent manner. TPL inhibited migration and tube formation of RAECs and decreased the expression of VEGFA and VEGFC in vitro. Furthermore, TPL suppressed alkali burn‐induced corneal angiogenesis and inhibited the expression of VEGFA and VEGFC in corneas in vivo. In conclusion, topical TPL as a pharmacological agent has the ability to reduce angiogenesis in cornea and may have clinical indications for the treatment of corneal angiogenesis diseases which have to be further explored. Anat Rec, 300:1348–1355, 2017. © 2017 Wiley Periodicals, Inc.     

TRPV1 involvement in inflammatory tissue fibrosis in mice

We examined whether absence or blocking of transient receptor potential vanilloid subtype 1 (TRPV1) affects the level of inflammation and fibrosis/scarring during healing of injured tissue using an alkali burn model of cornea in mice. A cornea burn was produced with 1 N NaOH instilled into one eye of TRPV1-/- (KO) (n = 88) or TRPV1+/+ (n = 94) mice. Examinations of the corneal surface and eye globe size suggested that the loss of TRPV1 suppressed inflammation and fibrosis/scarring after alkali burn, and this was confirmed by histology, IHC, and gene expression analysis. The loss of TRPV1 inhibited inflammatory cell invasion and myofibroblast generation in association with reduction of expression of proinflammatory and profibrogenic components. Experiments of bone marrow transplantation between either genotype of mice showed that KO corneal tissue resident cells, but not KO bone marrow-derived cells, are responsible for KO-type wound healing with reduced inflammation and fibrosis. The absence of TRPV1 attenuated expression of transforming growth factor β 1 (TGFβ1) and other proinflammatory gene expression in cultured ocular fibroblasts, but did not affect TGFβ1 expression in macrophages. Loss of TRPV1 inhibited myofibroblast transdifferentiation in cultured fibroblasts. Systemic TRPV1 antagonists reproduced the KO type of healing. In conclusion, absence or blocking of TRPV1 suppressed inflammation and fibrosis/scarring during healing of alkali-burned mouse cornea. TRPV1 is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing.     

The influence of substrate topography on the migration of corneal epithelial wound borders

Currently available artificial corneas can develop post-implant complications including epithelial downgrowth, infection, and stromal melting. The likelihood of developing these disastrous complications could be minimized through improved formation and maintenance of a healthy epithelium covering the implant. We hypothesize that this epithelial formation may be enhanced through the incorporation of native corneal basement membrane biomimetic chemical and physical cues onto the surface of the keratoprosthesis. We fabricated hydrogel substrates molded with topographic features containing specific bio-ligands and developed an in vitro wound healing assay. In our experiments, the rate of corneal epithelial wound healing was significantly increased by 50% in hydrogel surfaces containing topographic features, compared to flat surfaces with the same chemical attributes. We determined that this increased healing is not due to enhanced proliferation or increased spreading of the epithelial cells, but to an increased active migration of the epithelial cells. These results show the potential benefit of restructuring and improving the surface of artificial corneas to enhance epithelial coverage and more rapidly restore the formation of a functional epithelium.     

Improvement of herpetic stromal keratitis with fumaric acid derivate is associated with systemic induction of T helper 2 cytokines

Fumaric acid derivates have been shown to stimulate T helper-2-cytokines (interleukin (IL)-4, -5) without affecting the T-helper-1-cytokine (IL-2, interferon (IFN)-gamma)-response. Herein, the influence of systemic treatment with the fumaric acid derivate dimethylfumarate (DMF) on the secretion of T helper-cytokines and the development of HSV-1 stromal keratitis (HSK) was studied in mice. The corneas from BALB/c mice were infected with 10(5) PFU of HSV-1 (KOS strain). While one group of mice was treated intraperitoneally with PBS, another group of mice received DMF at 15 mg/kg of body weight. Expression of IL-2, -4, -10 and IFN-gamma was analysed in HSV-1 activated lymphocytes by ELISA. The severity of epithelial and stromal herpetic keratitis was investigated clinically. Corneas were studied for the inflammatory cell infiltration, and the CD3-, CD4- and CD8-positive cells were analysed by immunohistochemistry. The IL-2, -4, 10 and IFN-gamma content was measured in the corneas. Virus replication in the eyes was analysed by a plaque-assay. The DTH-response, the HSV-specific T cell proliferation and the serum neutralizing antibody-titres were investigated. DMF increased IL-4 and IL-10, but not IL-2 and IFN-gamma, secretion in activated lymphocytes from the spleen. Incidence and severity of stromal HSV-1 keratitis was reduced in the DMF group (P < 0.01). In the corneas from DMF-treated mice, the numbers of CD3+ and CD4+ cells were decreased and IL-4 was increased. Severity of epithelial disease and the virus-clearance from the eyes did not differ between the PBS and DMF group of mice. DTH, HSV-specific T cell proliferation and the neutralizing antibody-titres were not impaired. DMF increased the T helper-2-cytokine secretion in activated lymphocytes. After corneal HSV-1 infection, corneas from DMF treated mice had increased IL-4 content. This is associated with an improvement of herpetic stromal keratitis and reduced corneal T cell infiltration. DMF did not impair the systemic antiviral response.     

Hes1 regulates corneal development and the function of corneal epithelial stem/progenitor cells

Hes1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of Hes1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in Hes1-null mice. Hes1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, Hes1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of Hes1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced Hes1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that Hes1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.     

Albumin precursor and Hsp70 modulate corneal wound healing in an organ culture model

In order to investigate the role of albumin precursor and Hsp70 in corneal wound healing, we have analyzed the distribution of these proteins in wounded and non-wounded corneas of rabbits and the effects of topical applications of anti-albumin precursor and anti-Hsp70 antibodies on wound healing. Anti-albumin precursor and anti-Hsp70 antibodies were topically applied in healing corneal epithelium of rabbit eyes in organ culture. Corneas were allowed to heal in vitro for up to 120 h in serum-free medium with 5 and 10 μg/ml or without (migrating control) anti-albumin precursor/ or anti-Hsp70 antibodies. Fibronectin (Fb) (5 μg/ml) was used as a positive control. Immunofluorescence labelling was used to detect proteins in corneal epithelium at various time intervals following an epithelial defect. Delay in wound healing (p<0.005) was observed with 10 μg/ml anti-albumin antibody labelling. A similar pattern was observed when anti-fibronectin antibody (5 μg/ml) alone and in combination with anti-albumin (10 μg/ml) was ectopically added with wound closure occurring at 120 h. However with anti-Hsp70 antibody (5 μg/ml) slightly delayed (p<0.005) wound closure was observed at 96 h and considerable retardation >120 h with 10 μg/ml. Additionally, immunofluoresence showed a strong co-localization of Hsp70 and albumin precursor during the active phase of wound healing. The presence of albumin precursor and Hsp70 in the epithelial compartment of the cornea indicates a role for these proteins in modulating cell behavior such as epithelial growth, adhesion or regeneration, thus contributing to corneal epithelial wound healing.     

色素上皮衍生因子对角膜碱烧伤后新生血管形成的抑制

背景：角膜新生血管导致角膜透明性降低,造成严重的视觉障碍。色素上皮衍生因子是一种内源性血管生成抑制剂,其对于角膜新生血管是否具有抑制作用尚不清楚。 目的：探索局部运用色素上皮衍生因子对大鼠角膜碱烧伤后角膜新生血管的抑制作用。 方法：将20只大鼠随机分为生理盐水组与色素上皮衍生因子组,每组10只。用NaOH溶液将大鼠右眼角膜烧伤诱导产生新生血管。碱烧伤后2组每日分别给予生理盐水和色素上皮衍生因子点眼,并采用裂隙灯显微镜观察和测量各组角膜新生血管生长情况。碱烧伤后12 d处死大鼠,将角膜组织固定切片,行苏木精-伊红染色观察,并进行免疫组织化学染色检测各组大鼠角膜血管内皮生长因子和CD31的表达。 结果与结论：大鼠角膜碱烧伤后3,7,12 d,色素上皮衍生因子组大鼠角膜新生血管面积均小于生理盐水组(P < 0.05)。角膜碱烧伤后12 d,苏木精-伊红染色显示生理盐水组大鼠角膜产生大量新生血管,角膜组织结构紊乱;色素上皮衍生因子组新生血管较少,角膜组织结构趋于整齐。角膜碱烧伤后12 d,免疫组织化学染色示生理盐水组大鼠角膜上皮和基质层可见血管内皮生长因子大量表达,角膜基质层可见血管内皮生长因子和CD31大量表达;色素上皮衍生因子组新生血管稀少,CD31表达较弱。证实局部应用色素上皮衍生因子可有效抑制大鼠角膜化学伤后的血管新生。     

Heightened Resistance of Athymic, Nude (nu/nu) Mice to Experimental Pseudomonas aeruginosa Ocular Infection

BALB/c-derived athymic, nude (nu/nu) mice exhibited a heightened natural resistance to experimental corneal infection with Pseudomonas aeruginosa when compared with their heterozygote (nu/+) littermates. Stereomicroscopic examination of the eyes of nu/nu mice 24 h after corneal trauma and topical bacterial application revealed slightly cloudy corneas (iris visible), whereas nu/+ littermate corneas were opaque (iris not visible). Nu/+ mice failed to resolve the infection, and endophthalmitis and shrinkage of the infected eye occurred in these mice within 2 weeks after experimental Pseudomonas infection, as in the parent BALB/c strain. However, nu/nu mice, similarly infected, resolved the infection within 24 h and never exhibited full corneal opacity or eye shrinkage. Histological examination of the corneas of nu/nu mice 24 h after experimental wounding and bacterial application demonstrated subepithelial capillaries and a few polymorphonuclear neutrophils (with numerous intracellular bacteria) in the central cornea. In contrast, the equivalent corneal areas of infected nu/+ littermates, examined similarly, showed a more striking neutrophilic response (but with few intracellular bacteria) to similar bacterial infection, as well as a lack of blood vessels within the central cornea. The central corneas of uninfected and saline control nu/nu mice also were observed. This area in nu/nu mice exhibited an infrequent polymorphonuclear neutrophil (with no intracellular bacteria) and capillaries similar in size and location to those described for experimentally infected nu/nu mouse corneas. Untreated and saline control nu/+ mice, on the other hand, lacked both vessels and polymorphonuclear neutrophils in the central cornea.     

Transient downregulation of microRNA-206 protects alkali burn injury in mouse cornea by regulating connexin 43

Purpose: Chemical burn in cornea may cause permanent visual problem or complete blindness. In the present study, we investigated the role of microRNA 206 (miR-206) in relieving chemical burn in mouse cornea. Method: An alkali burn model was established in C57BL/6 mice to induce chemical corneal injury. Within 72 hours, the transient inflammatory responses in alkali-treated corneas were measured by opacity and corneal neovascularization (CNV) levels, and the gene expression profile of miR-206 was measured by quantitative real-time PCR (qPCR). Inhibitory oligonucleotides of miR-206, miR-206-I, were intrastromally injected into alkali-burned corneas. The possible protective effects of down-regulating miR-206 were assessed by both in vivo measurements of inflammatory responses and in vitro histochemical examinations of corneal epithelium sections. The possible binding of miR-206 on its molecular target, connexin43 (Cx43), was assessed by luciferase reporter (LR) and western blot (WB) assays. Cx43 was silenced by siRNA to examine its effect on regulating miR-206 modulation in alkali-burned cornea. Results: Opacity and CNV levels, along with gene expression of miR-206, were all transiently elevated within 72 hours of alkali-burned mouse cornea. Intrastromal injection of miR-206-I into alkali-burned cornea down-regulated miR-206 and ameliorated inflammatory responses both in vivo and in vitro. LR and WB assays confirmed that Cx43 was directly targeted by miR-206 in mouse cornea. Genetic silencing of Cx43 reversed the protective effect of miR-206 down-regulation in alkali-burned cornea. Conclusion: miR-206, associated with Cx43, is a novel molecular modulator in alkali burn in mouse cornea.     

Kinetics of serum, tear, and corneal antibody responses in resistant and susceptible mice intracorneally infected with Pseudomonas aeruginosa.

The studies described here are aimed at determining the kinetics of antibody responses specific to Pseudomonas aeruginosa ATCC 19660 in sera, tears, and corneas of naturally resistant DBA/2 mice and susceptible C57BL/6 mice after intracorneal infection. Immunoglobulin (IgG) and IgM responses in sera were significantly greater in DBA/2 mice for the first 2 weeks postinfection. Little or no IgA was detected in the sera of mice from either strain. IgG was the predominant immunoglobulin class present in the corneas of the infected eyes from both mouse strains. However, differences in both the magnitude and the kinetics of the corneal IgG responses were noted between mouse strains. The kinetics of the corneal IgG responses were more similar to those of the serum IgG response than to those of the tear IgG response. Tear antibody responses in DBA/2 mice differed from those of C57BL/6 mice in two ways. First, there was a sharp increase in tear IgG levels 2 weeks after infection in DBA/2 mice that was not present in C57BL/6 mice. Second, IgA levels present in tears from the infected eyes of C57BL/6 mice dropped to nearly preinfection levels after the first week, whereas in DBA/2 mice, IgA levels remained elevated in the infected eyes after the first week. Determination of P. aeruginosa-specific antibody responses in the uninfected, contralateral control eyes revealed that IgA was detectable in the tears but not in the corneas of DBA/2 mice. Very little IgA was detected in the tears of the uninfected eyes of C57BL/6 mice. IgG was the only immunoglobulin class present in the uninfected corneas in both mouse strains tested. These results suggest that ocular IgA was made locally, whereas most ocular IgG may have originated from the serum, with some possible local synthesis. These immunological results indicate that DBA/2 and C57BL/6 mice respond differently to corneal challenge with P. aeruginosa.     

大鼠角膜碱烧伤后热休克蛋白70的表达

背景：角膜碱烧伤后损伤修复受许多因素的影响,热休克蛋白可促进变性、损伤蛋白质的迅速恢复或清除。 目的：观察大鼠角膜碱烧伤后热休克蛋白70的表达及其与角膜损伤修复的关系。 方法：检查大鼠眼无炎症及其他病变后,奥布卡因滴眼液点眼2次,棉签吸除结膜囊液体,将统一规格直径5 mm的滤纸片浸泡于1 mol/L NaOH溶液中10 s,然后置于大鼠角膜中央30 s制作大鼠角膜碱烧伤模型。分别于碱烧伤后6 h,1,3,7,14,21 d取材。 结果与结论：RT-PCR、免疫组织化学染色、Western blot结果均显示热休克蛋白70 mRNA和蛋白在角膜碱烧伤后1 d即开始升高,7 d时达高峰,14 d后开始下降。苏木精-伊红染色及电镜观察显示角膜损伤在烧伤后6 h即较明显,烧伤后7 d逐渐恢复。提示大鼠角膜碱烧伤后热休克蛋白70的表达与碱烧伤后角膜损伤修复过程一致,参与了大鼠角膜碱烧伤后细胞的自我保护及修复过程。     

C-Jun NH2 terminal kinase (JNK) is an essential mediator of Toll-like receptor 2-induced corneal inflammation

TLRs play an important role in the host inflammatory response to bacteria and bacterial products by activating a cascade of intracellular events leading to production of proinflammatory and chemotactic cytokines. To determine the role of MAPKs in TLR- induced corneal inflammation, we stimulated human corneal epithelial (HCE) cells with TLR2 ligands, tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3Cys) or inactivated Staphylococcus aureus, and examined the time course of expression of MAPKs and the effect of MAPK inhibition on IkBalpha degradation and CXC chemokine production. We found that S. aureus and Pam3Cys stimulate phosphorylation of JNK, p38 MAPK, and ERK within 4 h and that blockade of JNK, but not p38 or ERK phosphorylation, had an inhibitory effect on IkBalpha degradation and CXC chemokine production. To determine if JNK is also important in TLR2-induced corneal inflammation in vivo, we examined JNK1(-/-) mice and pharmacological inhibitors in a murine model of TLR2-induced corneal inflammation which is characterized by neutrophil recruitment to the corneal stroma and development of corneal haze. We found that corneal inflammation was significantly impaired in JNK1(-/-) mice compared with control mice, and in mice treated with the JNK inhibitor compared with vehicle control. Taken together with results from HCE cells, these findings demonstrate that JNK has an essential role in TLR2-induced corneal inflammation.     

Effects of inhibition of urokinase-type plasminogen activator (u-PA) by amiloride in the cornea and tear fluid of eyes irradiated with UVB

The purpose of the present study was to test our hypothesis that amiloride, a specific u-PA inhibitor, effectively decreases u-PA activity in cornea as well as in tear fluid and favourably affects corneal healing. Therefore, comparative histochemical and biochemical studies of u-PA and the effects of amiloride were performed on rabbit corneas and tear fluid using the sensitive fluorogenic substrate Z-Gly-Gly-Arg-7-amino-4-trifluoromethylcoumarin. Rabbit eyes were repeatedly irradiated with UVB for 9 days and during the irradiation topically treated with amiloride (1 mg/ml saline) or placebo (saline) (dropwise, 5 times daily). Results show that in placebo-treated eyes, UVB evoked the appearance of u-PA activity in cornea and tear fluid in early stages of irradiation, and u-PA levels increased during irradiation. Corneal epithelium was gradually lost and remnants of the epithelium as well as keratocytes in the upper part of corneal stroma showed high u-PA activity. Finally, corneas lost their epithelium completely. In corneal stroma, numerous u-PA-containing inflammatory cells were present. Corneas were vascularized. When amiloride was dropped on the eye surface on the first day of irradiation and subsequently daily until the end of the experiment, u-PA activity in both cornea and tear fluid was strongly inhibited. Corneas were covered with a continuous epithelium until the end of the experiment. The number of inflammatory cells was significantly decreased. Corneal vascularization was reduced by 50%. In conclusion, early application of amiloride inhibited u-PA activity in UVB-irradiated corneas as well as in tear fluid and diminished the development of corneal pathology.