Statens Seruminstitut Rabbit Cornea Cells Research Articles

Solanum nigrum Toxicity and Its Neuroprotective Effect Against Retinal Ganglion Cell Death Through Modulation of Extracellular Matrix in a Glaucoma Rat Model.

Purpose: Glaucoma is a complex degenerative optic neuropathy characterized by loss of retinal ganglion cells (RGCs) leading to irreversible vision loss and blindness. Solanum nigrum has been used for decades in traditional medicine system. However, no extensive studies were reported on its antiglaucoma properties. Therefore, this study was designed to investigate the neuroprotective effects of S. nigrum extract on RGC against glaucoma rat model. Methods: High performance liquid chromatography and liquid chromatography tandem mass spectrometry was used to analyze the phytochemical profile of aqueous extract of S. nigrum (AESN). In vitro, {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} (MTT) and H2DCFDA assays were used to determine cell viability and reactive oxygen species (ROS) production in Statens Seruminstitut Rabbit Cornea cells. In vivo, AESN was orally administered to carbomer-induced rats for 4 weeks. Intraocular pressure, antioxidant levels, and electrolytes were determined. Histopathological and immunohistochemical analysis was carried out to evaluate the neurodegeneration of RGC. Results: MTT assay showed AESN exhibited greater cell viability and minimal ROS production at 10 μg/mL. Slit lamp and funduscopy confirmed glaucomatous changes in carbomer-induced rats. Administration of AESN showed minimal peripheral corneal vascularization and restored histopathological alterations such as minimal loss of corneal epithelium and moderate narrowing of the iridocorneal angle. Immunohistochemistry analysis showed increased expression of positive BRN3A cells and decreased matrix metalloproteinase (MMP)-9 activation in retina and cornea, whereas western blot analysis revealed downregulation of extracellular matrix proteins (COL-1 and MMP-9) in AESN-treated rats compared with the diseased group rats. Conclusions: AESN protects RGC loss through remodeling of MMPs and, therefore, can be used for the development of novel neurotherapeutics for the treatment of glaucoma.

A promising synthetic citric crosslinked β-cyclodextrin derivative for antifungal drugs: Solubilization, cytotoxicity, and antifungal activity

Effective antifungal therapy for the treatment of fungal keratitis requires a high drug concentration at the corneal surface. However, the use of natural β-cyclodextrin (βCD) in the preparation of aqueous eye drop formulations for treating fungal keratitis is limited by its low aqueous solubility. Here, we synthesized water-soluble anionic βCD derivatives capable of forming water-soluble complexes and evaluated the solubility, cytotoxicity, and antifungal efficacy of drug prepared using the βCD derivative. To achieve this, a citric acid crosslinked βCD (polyCTR-βCD) was successfully synthesized, and the aqueous solubilities of selected antifungal drugs, including voriconazole, miconazole (MCZ), itraconazole, and amphotericin B, in polyCTR-βCD and analogous βCD solutions were evaluated. Among the drugs tested, complexation of MCZ with polyCTR-βCD (MCZ/polyCTR-βCD) increased MCZ aqueous solubility by 95-fold compared with that of MCZ/βCD. The inclusion complex formation of MCZ/βCD and MCZ/polyCTR-βCD was confirmed by spectroscopic techniques. Additionally, the nanoaggregates of saturated MCZ/polyCTR-βCD and MCZ/βCD solutions were observed using dynamic light scattering and transmission electron microscopy. Moreover, MCZ/polyCTR-βCD solution exhibited good mucoadhesion, sustained drug release, and high drug permeation of porcine cornea ex vivo. Hen’s Egg test-chorioallantoic membrane assay and cell viability study using Statens Seruminstitut Rabbit Cornea cell line showed that both MCZ/polyCTR-βCD and MCZ/βCD exhibited no sign of irritation and non-toxic to cell line. Additionally, antifungal activity evaluation demonstrated that all isolated fungi, including Candida albicans, Aspergillus flavus, and Fusarium solani, were susceptible to MCZ/polyCTR-βCD. Overall, the results showed that polyCTR-βCD could be a promising nanocarrier for the ocular delivery of MCZ.

Thermogels containing sulfated hyaluronan as novel topical therapeutics for treatment of ocular surface inflammation.

The development of long lasting therapeutic agents is critically important for efficient treatment of chronic diseases. We herein report a rational strategy to develop a therapeutic thermogel featured with prolonged anti-inflammatory and corneal-protective effects. Specifically, a hyaluronic acid with different sulfation degrees and an amine-terminated poly(N-isopropylacrylamide) are conjugated to achieve the thermogels. In vitro studies reveal that the thermogels are highly biocompatible to statens seruminstitut rabbit cornea cells and their anti-inflammatory properties are strongly dependent on the sulfation degree. In a rabbit model of ocular inflammation, single-dose topical administration of a thermogel formulation could repair defects in corneal epithelium (∼99% thickness restored), prevent corneal cell apoptosis (∼68.3% cells recovered), and suppress ocular surface inflammation (∼4-fold decrease) for a follow-up period of 7 days. This high treatment efficacy of the thermogel can be attributed to its potent inhibition in selectin-mediated leukocyte infiltration as well as effective corneal protection. These findings show a great promise for topical treatment of ocular inflammation and advancement of ophthalmic formulations using the bioactive thermogel as a therapeutic component that is not rapidly cleared from the eye and thus considerably reduces administration times.

Sorafenib Repurposing for Ophthalmic Delivery by Lipid Nanoparticles: A Preliminary Study

Uveal melanoma is the second most common melanoma and the most common intraocular malignant tumour of the eye. Among various treatments currently studied, Sorafenib was also proposed as a promising drug, often administered with other compounds in order to avoid resistance mechanisms. Despite its promising cellular activities, the use of Sorafenib by oral administration is limited by its severe side effects and the difficulty to reach the target. The encapsulation into drug delivery systems represents an interesting strategy to overcome these limits. In this study, different lipid nanoparticulate formulations were prepared and compared in order to select the most suitable for the encapsulation of Sorafenib. In particular, two solid lipids (Softisan or Suppocire) at different concentrations were used to produce solid lipid nanoparticles, demonstrating that higher amounts were able to achieve smaller particle sizes, higher homogeneity, and longer physical stability. The selected formulations, which demonstrated to be biocompatible on Statens Seruminstitut Rabbit Cornea cells, were modified to improve their mucoadhesion, evaluating the effect of two monovalent cationic lipids with two lipophilic chains. Sorafenib encapsulation allowed obtaining a sustained and prolonged drug release, thus confirming the potential use of the developed strategy to topically administer Sorafenib in the treatment of uveal melanoma.

Formulation of Ocular In Situ Gels with Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation In Vitro.

Royal jelly is a natural substance produced by worker bees that possesses a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, and protective. Although fresh royal jelly is kept at low temperatures, to increase its stability, it needs to be incorporated into pharmaceutical formulations, such as in situ gels. The aim of this study was to formulate in situ ocular gels containing Lithuanian royal jelly for topical corneal use in order to increase the retention time of the formulation on the ocular surface and bioavailability. Gels were evaluated for physicochemical characteristics (pH, rheological properties, refractive index) and in vitro drug release measuring the amount of 10-hydroxy-2-decenoic acid (10-HDA). An ocular irritation test and cell viability tests were performed using the SIRC (Statens Seruminstitut Rabbit Cornea) cell culture line. Results indicated that all the in situ gels were within an acceptable pH and refractive index range close to corneal properties. Rheology studies have shown that the gelation temperature varies between 25 and 32 °C, depending on the amount of poloxamers. The release studies have shown that the release of 10-HDA from in situ gels is more sustained than royal jelly suspension. All gel formulations were non-irritant according to the short-time exposure test (STE) using the SIRC cell culture line, and long-term cell viability studies indicated that the formulations used in small concentrations did not induce cell death. Prepared in situ gels containing royal jelly have potential for ocular drug delivery, and they may improve the bioavailability, stability of royal jelly, and formation of non-irritant ocular formulations.

Preparation of Ophthalmic Microemulsions Containing Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation

Royal jelly is a natural substance secreted by worker honeybees that possesses antioxidant, anti-inflammatory, and other biological activities. The purpose of this study was to formulate microemulsions with incorporated Lithuanian royal jelly for possible ophthalmic delivery and to evaluate the quality of the microemulsions in vitro. The oil in water type microemulsions were prepared by the oil titration method, incorporating royal jelly, surfactant, co-surfactant, oil, and water. Physicochemical characteristics of the microemulsions and the quantity of 10-hydroxy-2-decenoic acid released in vitro were assessed. The in vitro assessment of prepared microemulsions formulations was performed with the Statens Seruminstitut rabbit cornea (SIRC) cell culture model. The results revealed that the droplet size of all microemulsion formulations was 67.88–124.2 nm and the polydispersity index was lower than 0.180. In the in vitro release study, the release of 10-hydroxy-2-decenoic acid depended on the amount of royal jelly incorporated and on the ratio of surfactant and co-surfactant in formulations. The in vitro tests with the SIRC cell culture line have shown that all formulations were found non-irritating.

Protective effect of lipidure on ocular surface and soft contact lens against desiccation damage

AbstractPurposeThe insertion of a contact lens (CL) disturbs the homeostasis of the tear film, favouring its evaporation and producing friction between the lens and the eyelids and the lens and the ocular surface. These events may induce or aggravate a pre‐existing condition of eye dryness. Lipidure is a biocompatible polymer of 2‐methacryloyloxi‐ethyl‐phosphorylcholine, showing a structure similar to the phosphatidylcholine polar bases; it is endowed with excellent antifouling and moisture retaining abilities. Aim of this study has been to address the protective effects of lipidure, with or without either hyaluronic acid (HA) or hydroxyl‐propyl‐methyl‐cellulose (HPMC), on desiccation of Statens Seruminstitut Rabbit Cornea (SIRC) cells and explanted porcine corneas. Moreover, the ability of lipidure to protect soft CL from desiccation damage and loss of transparency has also been evaluated.MethodsThe protective effect of lipidure was evaluated measuring viability on SIRC cells and explanted porcine corneas leaving them dried at 37°C, 5%CO2 and 95% humidity for 1 h. The ability of lipidure to protect soft CL from desiccation damage and loss of transparency has also been evaluated leaving them air‐dried at 26°C and 46% humidity.ResultsThe protective effect of 0.1% lipidure±HA (0.15%) or HPMC (0.3%), was compared to trehalose (3%), a sugar known in nature to protect cells from dessiccation damage. Results obtained indicate that lipidure either alone or in association with HA or HPMC is able to improve corneal epithelial cells survival under desiccation conditions, both in vitro and ex vivo, similar or even better than threalose alone. Moreover, lipidure in association with HPMC can better preserve morphology and transparency of CL from dehydration.ConclusionsIn conclusion, these studies support the use of lipidure both in artificial eye drops to protect the ocular surface form dryness, and as wetting agents for CL improve their tolerance over the eye surface.

Exploration of alternative test methods to evaluate phototoxicity of ophthalmic agents by using Statens Seruminstitut Rabbit Cornea cell lines and 3D human reconstituted cornea models.

Many chemicals have been reported to induce phototoxicity. The absorbance of light energy within the sunlight range is a common characteristic of phototoxicity. The 3T3 NRU phototoxicity test (PT) in 3T3 mouse skin fibroblasts has been used to identify the phototoxic potential induced by excited chemicals after exposure to ultra violet (UV). However, as phototoxicity may occur in ocular cells, it is necessary to develop a more suitable test for cornea-derived cells. In this study, we attempted to establish a new in vitro PT method in rabbit corneal cell lines (SIRC). We evaluated five ophthalmic agents, ciprofloxacin, levofloxacin, lomefloxacin, norfloxacin, and tetracycline, for their cytotoxic potential and in vitro phototoxicity. The results obtained using 3D human corneal models revealed that the UV-induced eye tissue toxicity by the test substances showed good correlation with those obtained using the in vitro phototoxicity test. However, the results from the 3D PT for ciprofloxacin, norfloxacin, and tetracycline in the 3D human cornea model were only partially comparable. Therefore, we suggest the SIRC cell line as a new phototoxicity test model; however, a sequential testing strategy, such as 3D PT, was also proposed to obtain relevant information for topical eye agents.

Phenotypic characterization of the SIRC (Statens Seruminstitut Rabbit Cornea) cell line reveals a mixed epithelial and fibroblastic nature

The aim of the present study was to investigate, in the Statens Seruminstitut Rabbit Cornea (SIRC) cell line, the presence of epithelial and fibroblastic markers, comparing their levels with those of the human Retinal Pigmented Epithelial (ARPE-19) cell line, and the Human Keratocyte (HK) cell line, respectively. SIRC cells, often described as of epithelial origin, are used as a corneal epithelial barrier model to study the permeability of ophthalmic drugs. However, they show a morphology that is more consistent with a fibroblastic cell phenotype, similar to corneal keratocytes. Our comparative analyses of cell type specific markers demonstrated that SIRC do not express cytokeratins 19 and 16 (typical of ARPE-19) and cytokeratin 9 (typical of HK); they do express cytokeratins 3 and 18 common to all three cell lines, and cytokeratin 12 typical of ARPE-19. Tight junction proteins were absent in HK, and lower in SIRC than in ARPE-19. All cell lines expressed the markers lumican and vimentin, with SIRC expressing intermediate levels between HK and ARPE-19; alpha-SMA was highly expressed in all lines. These markers, considered typical of fibroblasts, can be, however, expressed by epithelial cells during wound healing. These results might suggest that long-term in vitro cultivation of cell lines leads to a derangement of their specific phenotype, most likely due to genetic and epigenetic factors. This could be the reason why SIRC cells came to exhibit a hybrid nature between epithelial and fibroblastic cells.

Predictive performance and inter-laboratory reproducibility in assessing eye irritation potential of water- and oil-soluble mixtures using the Short Time Exposure test method

The Short Time Exposure (STE) test method is an alternative method for assessing eye irritation potential using Statens Seruminstitut Rabbit Cornea cells and has been adopted as test guideline 491 by the Organisation for Economic Co-operation and Development. Its good predictive performance in identifying the Globally Harmonized System (GHS) No Category (NC) or Irritant Category has been demonstrated in evaluations of water-soluble substances, oil-soluble substances, and water-soluble mixtures. However, the predictive performance for oil-soluble mixtures was not evaluated. Twenty-four oil-soluble mixtures were evaluated using the STE test method. The GHS NC or Irritant Category of 22 oil-soluble mixtures were consistent with that of a Reconstructed human Cornea-like Epithelium (RhCE) test method. Inter-laboratory reproducibility was then confirmed using 20 water- and oil-soluble mixtures blind-coded. The concordance in GHS NC or Irritant Category among four laboratories was 90%–100%. In conclusion, the concordance in comparison with the results of RhCE test method using 24 oil-soluble mixtures and inter-laboratory reproducibility using 20 water- and oil-soluble mixtures blind-coded were good, indicating that the STE test method is a suitable alternative for predicting the eye irritation potential of both substances and mixtures.

Effect of hydrophilic polymers on the wettability, static and dynamic, of solid substrate covered by confluent monolayer of air-damaged SIRC cells

The aim of this study was to evaluate the possible implementation of hydrophilic polymers as recovery agents in air-damaged corneal cells. The sessile bubble technique was implemented to measure the wetting properties of four selected polymers: hydroxyethyl cellulose (HEC), sodium chondroitin sulphate (SCS), hydroxypropyl-methylcellulose (HPMC) and poloxamer F127 (PO12), at equilibrium conditions and in the case of advancing and receding contact angle. For testing the wetting properties of the polymers, glass slides covered with a confluent monolayer of Statens Seruminstitut rabbit cornea (SIRC) cells were used. HEC showed best properties for a broad concentration range, as the polymer showed capability to maintain low values of the static (equilibrium) contact angle (average static contact angle – 36.07˚, compared to average static compact angles of HPMC – 38.44˚, PO12 – 38.92˚ and SCS – 37.85˚), i.e. better wettability. Sessile bubble technique provides quick, relatively simple and reliable approach for testing surface properties of the listed polymers. The nature of the surface damage produced by the exposition of SIRC cells was used as a plausible model of evaporative dry eye syndrome, and thus the results may have clinical implementation.

Rubella virus perturbs autophagy.

Autophagy is a cellular catabolic process implicated in numerous physiological processes and pathological conditions, including infections. Viruses have evolved different strategies to modulate the autophagic process. Since the effects of rubella virus (RV) on autophagy have not yet been reported, we evaluated the autophagic activity in the Statens Seruminstitut Rabbit Cornea cell line infected with the To336 strain of RV. Our results showed that RV lowered the levels of microtubule-associated protein 1 light chain 3 B-II (LC3B-II) and the autophagy-related gene 12-autophagy-related gene 5 conjugate, inhibited the autophagic flux, suppressed the intracellular redistribution of LC3B, decreased both the average number and the size of autophagosomes per cell and impeded the formation of acidic vesicular organelles. Induction of autophagy by using rapamycin decreased both the viral yields and the apoptotic rates of infected cultures. Besides its cytoprotective effects, autophagy furnishes an important antiviral mechanism, inhibition of which may reorchestrate intracellular environment so as to better serve the unique requirements of RV replication. Together, our observations suggest that RV utilizes a totally different strategy to cope with autophagy than that evolved by other positive-stranded RNA viruses, and there is considerable heterogeneity among the members of the Togaviridae family in terms of their effects on the cellular autophagic cascade.

Comparative Studies of N-Glycans and Glycosaminoglycans Present in SIRC (Statens Seruminstitut Rabbit Cornea) Cells and Corneal Epithelial Cells from Rabbit Eyes

ABSTRACTPurpose: We compared cultured Statens Seruminstitut rabbit cornea (SIRC) cells and corneal epithelial cells from rabbit eyes by analyzing their N-glycans and glycosaminoglycans (GAGs). This work is a fundamental study on the efficacy of using cultured cells instead of animals for drug development.Materials and methods: N-Glycans and GAGs from SIRC cell monolayers and corneal epithelial cells of rabbit eyes were analyzed by capillary electrophoresis (CE) and a combination of high-performance liquid chromatography (HPLC) and mass spectrometry.Results: High mannose-type glycans and a hybrid-type glycan were the common N-glycans in SIRC cells and corneal epithelial cells of rabbit eyes. Mono-fucosylated biantennary glycans with or without one N-acetylneuraminic acid residue were observed only in SIRC cells. Hyaluronic acid was the only measurable GAG in the corneal epithelial cells of rabbit eyes. In contrast, hyaluronic acid and chondroitin sulfates were abundantly present in SIRC cells.Conclusions: Profiles of both N-glycans and GAGs were conspicuously different between SIRC cells and corneal epithelial cells of rabbit eyes. This report will be useful for the evaluation of pharmaceutical candidates when animals or cultured cells are employed in drug development studies.

Heat Effect Induces Production of Inflammatory Cytokines Through Heat Shock Protein 90 Pathway in Cornea Cells

Purpose: Many studies have been conducted to elucidate the molecular consequences of ultraviolet irradiation, whereas little is known about the effect of infrared radiation on ocular disease. Heat is generated as a consequence of infrared irradiation, in addition to photons, and heat shock is widely considered to be an environmental stressor. Our previous study has demonstrated that heat shock (42 °C) induces apoptosis of Statens Seruminstitut Rabbit Cornea (SIRC) cells. However, the biological effect of mild heat shock (39 °C), which does not induce apoptosis, on SIRC cells has not been studied yet.Methods: The SIRC cells were treated with mild heat shock and the expression of inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), in the mRNA and protein levels were then assayed with reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The levels of heat shock protein 90 (HSP90), phosphorylated AKT, phosphorylated IκBα and nuclear NF-κB were assessed with immunoblot assays; the involvement of these molecules in the signaling pathway were further assessed by using specific inhibitor for HSP90, AKT or NF-κB.Results: Mild heat shock increased the production of inflammatory cytokines, IL-1β and IL-6. Mild heat shock increased expression of HSP90, which increased the phosphorylation of AKT. The activated AKT then increased the phosphorylation of IκBα, resulting in nuclear translocation of NF-κB, which up-regulated the expression of IL-1β and IL-6 in SIRC cells.Conclusions: These findings suggest a critical role for the HSP90-AKT-NF-κB signaling pathway in mild heat shock-induced inflammatory response of cornea cells.

Surface Chemistry Study of the Interactions of Pharmaceutical Ingredients with Human Meibum Films

To perform a surface chemistry study of the interactions between the benzalkonium chloride (BAC)-preserved eyedrops Travatan, the SofZia-preserved TravatanZ, and the Polyquad-preserved DuoTrav, and tear film (TF) constituents. The interactions of TF compounds with the individual preservatives, BAC, SofZia, and Polyquad, were also examined. Langmuir surface balance measurements were used to examine the interactions between the pharmaceuticals and films of human meibum and rabbit corneal cell lipid extracts. Surface pressure-area isocycles were used to assess the sample's capability to compress and spread during dynamic area changes. The dilatational rheologic properties of human meibum films, pure and in the presence of preservatives, were probed by stress-relaxation studies. Lipid film morphology was monitored by Brewster angle microscopy. The viability of SofZia- and Polyquad-treated Statens Seruminstitut Rabbit Cornea (SIRC) cell cultures was also evaluated. The interactions between BAC-preserved eyedrops and lipids resulted in impaired lipid spread, formation of discontinuous nonuniform surface layers, and increased surface pressure-area hysteresis during compression/expansion. In contrast, TravatanZ, DuoTrav, and the individual preservatives SofZia and Polyquad proved to be safe to the lipid film structure and isothermal reversibility. The stress-relaxation experiments revealed that the viscoelastic properties of meibomian film are impaired by BAC, and remain unaffected by SofZia and Polyquad. SIRC cells' viability and capability to form confluent cellular monolayer were also maintained after exposure to SofZia and Polyquad. Surface chemistry studies present criteria for preclinical in vitro molecular scale characterization of the interactions between eyedrop compounds and TF constituents.

Surface Chemistry Study of the Interactions of Benzalkonium Chloride with Films of Meibum, Corneal Cells Lipids, and Whole Tears

To perform a surface chemistry study of the interactions between benzalkonium chloride (BAC), a common preservative used in ophthalmic formulations, and tear film (TF) constituents. The interactions between BAC and human tears, meibum, and rabbit corneal cell lipid extracts at the air-water interface were examined in vitro during controlled compression-expansion of the film area by a Langmuir surface balance, surface potential measurements, and pendant drop-axisymmetric drop shape analysis (PD-ADSA). Surface pressure-area isotherms and isocycles were used to assess the sample's lateral elasticity and capability of compressing and spreading during dynamic area changes. Lipid film morphology was monitored by Brewster angle microscopy. The viability of BAC-treated Statens Seruminstitut rabbit cornea (SIRC) cell cultures was also examined. The BAC concentration was kept within the clinical range of 0.001% to 0.02%. In the Langmuir balance and PD-ADSA experiments, the interactions between BAC and lipids or tears resulted in (1) impaired lipid spread and formation of discontinuous nonuniform surface layers, (2) increased surface pressure-area hysteresis during compression and expansion, and (3) displacement of the lipids by BAC from the surface. A decrease (>50%) in SIRC cell viability was observed. The effects occurred within seconds after BAC exposure, and their magnitude increased with BAC concentration. The surface chemistry approach used in this study provided molecular-scale insights into the detrimental effect of BAC on TF, which well explain the TF instability and corneal epithelial barrier dysfunction after exposure to BAC in the in vivo human eye.

Corneal Re-epithelialization Stimulated by Diadenosine Polyphosphates Recruits RhoA/ROCK and ERK1/2 Pathways

To investigate the role of ERK1/2 and RhoA/ROCK intracellular pathways in the modification of corneal re-epithelialization when stimulated by the diadenosine polyphosphates Ap(4)A and Ap(3)A. In wounded confluent SIRC (Statens Seruminstitut rabbit cornea) cell monolayers and in the presence or absence of Ap(4)A or Ap(3)A 100 microM, a battery of P2 receptor antagonists and inhibitors of tyrosin kinases, MAPK, and cytoskeleton pathways (AG1478 100 microM, U0126 100 microM, Y27632 100 nM, and (-)-blebbistatin 10 microM; n = 8 each) were assayed. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with Ap(4)A and Ap(3)A (100 microM), with or without suramin, RB-2, U0126, and Y27632. The intracellular distribution of pERK and ROCK-I was examined in the presence of Ap(4)A or Ap(3)A (100 microM) with U0126 and Y27632 (100 nM). In the presence of Ap(4)A, U0126, Y27632, AG1478, and (-)-blebbistatin, reduced the migration rate compared to the effect of Ap(4)A alone (P < 0.0001, P < 0.001, P < 0.01, and P < 0.1 versus Ap(4)A, respectively). In the presence of Ap(3)A 100 microM, U0126 and Y27632 accelerated the migration rate when compared with the effect of Ap(3)A alone, whereas AG1478 and (-)-blebbistatin (P < 0.0001 versus Ap(3)A) slowed the migration rate. Western blot assays demonstrated that both dinucleotides activated the ERK1/2 pathway but only Ap(4)A activated the ROCK-I pathway. The intracellular distribution of pERK1/2 and ROCK-I reflected cross-talk between these two pathways. The activation of the Ap(4)A/P2Y(2) receptor, accelerates corneal epithelial cell migration during wound healing with the activation of MAPK and cytoskeleton pathways, whereas activation of the Ap(3)A/P2Y(6) receptor signals only the MAPK pathway.