Bovine Corneal Research Articles

Fabrication of a bioengineered corneal endothelial cell sheet using chitosan/polycaprolactone blend membranes

Our previous study has demonstrated cultivation of bovine corneal endothelial cells (BCECs) on the chitosan–polycaprolactone (PCL) blends. BCECs could grow well and express physiological phenotypes after PCL was introduced into chitosan by means of blending. The feasibility of using chitosan–PCL-blended membranes as scaffold and carrier for corneal endothelial cell (CEC) transplantation is worthy of more investigation. In this work, we attempt to manufacture various blended membranes to bring BCEC cultivation into harmony by hybridizing two polymers with fine adjustment. Therefore, chitosan, which does not promote BCECs maturation, and PCL, which supports, BCEC proliferation, are combined to prepare the blends. Analyses of transmittance, proliferative, abilities, phenotypic expressions, extracellular matrix (ECM) protein production, and hemotoxylin and eosin (H&E) staining were executed step-by-step. From our observations, the blended membranes united respective characteristics. The cultured BCECs on the blends illustrated normal appearance and good proliferative abilities. Immunohistochemical staining verified physiologically phenotypic expressions of ZO-1 and Na-K ATPase. Western blot analysis also confirmed the existence of collagen type IV proteins. Furthermore, the formation of a continuous monolayer of cells attached on the blended membrane was confirmed by H&E staining. These results suggested that chitosan–PCL blended membranes may be an optimized biomaterial to fabricate bioengineered corneal endothelium and show potential to facilitate CEC transplantation in the future.

Biocompatible Reverse Thermal Gel Sustains the Release of Intravitreal Bevacizumab In Vivo

We assessed the in vivo release profile of bevacizumab from and biocompatibility of poly(ethylene glycol)-poly-(serinol hexamethylene urethane), or ESHU, a thermoresponsive hydrogel administered intravitreally for drug delivery. The technical feasibility of injection was assessed quantitatively via mechanical testing. For in vivo studies, New Zealand White rabbit eyes were injected intravitreally with 0.05 mL of either: ESHU dissolved in 25 mg/mL bevacizumab, ESHU dissolved in PBS, or 25 mg/mL bevacizumab. Clinical examination included IOP measurements and examination with indirect ophthalmoscopy for signs of inflammation. Additionally, eyes were examined histologically following euthanasia. To quantify bevacizumab release, aqueous humor samples were obtained via anterior chamber paracentesis and ELISA was used to determine the concentration of drug weekly. In vitro cytotoxicity testing also was performed using bovine corneal endothelial cells. The ESHU was injected easily through a 31-gauge needle, was well tolerated in vivo, and caused minimal cell death in vitro when compared to other common materials, such as silicone oil. The long-term presence of the gel did not affect IOP, and there was no evidence of inflammation histologically or through indirect observation. The ESHU sustained the release of bevacizumab for over 9 weeks and maintained a drug concentration that averaged 4.7 times higher than eyes receiving bolus bevacizumab injections. To our knowledge, this is the first report demonstrating sustained bevacizumab release in vivo from an intravitreally injected hydrogel formulation, suggesting that this delivery system may be a promising candidate for ocular drug delivery.

Biochemical restoration of aged human Bruch's membrane: experimental studies to improve retinal pigment epithelium transplant survival and differentiation.

Suspensions of human embryonic stem cell-derived retinal pigment epithelium (hES-RPE) and human fetal RPE resurface aged and age-related macular degeneration (AMD) Bruch's membrane to a limited degree at day 21 in organ culture. Survival and differentiation of hES-RPE and human fetal RPE on aged or AMD Bruch's membrane are enhanced greatly (200%) if a biologically synthesized extracellular matrix (bovine corneal endothelial cell extracellular matrix) is laid down on Bruch's membrane prior to transplantation. Transplanted RPE survival is enhanced even more (400-1,000%) if Bruch's membrane is treated with bovine corneal endothelial cell-conditioned medium during organ culture of hES-RPE or fetal RPE on aged or AMD Bruch's membrane. Future efforts are focused on identifying the bioactive components of bovine corneal endothelial cell-conditioned medium, so that this material can be reconstituted for clinical use as an adjunct to improve RPE transplant survival and differentiation in AMD eyes.

Effects of bathing solution on tensile properties of the cornea

The cornea is a transparent tissue with the major functions of protecting the inner contents of the eye and refracting incoming light. The biomechanical properties of the cornea strongly depend on the microstructure and composition of the stromal layer, a hydrated bio-gel. The uniaxial strip testing is a convenient and well-accepted experimental technique for characterizing corneal material parameters. It is known that the water content of specimens in this method depends on the osmolality of the bathing solution. The present study was designed to investigate the effects of different bathing solutions on uniaxial tensile material properties of the cornea. The tensile behavior of bovine corneal samples was measured in six different bathing solutions, i.e. hypertonic solution (12% NaCl solution), common preserving isotonic solutions (e.g. phosphate buffer saline, ophthalmic balanced salt solution, and 0.9% NaCl solution), hypotonic solution (distilled water), and neutral solution (mineral oil). It was observed that the bathing solution had significant influence on the tensile behavior of the corneal samples. In particular, the specimens tested in bathing solutions causing less swelling had significantly stiffer tensile properties. Furthermore, a simple mathematical model based on Voigt composite material model was developed to represent the measured solution-dependent tensile properties. The present study suggests that extra attention should be paid to corneal thickness (hydration) in uniaxial tensile experiments. It also provides important data on tensile properties of the cornea; such information could significantly contribute to improving the accuracy of numerical predictions of corneal biomechanics.

Exploring Leptin Antagonism in Ophthalmic Cell Models

BackgroundEmerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions.Methods and ResultsThe experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca.ConclusionsOur data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models.

Confirmation of in vitro and clinical safety assessment of behentrimonium chloride-containing leave-on body lotions using post-marketing adverse event data

Behentrimonium chloride (BTC) is a straight-chain alkyltrimonium chloride compound commonly used as an antistatic, hair conditioning, emulsifier, or preservative agent in personal care products. Although the European Union recently restricted the use of alkyltrimonium chlorides and bromides as preservatives to ⩽0.1%, these compounds have been safely used for many years at ⩽5% in hundreds of cosmetic products for other uses than as a preservative. In vitro, clinical, and controlled consumer usage tests in barrier-impaired individuals were conducted to determine if whole body, leave-on skin care products containing 1–5% BTC cause dermal irritation or any other skin reaction with use. BTC-containing formulations were predicted to be non-irritants by the EpiDerm® skin irritation test and the bovine corneal opacity and permeability (BCOP)/chorioallantoic membrane vascular assay (CAMVA) ocular irritation test battery. No evidence of allergic contact dermatitis or cumulative dermal irritation was noted under the exaggerated conditions of human occlusive patch tests. No clinically assessed or self-reported adverse reactions were noted in adults or children with atopic, eczematous, and/or xerotic skin during two-week and four-week monitored home usage studies. These results were confirmed by post-marketing data for five body lotions, which showed only 0.69 undesirable effects (mostly skin irritation) reported per million shipped consumer units during 2006–2011; a value consistent with a non-irritating body lotion. No serious undesirable effects were reported during in-market use of the products. Therefore, if formulated in appropriate conditions at 1–5%, BTC will not cause dermal irritation or delayed contact sensitization when used in a whole-body, leave-on product.

The stimulatory effect of ROCK inhibitor on bovine corneal endothelial cells

Reagents which can promote the proliferation, adhesion and migration of cultured corneal endothelial cells (CECs) will be helpful for the treatment of reduced visual acuity due to CECs deficiency. The objectives of this study were to investigate the potential use of an inhibitor of Rho-associated protein kinase (ROCK), Y-27632, to cultured bovine corneal endothelial cells (B-CECs) and evaluated its effects on the proliferation, adhesion and migration of B-CECs. The proliferation of cultured B-CECs was moderately enhanced by 10 μM Y-27632. Y-27632 induced fibroblast-like morphological changes in the cultured B-CECs and normal cell morphology could recover after Y-27632 removal. In addition, Y-27632 was found to significantly enhance the adhesion and migration of B-CECs. Furthermore, the hanging drop aggregation assay showed that Y-27632 promoted B-CECs to form cellular networks and sheets, which proliferated along the liquid-air interface and migrated to the surface of the lid of dish. Our study demonstrated that Y-27632 is a potentially powerful reagent which can enhance the proliferation of cultured B-CECs. Y-27632 will be useful in CEC injection therapy and topical application for CEC deficiency.

Mechanical stimulation-induced calcium wave propagation in cell monolayers: the example of bovine corneal endothelial cells.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca(2+)-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca(2+) that initiate the propagation of the Ca(2+)-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca(2+)-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca(2+)-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca(2+)-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca(2+)-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Characteristics of the low density corneal endothelial monolayer

Corneal endothelial cells form a leaky barrier on the posterior surface of the cornea, allowing influx of nutrient-carrying aqueous humor through the paracellular space and efflux of excess fluid. Corneal edema arises when the density of these non-proliferative endothelial cells declines from endothelial disease or intraocular surgery. The cellular changes occurring at low densities are ill-defined. We therefore investigated the paracellular pathway of corneal endothelial cell monolayers of varying density to determine alterations occurring in paracellular permeability and monolayer morphology. Primary cultures of bovine corneal endothelial cells (BCECs) were passaged onto permeable supports under varying culture conditions to obtain confluent monolayers of <1000, 1000–1999 and >2000 cells/mm2. Culture growth was monitored by transendothelial electrical resistance measurements. Diffusional permeability to sodium fluorescein, FITC-dextran MW 4000 or FITC-dextran MW 20,000 was measured. Confluent cultures were also analyzed by immunofluorescence localization of the tight junction protein ZO-1 and by transmission electron microscopy. For comparison, we evaluated ZO-1 for low and high density human corneal endothelium. Our results showed that all BCEC cultures grew to the same final transendothelial electrical resistance regardless of final density. In the diffusional permeability assay, permeability increased significantly only for the smallest tracer molecule (sodium fluorescein) in the lowest density monolayers (<1000 cells/mm2). ZO-1 immunofluorescence distinctly localized to intercellular junctions in high density BCEC cultures but had more diffuse localization at lower densities. Transmission electron microscopy imaging revealed cells with thinner cross-sectional profiles and longer overlapping intercellular processes at low density relative to high density cultures. Low density human corneal endothelium lacked the diffuse ZO-1 distribution seen in BCECs. Our data supports the hypothesis that barrier integrity is the primary function disrupted in low density corneal endothelial monolayers and contradicts the idea of a linear decline in barrier function with decreasing cell density.

Ion Transport Function of SLC4A11 in Corneal Endothelium

Mutations in SLC4A11, a member of the SLC4 superfamily of bicarbonate transporters, give rise to corneal endothelial cell dystrophies. SLC4A11 is a putative Na⁺ borate and Na⁺:OH⁻ transporter. Therefore we ask whether SLC4A11 in corneal endothelium transports borate (B[OH]₄⁻), bicarbonate (HCO3⁻), or hydroxyl (OH⁻) anions coupled to Na⁺. SLC4A11 expression in cultured primary bovine corneal endothelial cells (BCECs) was determined by semiquantitative PCR, SDS-PAGE/Western blotting, and immunofluorescence staining. Ion transport function was examined by measuring intracellular pH (pHi) or Na⁺ ([Na⁺](i)) in response to Ringer solutions with/without B(OH)₄⁻ or HCO₃⁻ after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11. SLC4A11 is localized to the basolateral membrane in BCEC. B(OH)₄⁻ (2.5-10 mM) in bicarbonate-free Ringer induced a rapid small acidification (0.01 pH unit) followed by alkalinization (0.05-0.1 pH unit), consistent with diffusion of boric acid into the cell followed by B(OH)₄⁻. However, the rate of B(OH)₄⁻-induced pHi change was unaffected by overexpression of SLC4A11. B(OH)₄⁻ did not induce significant changes in resting [Na⁺(i)] or the amplitude and rate of acidification caused by Na⁺ removal. siRNA-mediated knockdown of SLC4A11 (∼70%) did not alter pHi responses to CO₂/HCO₃⁻-rich Ringer, Na⁺-free induced acidification, or the rate of Na⁺ influx in the presence of bicarbonate. However, in the absence of bicarbonate, siSLC4A11 knockdown significantly decreased the rate (43%) and amplitude (48%) of acidification due to Na⁺ removal and recovery (53%) upon add-back. Additionally, the rate of acid recovery following NH₄⁺ prepulse was decreased significantly (27%) by SLC4A11 silencing. In corneal endothelium, SLC4A11 displays robust Na⁺-coupled OH⁻ transport, but does not transport B(OH)₄⁻ or HCO₃⁻.

Effect of doxycycline on the proliferation of bovine corneal myofibroblast in vitro

Background Doxycycline is a broad spectrum antibiotic,and it is frequently used in the treatment of ocular surface diseases.Objective The purpose of the present study was to investigate the effect of doxycycline on the inhibition of cell proliferation in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repairing mechanism.Methods Six fresh bovine corneas were collected.The corneal stromal layer was isolated by two-step method of 1.0 g/L and 2.0 g/L collegenase-1.Isolated cells were plated at mantaryay culture flask in 10％ FBS of RPMI-1640.Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry,and the cells with influoresccence staining for vimentin and α-SMA were identified as the corneal myofibroblasts.Doxycycline at the concentrations of 10,20,40,60,80 mg/L was added to the medium,respectively,in different concentrations of doxycycline groups.Dexamethasone (120 mg/L)was used in the same way in the positive control group,and no drug was used in the negative control group.Cell proliferation was evaluated by MTT and the cell cycle was analyzed by BD FACScan flow cytometer assay 24 hours and 48 hours after addition of any drug.Results The cells grew well and showed the positive response for vimentin and α-SMA.MTT assay showed that the A570values of bovine corneal myofibroblasts were gradually declined with the increase of the concentration of doxycycline and lapse of active time,showing statistically significant difference (Fconcentration =1233.778,P＜0.001 ; Ftime =227.564,P ＜ 0.001).And the difference between the two factors was also statistically significant (Ftime*concentration =51.656,P＜0.001).Flow cytometry cell cycle analysis showed that 24 hours after 10,20,40,60,80 mg/L doxycycline treated,the perentage of of corneal myofibroblast cell in G0-G1 phase was 82.85％,84.36％,85.18％,87.12 ％ and 89.31％,showing significant increase in comparison with 63.89％ of the negative control group (all P＜0.05),and that of 40 mg/L doxycycline group was near the positive control group.Forty-eight hours after 10,20,40,60,80 mg/L doxycycline treated,the perentage of of corneal myofibroblast cell in G0-G1 phase was 82.78％,86.15％,88.23％,89.57％,93.00％,with significant increase in comparison with 70.17％ of the negative control group (all P ＜ 0.01),and that of 40 mg/L doxycycline group was near the positive control group.Conclusions The growth of the bovine corneal myofibroblasts is inhibited by doxycycline in time-and dosedependent manner in the range from 10 mg/L to 80 mg/L,and 40 mg/L of doxycycline has an obviously inhibitory action as 120 mg/L dexamethasone. Key words: Corneal myofibroblast; Cell proliferation; Doxycycline

ENaC contribution to epithelial wound healing is independent of the healing mode and of any increased expression in the channel

Previous work from our laboratory and others has shown that, in some epithelia, the epithelial sodium channel (ENaC) increases its expression during wound healing. In these cases, inhibition of the channel determines a decrease in the healing rate, a result suggesting a role for ENaC in the overall healing process. To understand further this role of ENaC in epithelia, we explored the participation of ENaC in wound healing in four cultured epithelial cell lines selected on the basis of their different embryonic origins, function and modality of healing, i.e., by lamellipodial cell crawling or by actin cable formation. Three of the cell lines (bovine corneal endothelial cells, rabbit corneal epithelial cells and Madin-Darby canine kidney cells) exhibited an increase in ENaC expression and consequent membrane potential depolarization and an increase in cytosolic sodium and calcium, whereas one line (bovine aortal endothelial cells, BAEC) did not exhibit any of these changes. In all of the cell lines, however, ENaC inhibition determined a similar decrease in the rate of wound healing. In BAEC monolayers, the increase in ENaC activity produced plasma membrane depolarization, increased cytosolic sodium and calcium, and augmented the velocity of healing. These novel findings contribute to the idea that ENaC plays a critical role in wound healing in various epithelia, independently of the modality of healing and of any increase in the expression of the channel.

Inhibitory effects of doxycycline on argyrophilic nucleolar organizing regions and a-smooth muscle actin expression in proliferative bovine corneal myofibroblasts in vitro

To investigate the effect of doxycycline on the nucleolar organizing regions and a-smooth muscle actin expression in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repair mechanisms. Cell culture and identification: bovine corneal fibroblasts were cultured after the stroma was incubated in 1.0 and 2.0 g/L type I collagenase in two stages.Isolated cells were plated at mantaryay culture flask in 10% of BSA RPMI-1640. Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry. The cells staining positive for Vimentin and α-SMA indicated the presence of corneal myofibroblasts. Bovine corneal myofibroblasts were treated with different concentrations of doxycycline (10, 20, 40, 60, 80 mg/L) , a bland control group and the dexamethasone group (120 mg/L) were set up, each group had 30 cases. The argyrophilic nucleolar organizing regions (AgNOR) staining and the immunohistochemistry for α-SMA were performed when the cells were treated for 24 hours and 48 hours. The AgNOR count (Ag-c), AgNOR area (Ag-a) and the expression of α-SMA in the bovine corneal myofibroblasts among each experiment group and control group were compared using one-way ANOVA, further pairwise comparisons using Independent-Samples t test. Cell culture techniques were successfully used to establish a method for the isolation and culture of bovine corneal myofibroblasts. Microscopic examination and immunohistochemical staining confirmed that the cells cultured were bovine corneal myofibroblasts. The Ag-c and Ag-a of bovine corneal myofibroblasts progressively decreased as the concentrations of doxycycline was increase. 24 h:bland control group Ag-c was 6.40 ± 0.6, 60 mg/L doxycycline group Ag-c was 2.23 ± 0.43;bland control group Ag-a was (34.80 ± 2.36) µm(2), 60 mg/L doxycycline hormone group Ag-a was (19.91 ± 2.15) µm(2). 48 h: bland control group Ag-c was 7.27 ± 0.6,60 mg/L doxycycline hormone group Ag-c was 2.80 ± 0.76, bland control group Ag-a was (36.27 ± 1.99) µm(2), 60 mg/L doxycycline group Ag-a was (13.75 ± 2.09) µm(2). The differences were statistically significant: in the same time intervention (FAg-c 24 h = 252.55, FAg-a 24 h = 202.16, P < 0.05, FAg-c 48 h = 169.38, FAg-a 48 h = 853.23, P < 0.05), in the same concentrations intervention (tAg-c = 6.98, tAg-a = 11.62, P < 0.05). And 60 mg/L of doxycycline had an obviously inhibitory action as 120 mg/L dexamethasone in the same treated hours (dexamethasone group Ag-a 24 h = 30.56 ± 3.66, dexamethasone group Ag-a 48 h = 28.35 ± 1.23 ),the differences were not statistically significant (tAg-a 24h = 1.182, P = 0.242,tAg-a 48 h = 0.21, P = 0.832). As the concentrations investigated, doxycycline can inhibit the expression of α-SMA in the bovine corneal myofibroblasts (189.90 ± 7.48, 140.20 ± 7.79, 113.20 ± 8.98, 98.00 ± 3.50, 85.50 ± 4.99), the difference was statistically significant (F = 761.79, P = 0.00). While dexamethasone had no significant role in the expression of α-SMA (bland control group was 225.10 ± 6.74, the dexamethasone group was 228.50 ± 7.12), and the statistically difference was not obvious (t = 1.096, P = 0.287). As the concentrations of doxycycline was increased from 10 mg/L to 80 mg/L, the AgNOR count and AgNOR area of bovine corneal myofibroblasts can be significantly reduced in vitro. Compared with dexamethasone, doxycycline significantly suppressed the expression of α-SMA in bovine corneal myofibroblasts in a dose-dependent positive trend.

Cell viability score (CVS) as a good indicator of critical concentration of benzalkonium chloride for toxicity in cultured ocular surface cell lines

Cytotoxicity of benzalkonium chloride (BAK) is a major factor affecting drug cytotoxicity. This study aimed to determine the critical concentration of BAK for cultured ocular cells, using SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). Cell viability was determined following the exposure of cells to 11 concentrations of BAK for 10, 30, or 60min using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and neutral red assays, and the cell viability score (CVS) was used to evaluate comprehensively the toxicity of BAK. The CVS system consists of two values. The CVS50 was determined by the number of measurements for viability ⩾50% of control. The CVS40/80 was calculated as follows: CVS40/80=(number of measurements for viability values >80%)−(number of measurements for viability values <40%). Both %CVS50 and %CVS40/80 decreased with concentrations of BAK. When BAK concentrations were 0.01% or higher, %CVS50 and %CVS40/80 became 0 and less than −90, respectively. Meanwhile, when BAK concentrations were 0.001% or lower, %CVS50 became 100. In the case of %CVS40/80, when the BAK concentrations were 0.002% or lower, the values reached 75 or more, and when 0.0005% or lower, the %CVS40/80 value reached 100. Accordingly, BAK induced very low cytotoxicity in the cultured ocular cell lines at concentrations of 0.002% or lower. The concentration-dependency confirmed that the CVS score is useful for expressing drug cytotoxicity in a simple and comprehensive manner.

Isolation of bovine corneal keratan sulfate and its growth factor and morphogen binding

Keratan sulfate (KS) is an important glycosaminoglycan that is found in cartilage, reproductive tissues, and neural tissues. Corneal KS glycosaminoglycan is found N-linked to lumican, keratocan and mimecan proteoglycans, and has been widely studied by investigators interested in corneal development and diseases. Recently, the availability of corneal KS has become severely limited, owing to restrictions on the shipment of bovine central nervous system byproducts across international borders in an effort to prevent additional cases of mad cow disease. We report a simple method for the purification of multi-milligram quantities of bovine corneal KS, and characterize its structural properties. We also examined its protein-binding properties, and discovered that corneal KS bound with high affinity to fibroblast growth factor-2 and sonic hedgehog, a growth factor and a morphogen involved in corneal development and healing.

The role of protein tyrosine phosphorylation in the cell–cell junctions and intercellular permeability of post-confluent bovine corneal epithelial cells

PurposeTo evaluate the role of protein tyrosine phosphatase (PTP) in controlling the integrity of cell–cell junction and intercellular permeability in postconfluent bovine corneal epithelial cells. MethodsConfluent cultures of bovine corneal epithelial cells were treated with different concentrations of general phosphate inhibitors andsodium orthovanadate for varying periods. An MTS assay was used to confirm no cellular death under the treatment profile. Immunocytochemical (ICC) analysis was performed to demonstrate protein tyrosine phosphorylation after treatment with sodium orthovanadate, and the effect of sodium orthovanadate on junctional proteins such as p120, α-catenin, occludin, ZO-1, and ZO-2. Western blot analysis was used to analyze the changes in p120, α-catenin, occludin, ZO-1, and ZO-2 after treatment. Paracellular permeability was evaluated by transepithelial electrical resistance (TER). ResultsDuring the whole course of treatment, no significant cellular death was noticed. Dose- and time-dependent effects of sodium orthovanadate on protein tyrosine phosphorylation were confirmed by ICC. ICC also demonstrated the dose- and time-dependent effect of sodium orthovanadate on the disruption of p120, α-catenin, occludin, ZO-1, and ZO-2. However, results of Western blot analysis showed no change in the expression levels of p120, α-catenin, occludin, ZO-1, and ZO-2. Dose- and time-dependent increase of paracellular permeability was evaluated by TER. ConclusionInhibition of protein tyrosine phosphatase activity can increase protein tyrosine phosphorylation. A dose- and time-dependent release of cell–cell contacts and increased transepithelial permeability were found in postconfluent culture of bovine corneal epithelial cells.

Prediction of eye irritation potential of liquid and granular laundry detergent formulas using the bovine corneal opacity and permeability (BCOP) assay

Evaluation of eye irritation potential is a routine part of consumer product testing. Increasingly, companies are using in vitro methods to perform these assessments. We have used the bovine corneal opacity and permeability (BCOP) assay for the prediction of eye irritation of liquid and granular laundry detergent formulas. The BCOP assay was selected because it can distinguish between moderate and severe irritants as required to evaluate these classes of formulations. Corneas were maintained in short-term culture and the exposure conditions were optimized using marketed product upper-end benchmark formulas for each product class. The primary endpoint was the loss of epithelium as measured by the change in permeability of the cornea to fluorescein and was complemented by histological evaluation of depth of injury. The opacity endpoint was not used, as the surfactants in these products do not induce opacity in proportion to the depth of injury induced. Liquid laundry detergents were diluted to 25% and exposed to the corneas for 20 min while the granular detergents were diluted to 10% and exposed for 30 min. These conditions were selected for each product type to induce OD490 values in the midrange (between 0.5 and 0.6 absorbance units) and so increased or decreased irritation potential in the test formulas could readily be observed. Seventeen liquid and eleven granular laundry detergents were tested and the OD490 values ranged from 0.278 to 2.193 for the liquid detergents and 0.267 to 0.856 for the granular detergents. Histological changes in the epithelium and stroma were consistent with the OD490 values. These data suggest that the OD490 provides an effective measure of epithelial cell loss (degree of cell lysis) and thus irritation potential for these surfactant-based formulas. The upper-end benchmark set a known upper range for acceptable irritation for the product class. Those formulas inducing lower OD490 values may be considered to fall within the acceptable range while those inducing greater OD490 values should receive further evaluation and perhaps reformulation.

RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho)physiological conditions of stress.

Conjunctival and corneal tolerability assessment of ocular naltrexone niosomes and their ingredients on the hen's egg chorioallantoic membrane and excised bovine cornea models

This study aimed at combining the hen's egg test-chorioallantoic membrane (HET-CAM), bovine corneal opacity and permeability (BCOP) test and histological examination of excised corneas to evaluate the conjunctival and corneal toxicity of niosomes and their ingredients. Various surfactant/lipid combinations and concentrations (1–10%, w/v) were investigated for the ocular delivery of an ambitious drug (naltrexone hydrochloride) for treatment of diabetic keratopathy. Four niosomal formulations were investigated and found to be non irritant to the 10 days old HET-CAMs (an acceptable conjunctival model). Only one of the tested ingredients (sodium cholate – CH) showed moderate irritation, however such an effect was diminished when incorporated into niosomes. Corneal opacity and fluorescein permeability scores for the test substances correlated well with the HET-CAM test results. Corneal erosion and stromal thickness were found to be in agreement with the HET-CAM and BCOP results, which discriminated well between moderately and mildly irritant test substances. Corneal histological examination revealed toxicity signs included epithelial erosion, stromal condensation and stromal vacuolisation, which allowed better discrimination between strong and moderate irritants. It is concluded that the prepared niosomes possess good ocular tolerability and minimal ocular tissue irritation. They can be further investigated as ocular delivery systems using appropriate animal models.

In Vitro Proliferation of Adult Human Beta-Cells

A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1) analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched (“beta-cells”) population were plated on extracellular matrix from rat (804G) and human bladder carcinoma cells (HTB9) or bovine corneal endothelial ECM (BCEC). Cells were maintained in culture+/−liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted) or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted), independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05).These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.