Rabbit Conjunctival Epithelial Cells Research Articles

Experimental study of the mechanism of induction of conjunctival goblet cell hyperexpression using CHIR-99021 in vitro

A component of the tear film, mucin is produced by conjunctival goblet cells and is crucial to preserving the tear film's stability. Severe thermal burns, chemical burns, and severe ocular surface diseases can cause extensive damage to the conjunctiva, destroy the secretory function of goblet cells, and affect the stability of the tear film and integrity of the ocular surface. Currently, the expansion efficiency of goblet cells in vitro is low. In this study, we observed that rabbit conjunctival epithelial cells exhibited dense colony morphology after stimulation with the Wnt/β-catenin signaling pathway activator CHIR-99021 and promoted the differentiation of conjunctival goblet cells and the expression of its specific marker Muc5ac, among which the best induction effect was observed after 72 h in vitro culture with 5 μmol/L CHIR-99021. Under optimal culture conditions, CHIR-99021 increased the expression levels of the Wnt/β-catenin signaling pathway factors Frzb, β-catenin, SAM pointed domain containing ETS transcription factor, and glycogen synthase kinase-3β and the levels of the Notch signaling pathway factors Notch1 and Krüppel-like factor 4 while decreasing the expression levels of Jagged-1 and Hes1. The expression level of ABCG2, a marker of epithelial stem cells, was raised to keep rabbit conjunctival epithelial cells from self-renewing. Our study showed that CHIR-99021 stimulation successfully activated the Wnt/β-catenin signaling pathway and conjunctival goblet cell differentiation was stimulated, in which the Notch signaling pathway played a combined role. Those results provide a novel idea for the expansion of goblet cells in vitro.

Hypoxia-Inducible Factor-Dependent Expression of Angiopoietin-Like 4 by Conjunctival Epithelial Cells Promotes the Angiogenic Phenotype of Pterygia.

PurposeDisappointing results from clinical studies assessing the efficacy of therapies targeting vascular endothelial growth factor (VEGF) for the treatment of pterygia suggest that other angiogenic mediators may also play a role in its development. We therefore explore the relative contribution of VEGF, hypoxia-inducible factor (HIF)-1α (the transcription factor that regulates VEGF expression in ocular neovascular disease), and a second HIF-regulated mediator, angiopoietin-like 4 (ANGPTL4), to the angiogenic phenotype of pterygia.MethodsExpression of HIF-1α, VEGF, and ANGPTL4 were examined in surgically excised pterygia, and in immortalized human (ih) and primary rabbit (pr) conjunctival epithelial cells (CjECs). Endothelial cell (EC) tubule formation assays using media conditioned by ihCjECs in the presence or absence of inducers/inhibitors of HIF-1 or RNA interference (RNAi) targeting VEGF, ANGPTL4, or both were used to assess their relative contribution to the angiogenic potential of these cells.ResultsHIF-1α and VEGF expression were detected in 6/6 surgically excised pterygia and localized to CjECs. Accumulation of HIF-1α in was confirmed in ihCjECs and prCjECs, including stratified prCjECs grown on collagen vitrigel, and resulted in expression of VEGF and the promotion of EC tubule formation; the latter effect was partially blocked using RNAi targeting VEGF mRNA expression. We demonstrate expression of a second HIF-regulated angiogenic mediator, ANGPTL4, in CjECs in culture and in surgically excised pterygia. RNAi targeting ANGPTL4 inhibited EC tubule formation and was additive to RNAi targeting VEGF.ConclusionsOur results support the development of therapies targeting both ANGPTL4 and VEGF for the treatment of patients with pterygia.

Functional Identification of a Novel Transport System for Endogenous and Synthetic Opioid Peptides in the Rabbit Conjunctival Epithelial Cell Line CJVE

To investigate whether conjunctival epithelial cells express transport processes for opioid peptides. We monitored the uptake of [(3)H]deltorphin II and [(3)H]DADLE, two hydrolysis-resistant synthetic opioid peptides, in the rabbit conjunctival epithelial cell line CJVE and elucidated the characteristics of the uptake process. CJVE cells express robust uptake activity for deltorphin II and DADLE. Both opioid peptides compete with each other for transport. Several endogenous and synthetic opioid peptides, but not non-peptide opioid antagonists, are recognized by the transport process. Though various peptides inhibit the uptake of deltorphin II and DADLE in a similar manner, the uptake of deltorphin II is partly Na(+)-dependent whereas that of DADLE mostly Na(+)-independent. The transport process shows high affinity for many endogenous/synthetic opioid peptides. Functional features reveal that this transport process may be distinct from the opioid peptide transport system described in the retinal pigment epithelial cell line ARPE-19 and also from the organic anion transporting polypeptides, which are known to transport opioid peptides. CJVE cells express a novel, hitherto unknown transport process for endogenous/synthetic opioid peptides. This new transport process may offer an effective delivery route for opioid peptide drugs to the posterior segment of the eye.

Efficient Cryopreservative Conditions for Cultivated Limbal and Conjunctival Epithelial Cells

To examine the effects of cryopreservation on the viability of cultivated corneal limbal and conjunctival epithelial cells and to evaluate the optimal conditions for cryopreservation. The cultivated human limbal epithelial cells (HLECs) were stored in media including 20%, 50%, and 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) at -196 degrees C for 1 week. The cultivated rabbit conjunctival epithelial cells were stored in 10%, 20%, and 50% FBS with 10% glycerol or DMSO as a cryoprotectant at -196 degrees C for 1 week. After thawing, cell viability was assessed using the trypan blue vital staining and 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay. Immunofluorescent staining was performed with cytokeratin 3/12 antibody. Colony-forming efficiency (CFE) was evaluated 2 weeks after culture. HLECs cryopreserved with 50% FBS showed the highest cell viability, whereas those with 20% FBS revealed the lowest survival rate (87.1% +/- 0.8% and 79.8% +/- 4.01%, respectively; P = 0.030). CFE of HLECs was 2.13 +/- 1.35%, 2.31 +/- 2.23%, and 1.94 +/- 0.72% in cells with 20%, 50%, and 90% FBS, respectively (P > 0.05). For conjunctival epithelial cells, the cell viability was the highest with 50% FBS and 10% glycerol (95.0% +/- 4.27%), and the lowest survival rate was observed in the condition of 10% FBS and 10% DMSO (80.0% +/- 5.49%). CFE of cryopreserved conjunctival epithelial cells was 14.1% +/- 1.9% in cells with 20% FBS and glycerol and 13.5% +/- 2.0% in those with 20% FBS and DMSO (P > 0.05). HLECs expressed CK3/12 after cryopreservation in all conditions examined. The best results were yielded by 50% FBS for cell viability in HLECs. Glycerol seems to be superior to DMSO in cell viability of the rabbit conjunctival epithelium after cryopreservation.

Multidrug Resistance Protein 1 (MRP1) in Rabbit Conjunctival Epithelial Cells: Its Effect on Drug Efflux and Its Regulation by Adenoviral Infection

To evaluate the expression, localization, function, and regulation of multidrug resistance protein (MRP1) in rabbit conjunctival epithelial cells (RCEC). MRP1 gene expression in RCEC was determined by reverse transcription-polymerase chain reaction (RT-PCR), and MRP1 protein expression and its localization were determined by Western blot analysis and immunofluorescence using an anti-MRP1 monoclonal antibody, MRPr1. The effect of MRP1 on the transport and uptake of fluorescein was evaluated in RCEC grown on Transwell filters. Moreover, the effect of adenovirus type 5 (Ad5)-infected RCEC, and cytokines (Interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha (TNFalpha)) on MRP1 expression and leukotriene C4 (LTC4) uptake were investigated. A 652 bp RT-PCR product from rabbit conjunctiva showed a 87% homology to human MRP1. Immunostaining with MRPr1 revealed a predominant basolateral localization of MRP1 in RCEC. Uptake of fluorescein, a MRP1 substrate, was increased (203-290%) in the presence of uricosuric drug probenecid at 100 microM, anti-inflammatory drug indomethacin at 10 microM and diclofenac, flurbiprofen, and ofloxacin at 1 mM, and by ATP depletion, but not influenced by the depletion of GSH, and the presence of antiviral cidofovir and anti-inflammatory drug cromolyn and prednisolone. Apical-to-basolateral facilitated transport of LTC4 was abolished in the presence of probenecid. Western blot analysis with MRPr1 revealed a distinct band at approximately 190 kDa for freshly isolated and cultured RCEC. Both Ad5 and cytokines (IL-1, IL-6, and TNF-alpha) up-regulated MRP1 expression, thereby reducing LTC4 uptake. MRP1 appears to be primarily localized in the basolateral membrane of RCEC and function in the efflux of certain organic anions and inflammatory factors out of cells from the basolateral membrane. The upregulation in the expression of MRP1 by Ad5-infection and cytokines suggests a role of MRP1 in the transport of inflammatory factors during ocular inflammation. Supported by NIH grants EY12578, EY10421, and EY12356.

Enhancement of the Mechanical and Biological Properties of a Biomembrane for Tissue Engineering the Ocular Surface

In this study, we have developed and optimised a novel gelatin-chitosan (GC) substrate for use as a cellular carrier for tissue-engineered conjunctival epithelium. The substrate was fabricated by casting and the mechanical properties of the substrate, including tensile strength and elongation, were measured. Using the MTT, cell proliferation assay with rabbit conjunctival fibroblasts, we optimised the G:C ratio to enhance cytocompatibility. Rabbit conjunctival epithelial cells were immunostained using monoclonal antibodies for keratin 4 and pancytokeratin to investigate the biological effects of the GC substrate on the proliferation and differentiation of epithelial cells. We found that increasing the amount of gelatin resulted in an increase in elasticity (from 1:9 to 1:1 ratio), reaching a maximum (101.89% +/- 7.13%) at a ratio of 1:1. The MTT assay showed that the proliferation of conjunctival fibroblasts significantly increased from 0.068 +/- 0.017 to 0.177 +/- 0.011 (P = 0.014) as the gelatin was increased from 20% (1:4) to 50% (1:1). Additional studies using tissue-cultured conjunctiva explants showed that these explants grew well on the substrate, forming a multilayered epithelium. Cell morphology on this substrate was similar to that of cells grown on culture dishes alone. Positive staining of keratin 4 and pancytokeratin indicated that the substrate supported normal differentiation of conjunctival epithelial cells. By enhancing the proportion of gelatin, both the mechanical and biological properties of the chitosan substrate were improved. The results also suggest that this GC biomembrane may be a useful candidate for reconstructive tissue engineering of the conjunctiva.

Regulation of L-cystine transport and intracellular GSH level by a nitric oxide donor in primary cultured rabbit conjunctival epithelial cell layers.

Metabolism and transport of cysteine are critical for maintenance of the intracellular glutathione (GSH) level. In this study, transport mechanisms of L-cystine and regulation of GSH biosynthesis in the absence or presence of NO-induced oxidant stress were investigated in primary cultured rabbit conjunctival epithelial cells (RCECs). RCECs were grown in membrane filters to exhibit tight barrier properties. Uptake and transepithelial transport of L-cystine were determined in the presence or absence of extracellular Na(+). Uptake was determined at 10 minutes after (14)C-L-cystine instillation into apical (a) or basolateral (b) bathing fluid. The effect of nitric oxide (NO) on L-cystine uptake, cellular GSH level, and expression level of two subunits of the rate-limiting enzyme gamma-glutamylcysteine synthetase (GCS) was examined after a 24-hour incubation of primary cultured RCECs with an NO donor, S-nitroso-N-acetylpenicillamine (SNAP; N-acetyl-3-(nitrosothio)-D-valine. Cellular uptake of L-cystine by RCECs occurred through both Na(+)-dependent and -independent mechanisms. Uptake from apical fluid was higher than that from basolateral fluid, except for the highest concentration of L-cystine tested (200 microM). Transepithelial permeability (P(app)) of L-cystine (at 2.5 microM) was three times higher in the a-to-b direction than in the b-to-a direction in the presence of Na(+), whereas the reverse was true in the absence of Na(+). Na(+)-dependent L-cystine uptake from apical fluid was significantly elevated in primary cultured RCECs treated for 24 hours with various concentrations (0.1-2.0 mM) of SNAP, with maximum uptake observed at 1 mM. A similar pattern of SNAP-induced increase of Na(+)-independent L-cystine uptake from apical fluid was observed, whereas no significant difference was observed for basolateral uptake. Concomitantly, a significant elevation of intracellular GSH (up to fivefold versus the control) was recorded, with the highest increase occurring at 0.1 to 0.25 mM SNAP. A parallel increase in the expression levels of both catalytic and regulatory subunits of GCS was observed by Western blot analysis of lysates from RCECs treated with 0.25 mM SNAP for 24 hours. L-Cystine is transported by both Na(+)-dependent and -independent amino acid transport systems in RCECs. At low substrate concentrations, L-cystine uptake was higher from apical than basolateral fluid. Permeability studies indicated net absorption of L-cystine across RCECs. SNAP caused significant increases in both L-cystine uptake and intracellular GSH level, which occurred concomitantly with elevation of both catalytic and regulatory subunits of GCS. Understanding sulfur amino acid precursor-dependent cellular mechanisms of GSH homeostasis would be of value in devising GSH-based treatment for conjunctival or other ocular disorders.

Pilocarpine permeability across ocular tissues and cell cultures: influence of formulation parameters.

In vitro permeation studies of drugs across biological barriers are promising tools for estimating the quality and quantity of drug transport in vivo. The objective of this work was to compare the permeability of the hydrophilic model drug pilocarpine-HCl (P-HCl) through different ocular tissues and cell cultures: isolated pig cornea (PCr) and sclera (PSc), rabbit conjunctiva (RCo), and rabbit conjunctival (RCoEC) or corneal epithelial cell culture (RCrEC). Furthermore, the study included investigations about the influence of the excipients benzalkonium chloride (BAC) and ethylene diamine tetra acetic acid disodium salt (EDTA) on the permeability of the small drug. In general, BAC caused a facilitated drug transport, while EDTA hardly influenced the P-HCl concentration on the acceptor side, except for RCoEC. Additionally, the impact of variation in buffer solution pH and tonicity on drug transport in both cell cultures was tested. The higher the tonicity of the buffer solution (80, 300, and 600 mOsm/kg) the lower the permeability coefficient (P(eff)). At different pH values (6.4, 7.4, and 8.4) the P(eff) showed a directly proportional demeanor. In summary, a good correlation between the isolated tissues and cell cultures with regard to P-HCl transport could be observed.

Characterization of cyclic AMP-regulated chloride conductance in the pigmented rabbit conjunctival epithelial cells.

We have previously reported that the pigmented rabbit conjunctiva is a Cl- secreting tissue, subject to cAMP, Ca2+, and PKC modulation. The present study was conducted to characterize, at the cellular and molecular levels, cAMP-regulated Cl- channels in rabbit conjunctival epithelial cells. cAMP-inducible Cl- channel properties were evaluated by monitoring the whole-cell currents using patch clamp techniques. Results showed that 10 microM forskolin significantly stimulated a glibenclamide-inhibitable whole-cell conductance by approximately five-fold. Furthermore, reduction of the Cl- concentration in the bathing solution through partial substitution of NaCl with Na-isethionate resulted in a rightward shift of the reversal potential for both baseline and forskolin-stimulated whole-cell currents from 0 to values close to the theoretical Cl- reversal potential predicted by the Nernst equation. Western blot analysis with a monoclonal antibody recognizing the epitope in the C-terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) showed a positive band at its molecular weight, approximately 170 kD. Immunostaining under confocal microscopy revealed a CFTR specific signal in the apical sections of primary conjunctival epithelial cells. In addition, RT-PCR detection amplified a cDNA fragment 100% identical to the predicted portion of the cloned rabbit CFTR message. The stage is thus set for determining the extent of CFTR contribution to cAMP-regulated Cl- conductance in pigmented rabbit conjunctival epithelial cells.

Expansion of Conjunctival Epithelial Progenitor Cells on Amniotic Membrane

Amniotic membrane (AM) reconstructed human conjunctival surfaces recover a goblet cell density higher than normal. Cultured rabbit conjunctival epithelial cells (RCE) on AM preferentially exhibit non-goblet epithelial differentiation. It was thus wondered if conjunctival progenitor cells that might have been preserved during ex vivo expansion on AM can still differentiate into conjunctival non-goblet epithelial and goblet cells under the influence of mesenchymal cells.Fourteen day old AM cultures of RCE were subcutaneously implanted in Balb/c athymic mice for 11 days and processed for PAS staining and immunostaining with monoclonal antibodies to conjunctival goblet cell mucin (MUC5AC, AM3), glycocalyx (AMEM2), cornea specific cytokeratins K3 (AE5) and K12 (AK2) and basal cell specific cytokeratin K14. Cell cycle kinetics were measured by BrdU labelling for 1 or 7 days. The 7 day labelled RCE were chased for 14 days in the same primary culture.After subcutaneous implantation, conjunctival non-goblet epithelial cells increased stratification and formed occasional cysts. The resultant epithelial phenotype was conjunctival with many PAS-positive, MUC5AC-positive, and AM3-positive goblet cells, AMEM2-positive suprabasal and superficial cells, and K14-positive basal cells, but was not corneal (negative to AE5 and AK2 staining). Twenty four hr BrdU labelling showed a labelling index of 42.5%. A higher labelling index or 69% was noted after continuous BrdU labelling for 7 days. A large number of label retaining basal cells with a labelling index of 84% were noted following 14 days of chase.Conjunctival epithelial progenitor cells for goblet and non-goblet cell differentiation are preserved by AM in vitro as evidenced by being able to differentiate into goblet cells in a permissive stromal environment, and being slow-cycling, and label retaining. This information is useful for future ex vivo expansion of conjunctival epithelial stem cells for conjunctival surface reconstruction.

Metabolism and transport of purinergic receptor agonists in rabbit conjunctival epithelial cells.

The role of extracellular nucleotides as signaling molecules is well established in a number of biological systems. Their function in the regulation of ion1, 2 and fluid3 transport has been investigated in the pigmented rabbit conjunctiva where active Cl− secretion has been implicated as the driving force for fluid secretion.3 These agonists interact with two classes of receptors located on the cell membrane: P2X ligand-gated ion channels and P2Y G-protein-coupled receptors.4 Receptor mapping techniques have revealed the presence of P2Y type receptors in ocular epithelial cells.5 KeywordsContact LensProtein ProfileProtein DepositionContact Lens WearLens MaterialThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Cyclic AMP-dependent Stimulation of Basolateral K+Conductance in the Rabbit Conjunctival Epithelium

The regulation of Na+transport by cAMP in freshly isolated rabbit conjunctival epithelium, a tissue exhibiting both Cl−secretion and Na+absorption, was examined. Bulbar-palpebral segments of conjunctiva were mounted between Ussing-type hemichambers under short-circuit conditions in Cl−free media. In this situation, the short-circuit current (Isc) measures an amiloride-resistant Na+absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 μm) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na+-dependent Iscby ≈3.5–4.5 μA cm−2(minimal 40% increase) and reduced transepithelial resistance (Rt) by at least 7% (P<0.05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 μm), which in itself inhibited the Iscby 0.5 μA cm−2, attenuated the Iscresponses of the cAMP-elevating agents (P< 0.05, unpaired data). In reverse, H-89 promptly decreased the Iscby 1.5–2.5 μA cm−2and increased Rtby 5% (P<0.05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX combination. Additions of epinephrine or rolipram to apically permeabilized preparations using amphotericin B, increased the Iscby 12 and 22% respectively over baseline and reduced Rtby 6% (P<0.05). Similarly, in the presence of a transepithelial K+gradient (apical to basolateral) and amphotericin B, cAMP elevation stimulated K diffusion across the preparation by at least 1.8 μA cm−2and decreased the Rtby 4% (P<0.05), changes that were reversed by subsequent H-89 addition. Under Cl−rich conditions, pretreatment with 5 m m Ba2+reduced the basal Iscby 59% and blocked the cAMP-induced Iscstimulations typically seen in the presence of the anion. The results provide evidence for a PKA-regulated, Ba2+-inhibitable (voltage insensitive) basolateral K+conductance in rabbit conjunctival epithelial cells. The action of Cl−secretogogues acting via cAMP on basolateral K+channel activity indicates that endogenous levels of cAMP may play a role in the regulation of Cl−secretion and Na+absorption in the conjunctiva.

Meeting future challenges in topical ocular drug delivery:: Development of an air-interfaced primary culture of rabbit conjunctival epithelial cells on a permeable support for drug transport studies

The purpose of this study was to develop and characterize a functional air-interfaced primary culture of rabbit conjunctival epithelial cells grown on a permeable support for drug transport studies. Conjunctival epithelial cells from the pigmented rabbit were isolated, seeded at 1.2×10 6 cells cm −2 on permeable Transwell filters, and cultured at the air interface using a modified PC-1 medium. Conjunctival epithelial cell layers showed a transepithelial resistance of 1.1±0.1 kΩ cm 2, a potential difference of 17.0±0.5 mV, and an equivalent short-circuit current ( I eq) of 16.1±0.4 μA cm −2. The I eq was reduced by 35% using 0.01 mM bumetanide, 66% using 0.1 mM ouabain, 46% using 2 mM barium chloride (all three in the basolateral fluid), and 63% using 0.3 mM NPAA in the apical fluid, consistent with active Cl −-secretion across the conjunctival epithelial barrier. Amiloride-sensitive Na + channels were absent. The permeability of the cell layers to polar solutes decreased with increased solute size, and the calculated equivalent pore size was about 8.0 nm. The Papp of β-blockers varied with lipophilicity in a sigmoidal fashion. Uridine transport showed temperature sensitivity and directionality, favoring transport in the apical-to-basolateral direction. Apical l-carnosine uptake was reduced by 46% in the absence of an inwardly directed proton gradient, and lowering the temperature to 4°C abolished direction-dependent l-carnosine uptake. Furthermore, uptake was inhibited by 73% using apical 10 mM glycyl sarcosine (a dipeptide transporter substrate) and by 60% using 1 mM l-valacyclovir (a dipeptide prodrug). In conclusion, a functional air-interfaced primary culture of rabbit conjunctival epithelial cell layers was established. This air-interfaced primary culture model may be useful for studying passive and active transport processes for ion and solute translocation in the mammalian conjunctival epithelial barrier in a defined experimental setting.

Role of P-glycoprotein in restricting propranolol transport in cultured rabbit conjunctival epithelial cell layers.

To determine the role of P-glycoprotein (P-gp) in propranolol transport in cultured rabbit conjunctival epithelial cell layers (RCEC). The localization of P-gp in the cultured RCEC as well as in the excised conjunctiva was determined by immunofluorescence technique. The role of P-gp in transepithelial transport and uptake of propranolol in conjunctival epithelial cells cultured on Transwell filters was evaluated in the presence and absence of P-gp competing substrates, an anti-P-gp monoclonal antibody (4E3 mAb), or a metabolic inhibitor, 2,4-dinitrophenol (2,4-DNP). Immunofluorescence studies revealed positive staining in the apical membrane of cultured RCEC and in the apical surface of the superficial cell layers in the excised conjunctiva, but not the basolateral membrane of cultured RCEC. Transport of propranolol showed preference in the basolateral-to-apical direction. The net secretory flux was saturable with a Km of 71.5 +/- 24.0 nM and a Jmax of 1.45 +/- 0.17 pmol/cm2/hr. Cyclosporin A, progesterone, rhodamine 123, verapamil, 4E3 mAb and 2,4-DNP all increased apical 50 nM propranolol uptake by 43% to 66%. On the other hand, neither beta-blockers (atenolol, metoprolol, and alprenolol) nor organic cation transporter substrates (tetraethylammonium (TEA) and guanidine), affected apical 50 nM propranolol uptake. The energy-dependent efflux pump P-gp appears to be predominantly located on the apical plasma membrane of the conjunctival epithelium. It may play an important role in restricting the conjunctival absorption of some lipophilic drugs.

Expression of nuclear retinoic acid receptor and retinoid X receptor mRNA in the cornea and conjunctiva

PURPOSE. The effects of retinoic acid in cells are mediated by the nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Although vitamin A is essential for the normal development and maintenance of the ocular surface, the RARs and RXRs have not been studied in cornea and conjunctiva. The purpose of this study was to identify the mRNA for these receptors in corneal and conjunctival cells in culture and to determine whether all- trans retinoic acid is able to induce expression of RAR mRNA. METHODS. Total RNA was extracted from cultured rabbit corneal stroma and conjunctival fibroblasts and rabbit corneal epithelial cells. RNA was analyzed by Northern blotting using the cDNA probes for RARa, RARß, RAR?, RXRa, RXRß and RXR? mRNA. To investigate induction of retinoid receptors, cells were exposed to 10 -6 M all- trans retinoic acid for 2–48 h before preparation of RNA. Effects of retinoic acid on cell proliferation were also investigated. RESULTS. RARa mRNA transcripts (3.7 kb), RARß mRNA transcripts (3.3 kb) and RAR? mRNA transcripts (3.3 kb) are expressed by all the cell types studied, as are the RXRa mRNA transcripts (5.0 kb) and RXRß mRNA transcripts (3.3 kb). RXR? mRNA is not detectable in corneal and conjunctival cells. All- trans retinoic acid induced RARß mRNA expression in corneal and conjunctival fibroblasts. Increased mRNA levels were detectable after 4–8 h and peaked by 24 h. RARß mRNA was not induced by retinoic acid in corneal epithelial cells. Retinoic acid also inhibited proliferation of conjunctival and corneal fibroblasts but had no effect on growth of corneal epithelial cells. CONCLUSIONS. The expression of RARs and RXRs in the cornea and conjunctiva is similar to that reported in other tissues. The identification of these receptors may lead to a better understanding of gene transcription pathways in the cornea and conjunctiva and of the mechanisms that control keratinization, differentiation and proliferation of the cells of these tissues. The data suggest a relationship between the induction of RARß mRNA expression and inhibition of cell proliferation by retinoic acid.

Co-cultivation of conjunctival epithelial cells and Chlamydia trachomatis: electron microscopic findings.

This study used primary culture of rabbit conjunctival epithelial cells to investigate the infection process of chlamydia. The epithelial cells isolated from conjunctiva of rabbit were initially cultured for three weeks. After attaining confluence they were infected with Chlamydia trachomatis (C. trachomatis) serotype D, and after co-cultivation for 24, 48, and 96 hours, electron microscopic study was performed. An inclusion body, a characteristic finding of chlamydial infection, was observed in the vicinity of the nucleus after 24 hours of co-cultivation. It contained a large number of elementary and reticulate bodies and their intermediate forms. Infectious particles known as elementary bodies were noted in the inclusion as 20 to 30 microns sized round bodies with an electron dense core. Reticulate bodies were also noted; they too were round but somewhat pleomorphic and larger than elementary bodies. Some reticulate bodies multiplied actively by means of binary fission. In this study, we observed the characteristic changes of C. trachomatis-infected cells; this in-vitro system might provide a suitable model for the study of some aspects of the pathogenesis of ocular chlamydia infection.

Collagen gel-embedding culture of conjunctival epithelial cells.

Collagen has effects on cell morphology, differentiation characteristics and function. Using collagen gel culture, several studies about cell differentiation were reported. In this study, the differentiation of rabbit conjunctival epithelial cells in a collagen gel-embedding culture system was investigated by electron microscope and lectin labeling. Rabbit bulbar conjunctival epithelial cells were cultured in type I collagen gel. After 1 and 2 weeks of culture, some of these cells were stained with PAS and seven kinds of lectins, and others were examined by transmission electron microscopy. The conjunctival epithelial cells cultured within collagen gel formed stratified cell layers and globules with cavities. The inner layer cells facing the cavities showed PAS and lectin staining patterns similar to those of conjunctival goblet cells in vivo, whereas the staining patterns of the outer layer cells on the collagen matrices resembled the patterns of non-goblet epithelial cells. Microvilli on the surface of the innermost cells, basement membranes beneath the outermost cells, tight junctions, adherent junctions, interdigitating folds and desmosomes between cells were identified on electron microscopic examination. These results indicate that cell junction structures of the conjunctival epithelial cells are well developed in collagen gel-embedding culture systems, and that the inner layer cells have carbohydrates similar to those of conjunctival goblet cells. Culture of conjunctival epithelial cells within collagen gel is a useful model for examining differentiation of these cells.

Conjunctival epithelial cells do not transdifferentiate in organotypic cultures: expression of K12 keratin is restricted to corneal epithelium.

The conjunctival epithelium is intrinsically different from the corneal epithelium in vivo, but sometimes can transform into an epithelium morphologically indistinguishable from the latter after healing of a total corneal epithelial defect. It remains unclear whether this morphologic transformation represents a process of extrinsic modultation or transdifferentiation of intrinsically divergent epithelium. In air-lifted organotypic cultures, rabbit conjunctival epithelial cells lost goblet cell differentiation and were stratified to the same extent as corneal epithelial cells, resembling the above in vivo morphologic transformation. Paired expression of K3 (64 kD) and K12 (55 kD) keratins has been regarded as a marker for corneal-type differentiation. Immunoblot analysis by monoclonal antibody AE5 revealed that K3 keratin was expressed by both submerged or air-lifted corneal and conjunctival cultures with or without 3T3 fibroblasts in collagen gel. In contrast, K12 keratin was expressed only by air-lifted corneal cultures with 3T3 fibroblasts using monoclonal antibody AK2 and two epitope-specific antibodies to N- and C- terminal oligopeptides deduced from the mouse K12 gene. This finding was also confirmed by Northern hybridization with a rabbit K12 cDNA probe. The expression of K12 keratin was more delayed than that of K3 keratin in air-lifted corneal cultures. This dissociated expression of these two keratins resembles that noted in vivo in the stem cell-containing limbal region. These results suggest that morphologic transformation of the conjunctival epithelium represents extrinsic environment modulation, and that differential expression of K12 but not K3 keratin signifies corneal epithelial differentiation.

Surfactant Cytotoxicity Potential Evaluated with Primary Cultures of Ocular Tissues: a Method for the Culture of Rabbit Conjunctival Epithelial Cells and Initial Cytotoxicity Studies

Rabbit conjunctiva, cornea, and iris have been used to evaluate and rank the ocular irritation potential of chemicals in the Draize eye irritancy test by subjective criteria. In order to rank the irritancy potential of chemicals objectively and biochemically, a primary culture method for rabbit conjunctival epithelial cells has been developed in our laboratory as a potential in vitro method for ocular toxicity testing of xenobiotics. Conjunctival epithelial cells were dispersed by Dispase II, followed by trypsin treatment. Cells were cultured in serum-free medium of 1:1 ratio of Dulbecco's modified Eagle's medium (DMEM) and F-12 nutrient mixture plus various concentrations of growth factors. At a plating density of 85,000 cells/cm2, cells grew to confluency in 3-4 days. Conjunctival cells showed a positive anti-keratin antibody stain which demonstrated their epithelial nature. These cells also showed positive periodic acid-Schiff (PAS) staining which is consistent with their goblet cell-containing and muc...