Changes in hydration, protein and proteoglycan composition of the collagen-keratocyte matrix of the bovine corneal stroma ex vivo in a bicarbonate-mixed salts solution, compared to other solutions

Many solutions have been used to investigate the swelling properties of the mammalian corneal stroma but few of the solutions resemble the expected extracellular matrix fluid of the corneal stroma, and little information is available on whether incubation ex vivo causes significant changes in the gross composition of the stroma. From quality-selected recent post-mortem eyes of adult cattle, stroma preparations were cut from the central part of the cornea. The time-dependent changes in wet mass were assessed over 9 h at 37 degrees C, and the preparations then dried. Various solutions of known pH (6.88-8.32) and osmolality (<50-327 mosmol/kg) were used, and were assayed for protein and proteoglycan after the incubation. The rates and extent of stromal swelling were lowest in a glucose-supplemented mixed salts solution containing 35 mM bicarbonate (0.5% CO2) solution, marginally greater in a mixed salts solution containing 35 mM bicarbonate (5% CO2) or similar non-bicarbonate mixed salts solutions (including BSS), and progressively greater in phosphate-buffered saline (PBS), various phosphate buffers (10-67 mM) and saline solutions (0.025-1%), and greatest in water. The initial rates of swelling ranged from 44 to 451 mg/h and the secondary rates from 9 to 106 mg/h. In all solutions, protein and proteoglycans were detected, but these ranged from around 1 to 10% of the samples with the bicarbonate-buffered solutions, to around 30% with the use of some phosphate buffers or saline.     

Changes in hydration,protein and proteoglycan composition of the collagen–keratocyte matrix of the bovine corneal stroma ex vivo in a bicarbonate–mixed salts solution,compared to other solutions

Many solutions have been used to investigate the swelling properties of the mammalian corneal stroma but few of the solutions resemble the expected extracellular matrix fluid of the corneal stroma, and little information is available on whether incubation ex vivo causes significant changes in the gross composition of the stroma. From quality-selected recent post-mortem eyes of adult cattle, stroma preparations were cut from the central part of the cornea. The time-dependent changes in wet mass were assessed over 9 h at 37°C, and the preparations then dried. Various solutions of known pH (6.88–8.32) and osmolality (<50–327 mosmol/kg) were used, and were assayed for protein and proteoglycan after the incubation. The rates and extent of stromal swelling were lowest in a glucose-supplemented mixed salts solution containing 35 mM bicarbonate (0.5% CO₂) solution, marginally greater in a mixed salts solution containing 35 mM bicarbonate (5% CO₂) or similar non-bicarbonate mixed salts solutions (including BSS), and progressively greater in phosphate-buffered saline (PBS), various phosphate buffers (10–67 mM) and saline solutions (0.025–1%), and greatest in water. The initial rates of swelling ranged from 44 to 451 mg/h and the secondary rates from 9 to 106 mg/h. In all solutions, protein and proteoglycans were detected, but these ranged from around 1 to 10% of the samples with the bicarbonate-buffered solutions, to around 30% with the use of some phosphate buffers or saline.     

Impact of hypotonic solutions on the stromal swelling, lactate dehydrogenase and aldehyde dehydrogenase activity of the keratocytes of the bovine cornea

The present studies were designed to explore the relationship between the swelling-related changes of the collagen-cell (keratocyte) matrix of the corneal stroma, and the integrity of the cells. From recent postmortem eyes of adult cattle, complete stroma preparations were dissected out and allowed to swell in solution (free swelling) or enclosed within a 12 kDa cut-off dialysis membrane with or without spacers. The swelling was at 4 degrees C with either water, a hypotonic phosphate-buffered saline (PBS, pH 7.0), a hypotonic mixed salt (MS) solution (pH 7.5), or an isotonic mixed salt solution with glucose (pH 7.5). Measures of tissue wet mass and thickness and analyses of the soluble protein, LDH and ALDH activity in the solutions were made. The relative swelling of the stroma preparations was greatest in water (to 624% of the original wet mass) > dilute PBS (to 404%) > dilute MS (to 381%) > MS with glucose (to 356%). The relative swelling was in the same order, but slightly less if the stroma preparations were enclosed in a dialysis tube with spacers, and substantially reduced when enclosed in a dialysis bag without spacers. With the use of hypotonic solutions, substantial quantities of proteinaceous material and enzyme activity were lost from the preparations, with the loss being proportional to the extent of swelling (p < 0.001). Swelling of an isolated corneal stroma, especially in hypotonic solutions, is associated with substantial loss of soluble protein and cytoplasmic enzyme activities, and so these solutions must be considered as cytotoxic to the keratocytes.     

Swelling of the collagen-keratocyte matrix of the bovine corneal stroma ex vivo in various solutions and its relationship to tissue thickness

AIM: The mammalian corneal stroma, like some other connective tissues, can absorb fluid, swell and become oedematous. Since studies on the corneal stroma have been carried out with different types of preparations and solutions, inter-study comparisons are very difficult. A study was thus undertaken on a standardised preparation to assess the relative magnitude of this swelling and its relationship to thickness of the preparations. METHODS: From selected recent post-mortem eyes of adult cattle, stroma preparations were cut from the central part of the cornea. These preparations were immersed in various solutions of known pH and osmolality, and the time-dependent changes in wet mass were assessed over 9 h at 37 degrees C. The relative rates and magnitude of the swelling of the tissue were then compared. RESULTS: A reference value for stromal swelling was obtained by incubation in a 35 mM bicarbonate-buffered mixed salts solution equilibrated with 5% CO2-air (pH 7.60) where a 3.39-fold increase in wet mass and a 4.58-fold increase in thickness was realised in 9 h, at an initial rate of 76 +/- 3%/h. The swelling was essentially the same in an organic buffer-mixed salt solution (pH 7.5) but progressively greater in phosphate-buffered saline (pH 7.5), a range of phosphate buffers (10-67 mM, pH 7.5), NaCl solutions (0.025-1%) and with gross swelling observed in water (where a 15.9-fold increase in wet mass occurred along with a 25-fold increase in thickness, at an initial rate of 643 +/- 62%/h). Overall, the wet mass changes were strongly related to thickness (P < 0.001). CONCLUSIONS: The results confirm that the selection of solution(s) for studies on corneal stromal swelling is critical. The swelling (oedema) is lower in a physiologically-relevant solution (similar to the aqueous humour of the eye). This indicates that the swelling tendency of the corneal stroma has been overestimated in the past, and that a similar discrepancy may also exist for studies on other connective tissues ex vivo when non-physiological experimental solutions are used.     

Re-assessment of the potential impact of physiologically relevant pH changes on the hydration properties of the isolated mammalian corneal stroma

The pH sensitivity of the swelling of the mammalian corneal stroma was reinvestigated to assess whether or not there were detectable differences in the hydration properties of this collagen-keratocyte matrix within a physiologically relevant range (as opposed to extremes of acid or alkaline pH) and at a physiologically relevant temperature. From recent post-mortem eyes of adult cows, square (8 x 8 mm) samples of corneal stroma were prepared and incubated in an isotonic, buffered (HEPES etc.), mixed salts solution with added glucose at 37 degrees C. The time-dependent changes in wet mass were assessed over 24 h. The rate and magnitude of stromal swelling were different within the range of pH 6.5-8.5. The wet mass of stromal samples increased almost 2-fold within 1 h, and then at lesser rates to realise 3.25-3.75-fold and 4-5-fold increases in wet mass by 9 h and 24 h respectively. The maximum increases were observed at pH 7.25-7.5, with most of the effect being the result of differences in the initial rate of swelling. The discontinuous swelling and the pH effect on the rates of swelling were also evident when the data were fitted to a previous kinetic model (Elliott et al., J. Physiol. (Lond.) 298 (1980) 453-470). It is concluded that pH changes in the physiological range can have a small but reproducible impact on the swelling kinetics of the isolated mammalian corneal stroma ex vivo.     

Benzalkonium Chloride Accelerates the Formation of the Amyloid Fibrils of Corneal Dystrophy-associated Peptides

Corneal dystrophies are genetic disorders resulting in progressive corneal clouding due to the deposition of amyloid fibrils derived from keratoepithelin, also called transforming growth factor β-induced protein (TGFBI). The formation of amyloid fibrils is often accelerated by surfactants such as sodium dodecyl sulfate (SDS). Most eye drops contain benzalkonium chloride (BAC), a cationic surfactant, as a preservative substance. In the present study, we aimed to reveal the role of BAC in the amyloid fibrillation of keratoepithelin-derived peptides in vitro. We used three types of 22-residue synthetic peptides covering Leu110-Glu131 of the keratoepithelin sequence: an R-type peptide with wild-type R124, a C-type peptide with C124 associated with lattice corneal dystrophy type I, and a H-type peptide with H124 associated with granular corneal dystrophy type II. The time courses of spontaneous amyloid fibrillation and seed-dependent fibril elongation were monitored in the presence of various concentrations of BAC or SDS using thioflavin T fluorescence. BAC and SDS accelerated the fibrillation of all synthetic peptides in the absence and presence of seeds. Optimal acceleration occurred near the CMC, which suggests that the unstable and dynamic interactions of keratoepithelin peptides with amphipathic surfactants led to the formation of fibrils. These results suggest that eye drops containing BAC may deteriorate corneal dystrophies and that those without BAC are preferred especially for patients with corneal dystrophies.     

The effect of hydration of the rabbit cornea on the histochemical demonstration of acid mucopolysaccharides

Summary The influence of hydration on the histochemical staining of acid mucopolysaccharides (AMPS) in the rabbit cornea was studied. The rabbit cornea was immersed in distilled water for 4, 8, 12 and 16 hour intervals, and the water content of the hydrated stroma was investigated. Simultaneously the hexosamine content was examined. For the histochemical identification of AMPS the corneae were fixed with formol-calcium chloride and embedded in paraffin.Various degrees of the corneal hydration were without influence on the content of AMPS determined by biochemical methods. On the other hand the results of all histochemical staining reactions for AMPS were extremely dependent on the state of hydration. The quantitative evaluation of the content of AMPS in the corneal stroma on the basis of staining reactions is thus impossible.     

Neutron and X-ray scattering by ox corneal stroma differentially loaded with bound anions

Ox corneas at near physiological hydration were subjected to two variables: the amount of chloride ions bound to them and exposure of various mixtures of H(2)O/D(2)O as solvent. The preparations were then exposed to a neutron beam and the contrast match points, at which the collagen fibrils of the corneal stroma most nearly matched the scattering density of the various H(2)O/D(2)O mixtures, were measured. In both cases of high and low bound chloride, the contrast match points of the collagen fibril were equal, indicating that there were no significant changes in the water of electrostriction at the fibril surface when chloride ions bind to the stroma. The data suggest that the ligands which bind anions to corneal stroma are not located at the collagen fibril surface. When the chloride binding ligands were extracted from the corneal stroma there were significant changes in the structure of the fibrils. We suggest that the chloride binding ligands may be located within the collagen fibril.     

Two different mechanisms are involved in the extremely high-level benzalkonium chloride resistance of a Pseudomonas fluorescens strain

A Pseudomonas fluorescens strain, PFRB, which we previously isolated as a contaminant in a batch of benzalkonium chloride (BAC) stock solution, exhibits high-level resistance, not only to BAC, but also to other cationic surfactants belonging to disinfectants classified as quaternary ammonium compounds (QACs). In this study, we analyzed the resistance mechanism of the strain to BAC and other disinfectants. We obtained results suggesting that two different mechanisms, reduced adsorption of BAC to the cell surface and an energy-dependent mechanism which is most probably an efflux system, were implicated in the high-level resistance to BAC. Reduced adsorption of BAC is likely due to the decreased negative cell surface charge of the strain. The putative efflux system seems to be unique in that it excretes only a certain range of cationic membrane-acting disinfectants belonging to QACs.     

Recovery and purification of thuringiensin from the fermentation broth of Bacillus thuringiensis

Thuringiensin is a heat stable -exotoxin from Bacillus thuringiensis with a great potential for replacing the traditional chemical pesticides. A process using micellar-enhanced ultrafitration method to recover thuringiensin was significantly improved by the use of a spiral-wound membrane, which could be operated at a low transmembrane pressure drop. This method was performed by adding a surfactant cetylpyridinium chloride (CPC) into the fermentation broth. After the surfactant-thuringiensin conjugates were formed, the broth then passed through the ultrafiltration membrane and the retentate was collected. The results indicated the optimal concentration of CPC for producing a maximal recovery up to 99.3% is 4%. For purification, the centrifuged broth was further filtered by a membrane filter. The filtered solution then was mixed with 50% of activated carbon. The supernatant then was injected into a preparative HPLC. The eluate was collected during thuringiensin peak formation. This eluate was then concentrated by vacuum evaporation and dialysis using an electrodialyzer to remove the excess salts. The dialyzed solution was then crystallized by lyophilization. The purity of the thuringiensin crystal was identified by HPLC, capillary electrophoresis, and mass spectrometry.This revised version was published online in October 2005 with corrections to the Cover Date.     

Control of corneal differentiation by extracellular materials. Collagen as a promoter and stabilizer of epithelial stroma production

The primary stroma of the cornea of the chick embryo consists of orthogonally arranged collagen fibrils embedded in glycosaminoglycan (GAG) produced by the epithelium under the early inductive influence of the lens. The experiments reported here were designed to test whether or not the collagen of the lens basement lamina is capable of stimulating corneal epithelium to produce primary stroma. Enzymatically isolated 5-day-old corneal epithelia were grown for 24 hr in vitro in the presence of ³⁵SO₄ or proline-³H on various substrata. Epithelia cultured on lens capsule synthesized 2.5 times as much GAG (as measured by incorporation of label into CPC precipitable material) and almost 3 times as much collagen (assayed by hot TCA extraction or collagenase sensitivity) as when cultured on Millipore filter or other noncollagenous substrata. A similar stimulatory response was observed when epithelium was combined with chemically pure chondrosarcoma collagen, NaOH-extracted lens capsule, vitreous humor, frozen-killed corneal stroma or cartilage, or tendon collagen gels; in the latter case, the magnitude of the effect can be shown to be related to concentration of the collagen in the gel. All of the collagenous substrata stimulate not only extracellular matrix production, but also polymerization of corneal-type matrix, as judged by ultrastructural criteria and by the association of more radioactivity with the tissue than the medium. Since purified chondrosarcoma collagen is as effective as lens capsule, the stimulatory effect on collagen and GAG synthesis by corneal epithelium is not specific for basal lamina (lens capsule) collagen.     

On the influence of fixations on the normal hydration of rabbit cornea and the total amount of acid mucopolysaccharides in the stroma

Summary The authors studied the influence of fixations on the normal hydration of the rabbit cornea and the total amount of acid mucopolysaccharides (AMPS) in the stroma. The following fixatives were used: formol-calcium chloride at 19° C for 24 hours, formolcetylpyridinium chloride (CPC) at 19 and 28° C for 48 hours, Lillie's fixation at 19° C for 24 hours and Carnoy's fluid at 19° C for 30 minutes. The sections of the corneae were stained with Alcian blue, colloidal Fe³⁺ in the modification according to Rinehart and Abu'l Haj and with toluidine and methylene blue. The amount of AMPS was determined with the method of Rondle and Morgan and the total hydration of the stroma by weighing the corneae before and after using different fixative fluids and by calculation of obtained values on dry weight.The best results were obtained by using formol-CPC at 28° C. At the ordinary room temperature (±19° C) it was the poorest fixation, however, as the corneae in this solution became hydrated. Formol-calcium chloride was the second in the row and it was much better than Lillie's and Carnoy's fluid.The amount of AMPS in the stroma was not essentially changed by the effect of fixatives. Within 24–48 hours formol-CPC at 28° C retained the normal content and formol-calcium chloride caused the 11% decrease of AMPS maximally. The loss of AMPS after other fixatives was minimal.The intensity of staining with cationic dyes in paraffin sections was different after individual fixatives and after the kind of their application and was dependent chiefly on the state of hydration of the corneal stroma: It is impossible to interpret the results of staining reactions in terms of the quantity of AMPS as it was hitherto done.     

A simple method for preserving environmental DNA in water samples at ambient temperature by addition of cationic surfactant

Environmental DNA (eDNA) analysis is a powerful tool within ecology for the study of the distribution or abundance of aquatic species, although the simplification of water sampling is required for enabling light and fast field sampling to expand further application of eDNA analysis. Here, certain candidate chemicals belonging to the group of cationic surfactants were examined for their effectiveness as preservatives for eDNA water samples by simply adding the chemicals to water samples to suppress the degradation of eDNA. The quaternary ammonium compound benzalkonium chloride (BAC) at a final concentration of 0.01% was effective to retain 92% of eDNA derived from the bluegill sunfish Lepomis macrochirus in an 8-h incubation test at ambient temperature, which assumed a transportation of water samples in 1-day field sampling during the daytime. Meanwhile, eDNA in water samples without BAC retained only 14% of the initial eDNA. Moreover, an additional long-term incubation test (up to 10 days) revealed BAC-treated samples retained ~70 and 50% of bluegill DNA compared to the initial amount after 1- and 10-day incubation at ambient temperature, respectively. Meanwhile, eDNA in naïve samples reduced to 20% after 1-day incubation and reached undetectable levels after 10 days. Up to now, many eDNA studies have adopted on-site filtration followed by filter fixation, which requires many pieces of equipment. Addition of BAC can protect eDNA in water samples with less effort and equipment resulting in an increase of measurement accuracy of the eDNA quantity and detection probability of rare species by preventing the disappearance of rare sequences in water samples.     

Size-Dependent Diffusion of Dextrans in Excised Porcine Corneal Stroma

Delivery of therapeutic agents to the eye requires efficient transport through cellular and extracellular barriers. We evaluated the rate of diffusive transport in excised porcine corneal stroma using fluorescently labeled dextran molecules with hydrodynamic radii ranging from 1.3 to 34 nm. Fluorescence correlation spectroscopy (FCS) was used to measure diffusion coefficients of dextran molecules in the excised porcine corneal stroma. The preferential sensitivity of FCS to diffusion along two dimensions was used to differentially probe diffusion along the directions parallel to and perpendicular to the collagen lamellae of the corneal stroma. In order to develop an understanding of how size affects diffusion in cornea, diffusion coefficients in cornea were compared to diffusion coefficients measured in a simple buffer solution. Dextran molecules diffuse more slowly in cornea as compared to buffer solution. The reduction in diffusion coefficient is modest however (67% smaller), and is uniform over the range of sizes that we measured. This indicates that, for dextrans in the 1.3 to 34 nm range, the diffusion landscape of corneal stroma can be represented as a simple liquid with a viscosity approximately 1.5 times that of water. Diffusion coefficients measured parallel vs. perpendicular to the collagen lamellae were indistinguishable. This indicates that diffusion in the corneal stroma is not highly anisotropic. Our results support the notion that the corneal stroma is highly permeable and isotropic to transport of hydrophilic molecules and particles with hydrodynamic radii up to at least 34 nm.     

Aquaporin deletion in mice reduces corneal water permeability and delays restoration of transparency after swelling

Two aquaporin (AQP)-type water channels are expressed in mammalian cornea, AQP1 in endothelial cells and AQP5 in epithelial cells. To test whether these aquaporins are involved in corneal fluid transport and transparency, we compared corneal thickness, water permeability, and response to experimental swelling in wild type mice and transgenic null mice lacking AQP1 and AQP5. Corneal thickness in fixed sections was remarkably reduced in AQP1 null mice and increased in AQP5 null mice. By z-scanning confocal microscopy, corneal thickness in vivo was (in microm, mean +/- S.E., n = 5 mice) 123 +/- 1 (wild type), 101 +/- 2 (AQP1 null), and 144 +/- 2 (AQP5 null). After exposure of the external corneal surface to hypotonic saline (100 mosm), the rate of corneal swelling (5.0 +/- 0.3 microm/min, wild type) was reduced by AQP5 deletion (2.7 +/- 0.1 microm/min). After exposure of the endothelial surface to hypotonic saline by anterior chamber perfusion, the rate of corneal swelling (7.1 +/- 1.0 microm/min, wild type) was reduced by AQP1 deletion (1.6 +/- 0.4 microm/min). Base-line corneal transparency was not impaired by AQP1 or AQP5 deletion. However, the recovery of corneal transparency and thickness after hypotonic swelling (10-min exposure of corneal surface to hypotonic saline) was remarkably delayed in AQP1 null mice with approximately 75% recovery at 7 min in wild type mice compared with 5% recovery in AQP1 null mice. Our data indicate that AQP1 and AQP5 provide the principal routes for corneal water transport across the endothelial and epithelial barriers, respectively. The impaired recovery of corneal transparency in AQP1 null mice provides evidence for the involvement of AQP1 in active extrusion of fluid from the corneal stroma across the corneal endothelium. The up-regulation of AQP1 expression and/or function in corneal endothelium may reduce corneal swelling and opacification following injury.     

Genetics of corneal endothelial dystrophies

The corneal endothelium maintains the level of hydration in the cornea. Dysfunction of the endothelium results in excess accumulation of water in the corneal stroma, leading to swelling of the stroma and loss of transparency. There are four different corneal endothelial dystrophies that are hereditary, progressive, non-inflammatory disorders involving dysfunction of the corneal endothelium. Each of the endothelial dystrophies is genetically heterogeneous with different modes of transmission and/or different genes involved in each subtype. Genes responsible for disease have been identified for only a subset of corneal endothelial dystrophies. Knowledge of genes involved and their function in the corneal endothelium can aid understanding the pathogenesis of the disorder as well as reveal pathways that are important for normal functioning of the endothelium.     

Mixed micelles and other structures in the solubilization of bilayer lipid membranes by surfactants

The solubilization of lipid bilayers by surfactants is accompanied by morphological changes of the bilayer and the emergence of mixed micelles. From a phase equilibrium perspective, the lipid/surfactant/water system is in a two-phase area during the solubilization: a phase containing mixed micelles is in equilibrium with bilayer structures of the lamellar phase. In some cases three phases are present, the single micelle phase replaced by a concentrated and a dilute solution phase. In the case of non-ionic surfactants, the lipid bilayers reach saturation when mixed micelles, often flexible rod-like or thread-like, start to form in the aqueous solution, at a constant chemical potential of the surfactant. The composition of the bilayers also remains fixed during the dissolution. The phase behavior encountered with many charged surfactants is different. The lamellar phase becomes destabilized at a certain content of surfactant in the membrane, and then disintegrates, forming mixed micelles, or a hexagonal phase, or an intermediate phase. Defective bilayer intermediates, such as perforated vesicles, have been found in several systems, mainly with charged surfactants. The perforated membranes, in some systems, go over into thread-like micelles via lace-like structures, often without a clear two-phase region. Intermediates in the form of disks, either micelles or bilayer fragments, have been observed in several cases. Most noteworthy are the planar and circular disks found in systems containing a large fraction of cholesterol in the bilayer. Bile salts are a special class of surfactants that seem to break down the bilayer at low additions. Originally, disk-like mixed micelles were conjectured, with polar membrane lipids building the disk, and the bile salts covering the hydrophobic rim. Later work has shown that flexible cylinders are the dominant intermediates also in these systems, even if the disk-like structures have been re-established as transients in the transformation from mixed micelles to vesicles.     

Corneal alternations induced by topical application of benzalkonium chloride in rabbit

Benzalkonium chloride (BAC) is the most common preservative in ophthalmic preparations. Here, we investigated the corneal alternations in rabbits following exposure to BAC. Twenty-four adult male New Zealand albino rabbits were randomly divided into three groups. BAC at 0.01%, 0.05%, or 0.1% was applied twice daily to one eye each of rabbits for 4 days. The contralateral untreated eyes were used as control. Aqueous tear production and fluorescein staining scores of BAC-treated eyes were compared with those of controls. The structure of the central cornea was examined by in vivo confocal microscopy. Expression of mucin-5 subtype AC (MUC5AC) in conjunctiva was detected by immunostainig on cryosections. Corneal barrier function was assessed in terms of permeability to carboxy fluorescein (CF). The distribution and expression of ZO-1, a known marker of tight junction, and reorganization of the perijunctional actomyosin ring (PAMR) were examined by immunofluorescence analysis. Although there were no significant differences between control and BAC-treated eyes in Schirmer scores, corneal fluorescein scores and the number of conjunctival MUC5AC staining cells, in vivo confocal microscopy revealed significant epithelial and stromal defects in all BAC-treated corneas. Moreover, BAC at 0.1% resulted in significant increases in central corneal thickness and endothelial CF permeability, compared with those in control eyes, and endothelial cell damage with dislocation of ZO-1 and disruption of PAMR. Topical application of BAC can quickly impair the whole cornea without occurrence of dry eye. A high concentration of BAC breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR and remodeling of apical junctional complex in vivo.