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.     

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.     

Assessment of the effects of cetylpyridium chloride on water content of the collagen-keratocyte matrix of the mammalian corneal stroma ex vivo

The effects of cationic surfactants on the time-dependent increases in hydration of the corneal stroma were investigated to assess if the contribution of the proteoglycans could be titrated and how it might relate to the maximum and minimum swelling properties of the corneal stroma. From recent post-mortem eyes from adult sheep, square (8 x 8 mm) samples of corneal stroma were prepared and incubated in isotonic neutral pH mixed salts solution with added glucose, or pure water, at 37 degrees C. The time-dependent changes in wet mass were assessed over 24 h in the absence or presence of 0. 001-2% w/v cetylpyridium chloride (CPC) or benzalkonium chloride (BAC). The rate and magnitude of stromal swelling was reduced in a concentration-dependent fashion by the surfactants. In mixed salts solution, 100% inhibition of swelling could be achieved at 2% CPC and BAC. In pure water, the relative swelling was much more substantial and could only be attenuated by CPC.     

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.     

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.     

Acetylcholinesterase activity as an indicator of the content of stroma in infusable hemoglobin solutions in relation to the manner of their preparation

Acetylcholinesterase as an integral membrane part is a suitable indicator for characterizing the stroma in solutions of haemoglobin. The erythrocyte stroma cannot be fully eliminated because of the procedures of preparations for infundable haemoglobin solutions. The most favourable conditions with a stroma rest content of approximately 1% are presented by hypotonic haemolysis.     

Osmotic Properties of Amphibian Muscles

Changes in the volume of fiber water in hypotonic and hypertonic Ringer's solution were determined for the sartorius, stomach, and cardiac muscle of the frog using two methods. Loss of water in hypertonic solutions was nearly the same in all muscles, but swelling in hypotonic solutions was greatest in the sartorius, smallest in the heart. For the sartorius the deviation from the properties of an osmometer can be accounted for by a loss of electrolyte and by assuming that a small part of the fiber water is bound, but this appears insufficient to explain the behavior of stomach and cardiac muscle in hypotonic solutions. In very dilute solutions of CaCl₂ and MgCl₂ a large difference in concentration of electrolytes is maintained between the fibers and the medium. Under these conditions divalent cations, accumulating in the fibers, produce a change in physical properties which indicates increased internal cross-linking. It is suggested, therefore, that swelling is limited as in a gel and that a considerable hydrostatic pressure may develop within the fibers.     

Heat aggregation studies of phycobilisomes, ferritin, insulin, and immunoglobulin by dynamic light scattering.

Dynamic laser light scattering studies on the heat aggregation behavior of phycobilisomes (PBS), ferritin, insulin, and immunoglobulin (IgG) in dilute aqueous solutions has been reported. Except for PBS, results are reported for heat aggregation trends in these proteins for three different pH environments (4.0, 7.5, 9.1). For PBS, studies were performed only in the neutral buffer medium (pH 7.5). The experiments were performed in the very dilute concentration regime (between 0.23 and 1.8 gL-1). For all these samples heat aggregation and dissociation trends were found to be linear with temperature. Upon temperature reversal (self-cooling), hysteresis-like behavior observed in insulin was found to be predominantly large at pH 7.5. PBS, ferritin, and IgG showed no such behavior at any of three pH values, and retraced their path of aggregation while dissociating on temperature reversal. Heat aggregation and dissociation processes in ferritin were found to be independent of pH. The IgG samples showed smooth aggregation tendency only up to 35 degrees C in the buffer media pH 4.0 and 9.1, whereas for pH 7.0 the same could be observed until 60 degrees C. Low polydispersity in the correlation spectra was observed in case of all these samples.     

An Apparatus for the Concentration of Large Volumes of Dilute Protein Solutions to A Predetermined Volume

An apparatus was designed and manufactured that can be used for the concentration of large quantities of dilute protein solutions to a predetermined volume. The method is based on the osmotic transfer of water to a concentrated hydrophilic polymer (poly ethylene glycol, PEG) through a protein-stopping dialysis membrane and is a refinement of a method previously reported by van Oss. The apparatus is made of perspex. Concentration takes place through a commercially available dialysis tube and is aided by a 40% polyethylene glycol solution. Large volumes (5) of dilute protein solution could be reduced to 50 ml at a rate of 30 ml per hour with no significant loss in biological activity.     

An apparatus for the concentration of large volumes of dilute protein solutions to a predetermined volume

An apparatus was designed and manufactured that can be used for the concentration of large quantities of dilute protein solutions to a predetermined volume. The method is based on the osmotic transfer of water to a concentrated hydrophilic polymer (poly ethylene glycol, PEG) through a protein-stopping dialysis membrane and is a refinement of a method previously reported by van Oss. The apparatus is made of perspex. Concentration takes place through a commercially available dialysis tube and is aided by a 40% polyethylene glycol solution. Large volumes (5l) of dilute protein solution could be reduced to 50 ml at a rate of 30 ml per hour with no significant loss in biological activity.     

Neutralization of diphtheria toxin in living cells by microinjection of antifragment A contained within resealed erythrocyte ghosts

When human erythrocytes suspended in phosphate-buffered saline (PBS) containing lgG were first dialyzed against a hypotonic solution and then dialyzed against PBS, lgG molecules were entrapped within resealed erythrocyte ghosts. The concentration of lgG inside the ghosts was about 33% of its concentration in the dialysis bag. With the aid of HVJ (Sendai virus), ghosts containing rabbit lgG antibody against fragment A of diphtheria toxin were fused with toxin-sensitive FL cells. The fused FL recipients were found to be resistant to the action of diphtheria toxin. Clones derived from the resistant recipient cells, however, became sensitive to the toxin again. Antifragment A neutralized the enzymic activity of isolated fragment A in vitro, but did not protect FL cells or rabbit skin against the complete toxin.     

The Erythrocyte Ghost Is a Perfect Osmometer

The osmotic swelling of intact erythrocytes in hypotonic solutions was measured using microhematocrit tubes, Van Allen tubes, and a calibrated Coulter counter. In agreement with earlier workers the intact cells did not behave as perfect osmometers, the cells swelling less than predicted by the Boyle-van't Hoff law. Erythrocyte ghosts were prepared from fresh intact erythrocytes by one-step hemolysis in 0.25% NaCl at an extremely dilute concentration of cells and the membranes were sealed at 37°. The ghosts were mixed with NaCl solutions of different osmolarities and the MCV (mean cell volume) of the shrunken cells immediately monitored by a calibrated Coulter counter. It was found that the MCV values of the shrunken ghosts were accurately predicted by the Boyle-van't Hoff law. These results indicate that these erythrocyte ghosts behaved as perfect osmometers.     

Kir4.1 channels regulate swelling of astroglial processes in experimental spinal cord edema

In glial cells, inwardly rectifying K⁺ channels (Kir) control extracellular [K⁺]_o homeostasis by uptake of K⁺ from the extracellular space and release of K⁺ into the microvasculature. Kir channels were also recently implicated in K⁺-associated water influx and cell swelling. We studied the time-dependent expression and functional implication of the glial Kir4.1 channel for astroglial swelling in a spinal cord edema model. In this CNS region, Kir4.1 is expressed on astrocytes from the second postnatal week on and co-localizes with aquaporin 4 (AQP4). Swelling of individual astrocytes in response to osmotic stress and to pharmacological Kir blockade were analyzed by time-lapse-two-photon laser-scanning microscopy in situ . Application of 30% hypotonic solution induced astroglial soma swelling whereas no swelling was observed on astroglial processes or endfeet. Co-application of hypotonic solution and Ba²⁺, a Kir channel blocker, induced prominent swelling of astroglial processes. In Kir4.1−/− mice, however, somatic as well as process swelling was observed upon application of 30% hypotonic solutions. No additional effect was provoked upon co-application with Ba²⁺. Our experiments show that Kir channels prevent glial process swelling under osmotic stress. The underlying Kir channel subunit that controls glial process swelling is Kir4.1, whereas changes of the glial soma are not substantially related to Kir4.1.     

Role of potassium channels, the sodium-potassium pump and the cytoskeleton in the control of dog sperm volume

Response to osmotic shock is an important aspect of mammalian sperm physiology. In this study we recorded volume changes of dog spermatozoa at 39, 33, and 25 degrees C under isotonic conditions and following hypotonic shock. Cell volume measurements were performed electronically in saline solutions of 300 and 150 mOsmol kg(-1), and Percoll-washed preparations were compared with unwashed samples. The involvement of potassium channels in volume control was tested by treatment with quinine, while the involvement of the plasma membrane Na(+)-K+ pump was tested by treatment with ouabain. The role of the cytoskeleton was investigated by treatment with colchicine and cytochalasin D. The number of cell populations observed varied with temperature and tonicity. In both types of sperm preparations, between two and three populations were present under isotonic conditions at 25 degrees C whereas at 39 and 33 degrees C only one population was detected. Hypotonic stress at the higher temperatures caused the single population to swell, whereas at 25 degrees C it resulted in a population of cells whose modal volume was similar to that of the middle isotonic sub-population. Both quinine and the cytoskeletal inhibitors markedly increased swelling both under hypotonic conditions at 39 degrees C and under isotonic conditions at 25 degrees C. However, little or no effect of ouabain was observed. We conclude that in dog spermatozoa swelling in response to hypotonic conditions is minimised through the activity of potassium channels and the presence of an intact cytoskeletal network. Under isotonic conditions at 25 degrees C, a considerable proportion of the sperm population is already swollen; this swelling varies between individual males and appears to be due to lowered cytoskeletal and potassium channel activity.     

A model of epithelial water transport. The corneal endothelium.

To try to understand how an epithelial tissue can transport water between bathing solutions of equal tonicity and how intracellular solute and protein concentration are related to the structural specialization of the cell membrane at its apical, basal, and lateral margins, we have formulated and solved, using approximate analytical techniques, a new model which combines the detailed transport of local osmotic flow in extracellular channel with the multicompartment approach of thermodynamic models requiring the overall conservation of water and solute for the entire cell layer. Thus, unlike most previous models, which dealt exclusively with either the average properties of the cell layer or the local transport in the extracellular channel, we are able to solve simultaneously for the interaction of the cell with its environments across its apical, basal, and lateral cell membranes as well as the detailed transport in the extracellular channel. The model is then applied to corneal endothelium to obtain new insight into the water flow movement in this tissue under in vitro and in vivo conditions. Then in vitro solution shows that the cell at 297 mosmol/liter is slightly hypotonic to the 300-mosmol/liter external bathing solutions which drive water equally out both the aqueous (apical) and stromal (basal) cell faces. This water is replaced from the extracellular channel. There is a net flow of water because more water enters the channel through its open stromal end than through the higher resistance tight junction. In vivo, the solution predicts that the stromal swelling pressure forces water through the tight junctions towards the stroma so that there is no net flow. The interesting new features of our solution are the water recirculation pattern and the role of the osmotically active proteins in making the cell hypertonic relative to the channel.     

The protective role of topical propolis on experimental keratitis via nitric oxide levels in rabbits

The aim of this study was to investigate antioxidant, anti-inflammatory, and antibacterial properties of propolis in the treatment of experimental Staphylococcus aureus keratitis. Twenty young New Zealand white rabbits were used in this experiment. Staphylococcus aureus were given by intrastromal injection to 16 rabbits and 4 rabbits were used as control group (Group 1). Group 2 was treated with phosphate-buffered solution drops; Group 3 was administered ethanolic extract of propolis drops; Group 4 received topical ciprofloxacin drops; Group 5 was treated with topical ciprofloxacin drops along with ethanolic extract of propolis drops. The eyes were examined by slit lamp to assess corneal opacity. And then, corneas were removed to determine nitric oxide (NO) levels and count bacteria. Corneas were also evaluated histologically. Corneal NO concentration in gruop 5, treated with a combination of propolis and ciprofloxacin was determined significantly lower (10.0± 1.8 μmol/g wet tissue) than in Group 4, treated with ciprofloxacin (24.0± 3.1 μmol/g wet tissue), from Group 3, treated with propolis (15.6± 1.8 μmol/g wet tissue), and treated with PBS (44.7± 7.8 μmol/g wet tissue). There were significantly fewer bacteria in eyes that received propolis plus ciprofloxacin than in eyes treated with ciprofloxacin (p = 0.0001) or propolis (p = 0.0001) or eyes treated with PBS (p = 0.0001). The light microscopic examination revealed that the control group showed normal corneal morphology. In the nontreated group, sections of the stromal infiltration revealed the presence of inflammatory cells, which were diffusely distributed (p < 0.05). Administrations of ciprofloxacin plus propolis resulted in a significantly reduced histological damage with fewer bacterial inoculation of the corneal stroma in comparison with the other groups (p < 0.05). Based on these findings, we suggest that ethanolic extract of propolis has antioxidant, anti-inflammatory, and antibacterial properties for S. aureus keratitis in rabbits.     

Calcium in the toad (Bufo marinus) cornea: binding by the stroma

1. The Ca concentration in the toad (Bufo marinus) cornea was 2.6 mmol/kg wet wt compared at 1.0 mmol/l in the bathing aqueous humor and 2.8 mmol/kg wet wt in the separated corneal stromal layer. Cell Ca content was calculated to be about 1.8 mmol/kg wet wt. 2. About 80% of the total Ca appears to be sequestered or bound to tissue components most of which (68% of the total) is associated with the stroma (2.2 mmol/kg wet wt stroma). 3. About 85-90% of the Ca in the stroma is readily exchangeable with external 45Ca. 4. The loss of accumulated 45Ca from the stroma was measured in vitro. This efflux of the isotope was enhanced by multivalent ions and was greatest when Ca2+ or La3+ was present in the external media. Other alkaline earth metal ions were not as effective. The relative effectiveness of this displacement of 45Ca was Ca = La greater than Sr greater than Ba greater than Mg. 5. The results suggest that the Ca2+ is bound by the amphibian stroma at sites that have a preference or specificity for this divalent ion as compared to the other alkaline earth metals. 6. The possible functional role of this bound Ca is discussed.     

In vitro effects of polyvinylpyrrolidone and sucrose on the acetylcholinesterase,succinate dehydrogenase and lactate dehydrogenase activities in the brain

Summary The supernatant prepared from the brain tissue homogenate incubated in vitro in the presence of PVP or sucrose exhibits a decrease of AChE, SDH as well as of LDH activity. A 0.75% PVP solution inhibits AChE activity by 30%, LDH activity is inhibited by 35% and SDH activity by 40%. A two hours lasting effect of a 7.5% PVP solution at 3° C on enzymatic preparations induces in AChE 20% inhibition of its activity, in LDH an inhibition of 44% and in SDH the inhibition of its activity amounts to 74%. 1 M Sucrose inhibits AChE activity by 34%, LDH activity by 41% and SDH activity is inhibited by 31%. After two hours lasting effect of 1.4 M sucrose at 3° C on the supernatant the AChE activity is inhibited by 22% and that of LDH by 30%. The SDH activity was after a two hours lasting effect of 1 M sucrose at 3° C inhibited by 34%. The inhibition of activity of the above mentioned enzymes localized in brain cortex preparations was compared with the inhibition of activity of the isolated serum cholinesterase. 0.25 M Sucrose inhibited the activity of this enzyme by 25% and 0.75% PVP by 45%. A two hours lasting effect of 7.5% PVP or 1 M sucrose at 3° C on the cholinesterase induced a 40% and 22% inhibition respectively. After double washing of the brain cortical minced tissue, prepared in a 7.5% PVP containing solution, AChE activity was constant. By triple washing of the brain cortical crude mitochondrial fraction, exposed for two hours at 3° C to the effect of 1 M sucrose, SDH activity was also constant.Abbreviations AChE acetylcholinesterase (EC 3.1.1.7.) - INT 2(p-iodophenyl)3-p-nitrophenyl-5-phenyl tetrazolium chloride - LDH lactate dehydrogenase (EC 1.1.1.27.) - PMS phenazine methosulfate - PVP polyvinylpyrrolidone - SDH succinate dehydrogenase (EC 1.3.99.1.)     

A fixed charge model of the transverse tubular system of frog sartorius

Volume changes of the transverse tubular system (T system) of frog sartorius in different solutions can be explained by a model which assumes fixed negative charges in the T system lumen, an open T system mouth, and a Donnan equilibrium between the T system and external solution. The T system volume is regulated by the osmotic pressure difference between the lumen and external solution, as well as by constraining forces whose nature is as yet unclear. The decreased swelling tendency produced by hypotonic solutions and increased tendency produced by some hypertonic solutions are ascribed to changes in the pressure constraint from the sarcoplasm. Fixed charge concentration was estimated tentatively from swelling and resistivity data to be between 0.1 and 0.4 M.