Biological oxygen apparent transmissibility of hydrogel contact lenses with and without organosilicon moieties

The instrument oxygen transmissibility (IOT) of organosilicon hydrogels, measured by electrochemical procedures, is 5-10 times larger than that of conventional hydrogels. A method is described that allows the estimation of the oxygen tension at the lens-cornea interface for closed- and open-eyelids situations by combining the IOT of the hydrogels and corneal parameters such as corneal thickness, corneal permeability and oxygen flux across the cornea. From these results the biological oxygen apparent transmissibility (BOAT) is obtained, an important parameter which an multiplication with the pressure of oxygen on the external part of the lens gives the oxygen flux onto the cornea. Contact lenses with oxygen transmissibility higher than 100 Dk/t units [1 Dk/t unit=10(-9) [cm(3) O(2) (STp) cm(-2)s(-1)(mmHg)(-1)] posses a large oxygen tension at the lens-cornea interface that substantially reduces the oxygen flux onto the cornea. Lenses whose oxygen transmissibility is lower than 50 Dk/t units allow a rather small oxygen flux onto the cornea under closed eyelids condition that prevent their use for extended wear.     

The regulation of corneal hydration by a salt pump requiring the presence of sodium and bicarbonate ions

1. The use of polyacrylamide gel salt bridges enables trans-membrane potentials to be measured to an accuracy of 20 muV over long periods.2. The technique is applied to measure electrical potentials across corneal endothelia of rabbits.3. In de-epithelialized corneas which translocate water, a spontaneous potential of 550 muV is found across the endothelium (tissue resistance 20 Omega cm(2)).4. This electrical potential (and water translocation) is reduced to zero when sodium is absent from the Ringer, and by about 80% when bicarbonate ions are absent. Removal of chloride has no such effect.5. Under a variety of conditions, the potential correlates with the observed translocation of fluid across corneal endothelium. The translocated fluid is shown to be isotonic with sodium in the Ringer and therefore the potential correlates with ;active' sodium transport.6. The potential and water translocation are abolished in the presence of ouabain at concentrations greater than 10(-5)M.7. The potential (lens-side negative) is of the wrong polarity to explain the net sodium transport (into the lens-side) by a sodium ion ;pump'.8. The current does not equal the net sodium flux under short circuit conditions. They differ in magnitude and polarity.9. A model is proposed where the endothelium ;pumps' salt out of the corneal stroma into the aqueous humour.10. Flux equations are derived for a condition where the membrane (corneal endothelium) separates an ion exchanger (corneal stroma) from free solution (aqueous humour), where the usual relationship for free-free solutions Deltapi = c(s)Deltamu(s) does not apply.11. The model is of use only when the stroma is well stirred. It may be used in whole corneas retaining their epithelium but it may not be used in de-epithelialized corneas.12. The model predicts that the presence of an ;active' salt flux out across the endothelium would create passive water and salt fluxes. The passive water flux would also travel out of the stroma across the endothelium; the passive salt flux would travel, in the opposite direction, into the stroma across the endothelium.13. The kinetics of the passive water efflux, as a swollen cornea reverts to physiological hydration (the temperature reversal phenomenon) are predicted extremely well if the ;active' salt flux is chosen at 3.3 x 10(-7) m-mole. cm(-2) sec(-1).14. The value of the active salt flux which cannot be measured directly is extrapolated to be somewhat greater than 2.8 x 10(-7) m-moles. cm(-2) sec(-1); in good agreement with that required by the model to explain the temperature reversal phenomenon.15. The model is further used to calculate the salt concentration difference across the endothelium (which drives salt passively into the stroma) at various stromal hydrations.16. When an appropriate salt concentration is applied across the endothelium of de-epithelialized cornea, it generates a potential of the same polarity and similar magnitude to that found across the endothelium of equilibrated whole cornea. The endothelium acts like a cation exchange membrane.17. Additionally the calculated salt concentration difference across the endothelium correlates well with the measured transendothelial potentials in whole cornea as the corneal hydration varies.18. It is concluded that the model of an endothelial neutral salt ;pump' regulating corneal hydration is self consistent. The spontaneous potential found across the endothelium could be caused by the consequential passive flux of salt in the opposite direction.     

The mucinous layer of corneal endothelial cells

PURPOSE: The goal of this study was to characterize the morphology of the mucinous layer on rabbit, bovine, owl, and human corneal endothelial cells. METHODS: Corneoscleral buttons were fixed using cetylpyridinium chloride to stabilize "mucus" and the tissue was prepared for transmission electron microscopy. Photomicrographs were measured to determine the thickness of the endothelial and epithelial mucinous layer in the central cornea. RESULTS: The endothelial mucinous layer was seen as a nearly uniform electrodense region on the apical aspect of the endothelium. It was found to be 0.9 microm, 0.9 microm, 0.9 microm, and 0.5 microm thick in rabbit, bovine, owl, and human, respectively. The owl endothelium had an additional less electrodense layer with a granular appearance and a thickness of about 200 microm. The mucinous layer on the epithelium was similar in appearance to that on the endothelium and across species. CONCLUSIONS: The morphologic similarity of the endothelial and epithelial mucinous layers is a serendipitous finding that should prove valuable in experimental design. Ultimately, it is hoped that studies of the posterior corneal surface will deepen our knowledge of endothelial protection.     

The location of the fluid pump in the cornea

1. Fluid transport across rabbit corneal tissue has been investigated by observing the movement of fluid interfaces under the microscope, or by mounting the tissue between two chambers and observing the displacement of menisci within capillary tubes.2. In both cases, the endothelial layer supported on a thin sheet of connective tissue is capable of pumping fluid in a direction out of the cornea, against a head of pressure. The volume of fluid moved may amount to twelve times the thickness of the endothelial cells in an hour.3. This active fluid movement accounts for the prevention of swelling of the normal corneal stroma. The hypothesis that corneal hydration is regulated by the sodium pump in the epithelial layer is not supported by these experiments.     

Oxygen consumption by the component layers of the cornea

1. The structural integrity of the cornea is maintained by an active fluid transport system which depends on metabolism. Experiments were designed to establish the respiratory activity of each corneal layer.2. A rapid micropolarographic technique was used to determine the oxygen consumptions of the individual layers of the rabbit cornea.3. Inherent problems of the determinations have been made minimal by the use of both denuded and whole corneal tissue preparations. Four independent measures were obtained for each limiting layer (epithelium and endothelium).4. Results show that the endothelium, epithelium and stroma use 21, 40 and 39% respectively of the total oxygen consumption of the cornea. On the basis of volumes of oxygen per unit volume tissue, epithelial oxygen utilization is about ten times that of the stroma and approximately 0.2 that of the endothelium.5. The endothelium has a larger oxygen uptake than previously reported.6. The present results, in conjunction with other studies, indicate that the ratio of glycolytic to oxidative activity in the rabbit cornea is 0.87:0.13.     

Physiology of epithelial transport in the human eye

Summary Epithelia of the eye are located in the cornea, the lens, ciliary body, and pigment epithelium. In this review the relevant aspects of electrolyte transport properties in the isolated human cornea and lens are described. The main physiological function of both the cornea and the lens is the conservation of the transparency of these structures.(1) In the isolated human cornea sodium and chloride ions are pumped towards the tear side. Both net transports are sensitive to pH-changes and can be stimulated by cyclic AMP, epinephrine, and norepinephrine. The response to adrenergic stimulation is mainly mediated by ₂ receptors. Propranolol, a -blocking agent widely used for treatment of glaucoma, hypertension, and angina pectoris, inhibits net sodium transport. By measuring intracellular membrane potentials and ion activities active transport mechanisms can be localized within the epithelium. In addition, an active transport across the endothelial cell layer has to be postulated. The discrepancy between the measured net ion flux and the short circuit current indicates that other ions but Na⁺ and Cl^– ions are transported across the human cornea.(2) In the human lens the epithelial layer is localized on the anterior side of the tissue. In the isolated human lens short circuit current can only be inhibited by ouabain when the glycoside is added to the anterior side of the lens. From the effect of ouabain and ion substitution experiments it can be concluded that about 30% of the translenticular ion transport is maintained by Na⁺ transport. In addition, short-circuit current is also dependent on K⁺, Cl^–, and HCO₃ ^–. Measurements of intracellular membrane potential and potassium activity reveal that the interior of the lens is an electrically well connected tissue. Translenticular electrolyte transport, ionic content, and bioelectric properties of the whole lens are controlled by the epithelial monolayer of the anterior lens side.(3) Some disorders of cornea and lens in patients with chronic renal failure are discussed.Supported by Deutsche Forschungsgemeinschaft (Grant Wi 328)     

The effects of theophylline and choleragen on sodium and chloride ion movements within isolated rabbit ileum.

1. Theophylline (10 mM) and choleragen change the direction of net Cl- movements across rabbit ileum, in the short-circuit current condition, from absorption to secretion. The specific activity ratio R of Cl- tracers within the tissue coming from mucosal and serosal solutions respectively is increased, which is consistent with an increase in Cl- exchange flux across the mucosal border. 2. Net Na+ movement is also changed from net absorption to secretion by theophylline and choleragen; the specific activity ratio R of Na+ tracers is raised by theophylline. Because of the large paracellular component to transepithelial Na+ movements, an increase in Na+ exchange flux across the mucosal border is not detected. 3. 2,4,6-Triaminopyrimidine (20 mM) which has been previously shown to block paracellular Na+ movements, blocks both the theophylline and choleragen-dependent reversal of net Na+ movement by preventing the decrease in m-s Na flux. The theophylline-dependent increase in the ratio R of Na+ is still present, and is consistent with an increase in Na+ exchange flux across the mucosal border--unmasked by removal of the paracellular flux components. 4. Ouabain (0.1 mM) abolishes net absorption of Na+ and Cl- in control and net secretion of Na+ and Cl- in theophylline-treated tissue. Ouabain does not affect the theophylline-dependent increase in Cl- exchange across the mucosal border. 5. Replacement of Ringer Cl- with SO24- or Na+ by choline prevents the effects of theophylline and choleragen on Na+ and Cl- fluxes respectively. 6. Ethacrynate (0.1 mM) prevents the theophylline-dependent effects on net Na+ movement. Raising ethacrynate to 0.2 mM abolishes the effects of theophylline on Cl- exchange. An interpretation of these results is that theophylline and choleragen raise the Cl- permeability of the brush border. This increases NaCl leakage from the hypertonic lateral intercellular space into the mucosal solution thereby causing secretion. The selective action of triaminopyrimidine and ethacrynate (0.1 mM) on Na+ flux indicates that Na+ and Cl- move via separate transport pathways across the mucosal border.     

Linkage of posterior polymorphous corneal dystrophy to 20q11

Posterior polymorphous dystrophy (PPMD) is an autosomal dominantdisorder of the cornea that is clinically recognized by thepresence of vesicles on the endothelial surface of the cornea.The corneal endothelium is normally a single layer of cellsthat lose their mitotic potential after development is complete.In PPMD, the endothelium is often multi-layered and has severalother characteristics of an epithelium including the presenceof desmosomes, tonofilaments, and microvilli. These abnormalcells retain their ability to divide and extend onto the trabecularmeshwork to cause glaucoma in up to 40% of cases. A large familywith 21 members affected with PPMD was genotyped with shorttandem repeat polymorphisms distributed across the autosomalgenome. Linkage was established with markers on the long armof chromosome 20. The highest observed LOD score was 5.54 (     

The bicarbonate ion pump in the endothelium which regulates the hydration of rabbit cornea.

1. Studies were made on the short-circuit current (s.c.c.) and fluid transport across rabbit corneal endothelium. 2. Normal s.c.c. of 27 muA.cm-2 is reduced to 19 muA.cm-2 in CO2-free Ringer, to 9 muA. cm-2 in HCO-3 -free Ringer and to zero in CO2 and HCO-3 -free Ringer. 3. Carbonic anhydrase inhibitors reduce s.c.c. from 27 to 19 muA.cm-2. Removal of exogenous CO2 causes no further reduction in s.c.c. 4. In CO2-free Ringer, net exogenous bicarbonate translocation is equal to s.c.c. 5. In all cases studied, net fluid transport across the endothelium in open circuit is directly proportional to s.c.c. 6. It is concluded that the endothelial 'pump' which regulates corneal hydration operates by 'pumping' bicarbonate ions into the aqueous humour. 7. Under physiological conditions, two thirds of the substrate is supplied by exogenous bicarbonate ions and one third is supplied by conversion of exogenous CO2 by intracellular carbonic anhydrase. 8. Metabolic CO2 does not participate significantly in the process, probably because it is at too low a concentration to compete effectively with exogenous CO2. 9. Electron histochemical studies suggest that carbonic anhydrase is located immediately underneath the posterior membrane of the endothelium, across which the active bicarbonate ion flux passes.     

Sodium and chloride transport in the isolated human cornea

²²Na and³⁶Cl fluxes in isolated human cornea preparations were measured under short-circuited conditions. At pH 7.3–pH 7.6 sodium net flux was directed from aqueous humour to tear side, chloride net flux was statistically not different from zero. Alkalinization of the bathing solution to pH 8.6 stimulated unidirectional sodium and chloride fluxes from aqueous humour to tear side resulting in net sodium and chloride fluxes towards the tear side which both were statistically different from zero. Cyclic AMP (10^–3 M) was found to stimulate sodium and chloride uridirectional fluxes from aqueous humour to tear side, thus leading to significant net sodium and chloride fluxes in the same direction. Epinephrine (10^–4 M) increased the unidirectional chloride flux from aqueous humour to tear side more pronounced than in the opposite direction, producing a significant net chloride flux towards the tear side. The results are consistent with the hypothesis that the electrolyte pumps may under certain conditions contribute to the dehydration of the stroma.Abbreviations PD transcorneal potential difference - SCC short circuit current - R dc electrical resistance - TA unidirectional tear to aqueous humour side flux - AT unidirectional aqueous humour to tear side flux - net net flux Supported by Deutsche Forschungsgemeinschaft (Wi 328)     

小鼠角膜的发生与细胞凋亡

目的 通过观察小鼠角膜的发生过程,探讨角膜细胞的增殖与凋亡对角膜结构修复与塑形的作用。方法 各日龄共计120只小鼠,用HE染色或4’,6-二脒基-2-苯基吲哚（DAPI）染色对小鼠角膜的一般结构进行观察;用5′-溴脱氧尿嘧啶核苷（BrdU）技术标记角膜增殖细胞和免疫荧光法标记干细胞和凋亡细胞。结果 胚胎发育及出生后早期,角膜以实质层的发育为主。出生14d（P14）左右,角膜上皮细胞层开始增殖分化为两层细胞,同时内皮细胞也开始分化。至P30时,我们可以辨别出角膜的6层结构。BrdU阳性细胞主要存在实质层中的成纤维细胞,出生以后也可见于角膜上皮细胞层和内皮细胞层。随着角膜发育,P10左右,其他层的BrdU阳性细胞都消失,仅存在于角膜上皮细胞层。增殖细胞核抗原（PCNA）阳性细胞在发育早期散在分布于角膜的各层,P14以后PCNA阳性细胞均匀的分布于角膜上皮细胞的基底层,并维持在稳定状态。在角膜发育早期,在各层可见许多细胞凋亡。结论 角膜的发育与其感光功能形成的过程相一致,角膜干细胞的增殖与其修复有关;有大量的凋亡细胞参与角膜结构的塑形。     

层粘连蛋白对兔角膜内皮细胞bcl-2表达的影响

目的：观察层粘连蛋白（LN）对兔角膜内皮细胞bcl-2表达的影响。 方法： 将体外培养的兔角膜内皮细胞分成3组：正常对照组、层粘连蛋白(LN)处理组、阴性对照组,采用免疫组化法和酶联免疫法分别检测3组的染色深浅及吸光度大小，以显示各组间bcl-2表达的水平。 结果： 免疫组织化学染色及评分结果显示LN组bcl-2为强阳性表达，正常对照组bcl-2为弱阳性表达，阴性对照组bcl-2为阴性表达。酶联免疫吸附试验检测3组吸光度分别为1.21±0.18（LN组）、1.05±0.14（正常对照组）和0.04±0.01（阴性对照组）。 结论： 层粘连蛋白能促进兔角膜内皮细胞bcl-2基因表达，从而可以有效地抗细胞凋亡。     

Localization of hyaluronic acid, chondroitin sulfate, and CD44 in rabbit cornea.

To demonstrate the localization of hyaluronic acid (HA) in rabbit cornea, the biotinylated HA-binding region, which specifically binds to the HA molecule, was applied to the tissue. Localization of chondroitin sulfate (CS) and CD44, a possible cell surface receptor for HA, were also examined by immunohistochemistry. The stainability of HA changed depending on the fixatives used. Reaction products for HA were distinctly detected in epithelial cells and stromal keratocytes, but faintly in the extracellular matrix of the stroma when unfixed cryosections were applied. No positive reaction was found in the endothelium, except that the positive deposits formed a continuous layer on the apical surface of the endothelium. Electron microscopy using samples fixed with 2% paraformaldehyde revealed gold particles indicating HA labeling the intercellular space of the epithelium and stromal extracellular matrix. No intracellular deposition was detected in epithelial cells, whereas the gold labeling was seen in vacuolar structures of stromal keratocytes. Immunodeposits for CS were intensely localized in the epithelium and stroma, and weakly in the endothelium. Immunoreactivity for CD44 was found in the epithelial, endothelial and stromal cells. In particular, immuno-deposits for CD44 were detected in basal parts of epithelial cells, while they were localized in the apical surface of endothelial cells. These results suggest that HA is synthesized in and secreted from epithelial and stromal cells of rabbit cornea, while the localization of HA in the apical surface of the endothelium is closely associated with that of CD44. Moreover, the presence of CS in corneal tissue may play a role in its transparency, as has been suggested for keratan sulfate and dermatan sulfate.     

Transport of Na, Cl, and water by the rabbit corneal epithelium at resting potential.

Theophylline (1 mM) produced a net transport of Na and Cl from aqueous humor to tears (.02-.04 mumol/cm2 h) in the isolated rabbit cornea denuded of endothlium and in the presence of normal resting potential (25-35 mV). The active transport of Na (tears to aqueous) and of Cl (aqueous to tears), estimated with the Goldman constant-field equations, was confirmed. A 10 degrees C rise in temperature produced changes close to those predicted for passive processes in both unidirectional fluxes of Na and in the tears-to-aqueous flux of Cl, but not for the aqueous-to-tears flux of Cl. Theophylline treatment doubled Cl permeability but did not significantly affect Na or urea permeability, suggestingspecificity of affect. In separate experiments it was shown that stromal thinning occurred in previously swollen corneas when the endothelium was blocked by silicone oil and the epithelium was treated with theophylline. These findings provide further support for the argument that the mammalian epithelium could have an active role in the regulation of corneal thickness in situ.     

Voltage dependence of calcium transport in the thick ascending limb of Henle's loop.

Thick ascending limbs of Henle's loop were dissected from rabbit kidneys and perfused in vitro. Unidirectional transepithelial calcium fluxes from lumen-to-bath and bath-to-lumen were measured with 45Ca using different solutions that caused the transepithelial voltage to vary over a wide range. With lumen-positive voltages there was net calcium absorption from lumen to bath which varied directly with the voltage. With voltage near zero there was no measurable net flux. When the voltage was made negative, the direction of net calcium transport reversed (i.e., secretion from bath to lumen). The presence or absence of bicarbonate in the lumen did not affect the calcium fluxes. Calcium permeability, calculated from the dependence of net flux on voltage, was 7.7 x 10(-6) cm/s, which is approximately 25% of the sodium permeability previously determined in this segment. Analysis of the calcium flux ratios revealed interdependence of the bidirectional fluxes consistent with single-file diffusion but no evidence for active calcium transport. We conclude that there is an important component of passive net calcium transport driven by the voltage in this segment.     

Direct measurements of membrane unstirred layers

1. The unstirred layer of fluid bounding the posterior surface of the rabbit cornea and contact lens has been directly measured using an optical apparatus.2. Small polystyrene latex spheres (< 0.25 mu diameter) were used as an indicator because of the excellent light-reflective properties; other indicators were used (e.g. carmine particles) and had a similar behaviour.3. With the bulk solution unstirred the layer was 350 mu thick on the cornea and 150 mu thick on the contact lens. Following vigorous stirring the layer was reduced to 65 mu on the cornea and < 20 mu on the contact lens.4. The present direct measurements of the unstirred layers thickness confirm previous indirect measurements.     

Measurement of corneal endothelial impedance with non-invasive external electrodes--a theoretical study

The corneal endothelial cell layer function is critical for the maintenance of hydration and transparency of the cornea. Recent advances in corneal lamellar transplantation point to the need for reliable, non-invasive and rapid endothelial function assessment. Findings using an invasive electrode in an experimental animal model have suggested an association between bioimpedance parameters and endothelial cell function. Currently, however there is no clinical device that allows for non-invasive measurements of endothelial layer electrical impedance. This report is a finite element simulation study that models the human eye. It evaluates the feasibility of using external non-invasive electrodes to detect changes in endothelial layer electrical properties as a function of electrode location and measurement frequencies. The findings show that the ratio between the potential recorded at low and high frequencies is sensitive to changes in endothelial resistivity as well as endothelial capacitance. Moreover, the optimal electrode configuration yielding the highest sensitivity is one where the current injecting electrodes are oppose to each other and the voltage recording electrodes are adjacent to the current injecting electrodes. This first-order theoretical study suggests that a non-invasive device which measures electrical properties of the endothelial layer from the exterior of the eye could be developed. Clearly further animal and human studies are required to develop a reliable clinical tool.     

Inhibition of blood vessel formation by a chondrocyte-derived extracellular matrix

In this study, the chondrocyte-derived extracellular matrix (CECM) was evaluated for its activity to inhibit vessel invasion in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) and rabbit chondrocytes were plated on a bio-membrane made of CECM or human amniotic membrane (HAM). The adhesion, proliferation, and tube formation activity of HUVECs and chondrocytes were examined. The CECM and HAM powders were then mixed individually in Matrigel and injected subcutaneously into nude mice to examine vessel invasion in vivo after 1 week. Finally, a rabbit model of corneal neovascularization (NV) was induced by 3-point sutures in the upper cornea, and CECM and HAM membranes were implanted onto the corneal surface at day 5 after suture injury. The rabbits were sacrificed at 7 days after transplantation and the histopathological analysis was performed. The adhesion and proliferation of HUVECs were more efficient on the HAM than on the CECM membrane. However, chondrocytes on each membrane showed an opposite result being more efficient on the CECM membrane. The vessel invasion in vivo also occurred more deeply and intensively in Matrigel containing HAM than in the one containing CECM. In the rabbit NV model, CECM efficiently inhibited the neovessels formation and histological remodeling in the injured cornea. In summary, our findings suggest that CECM, an integral cartilage ECM composite, shows an inhibitory effect on vessel invasion both in vitro and in vivo, and could be a useful tool in a variety of biological and therapeutic applications including the prevention of neovascularization after cornea injury.     

Localization and expression of chondromodulin-I in the rat cornea

The localization and expression in the rat cornea of chondromodulin-I (ChM-I), an inhibitory angiogenesis factor, were examined by immunohistochemistry, Western blot analysis, ribonuclease protection assay, and real-time PCR assay. We found immunoreactivity for ChM-I in the epithelial layer but not the stromal layer or endothelial layer in the cornea, in addition to the positive ChM-I immunoreactivity in other sites in the eye such as the sclera, retina, and ciliary body. The ChM-I immunoreactivity was most intense at the outside of the basal cells and in their cytoplasm while the intensity of the immunoreactivity decreased gradually from the wing cells to the superficial cells in the corneal epithelial layer. No reactivity however, was detected in the Bowman's membrane or conjunctival epithelial cells which had continuity with the corneal epithelial cells. The expression of ChM-I mRNA was demonstrated in the cornea at one-third less intensity than that in the sclera with choroids and retinal pigment epithelium by ribonuclease protection assay and real-time PCR. ChM-I in the corneal epithelial layer may prevent neovascularization and maintain avascularity in the cornea.