In vitro incubation paralleling changes occurring during mouse cataract formation

Certain biochemical and morphological changes involved in the formation of osmotic cataracts in the Nakano mouse and galactosemic rat lenses also occur in vitro. Incubation of young, normal, albino mouse lenses in glucose-free medium for 48 hr resulted in a gain in wet weight due to dramatic hydration. The glucose deprived lenses showed a marked decrease in the soluble protein content as is noted in cataract formation in vivo. Chromatography of the soluble protein demonstrated a substantial increase in heavy molecular weight material in the lenses incubated without glucose in comparison to the fresh lenses. A decrease in β_H-crystallin was also striking and might be correlated with the disappearance of a 31 000 mol. wt polypeptide band seen by SDS-PAGE gel. Interestingly, the membrane polypeptides in the in vitro incubated lenses mimicked changes observed during cataract formation in vivo. A 23 000 mol. wt polypeptide band became prominent in the membrane fraction of these lenses and there was a concomitant decrease in the 26 000 mol. wt component. Messenger RNAs were present in the glucose deprived lenses as shown by cell-free translation in vitro, although no [³⁵S]methionine incorporation into crystallin was noted. Both the hexokinase and glyceraldehyde phosphate dehydrogenase activities in the glucose deprived lenses fell rapidly and were virtually absent at the end of 48-hr incubation period. The data in this report suggest that the glucose deprived incubated mouse lens may serve as an in vitro model to the study of some biochemical and morphological changes occurring in the development of osmotic cataract in vivo.     

Composition and synthesis in vivo of water-soluble proteins in the rat ocular lens: normal and cataractous

This study describes the composition and synthesis in vivo of the water-insoluble proteins in normal and cataractous rat ocular lens. Cataracts of the lens nucleus were induced by treatment of the young rat with U18666A, 3β-(2-diethylaminoethoxy)androst-5-en-17-one. HCl. Opaque lens contained more insoluble proteins and incorporated more intraocularly injected [³H]leucine into these proteins than clear lens. Insoluble proteins from both clear and opaque lens consisted of classes in three mol. wt. ranges; > 68 000, 35 000–60 000 and 16 000–22 000. Treatment with mercaptoethanol produced changes in the electrophoretic profiles of the 35 000–60 000 and 16 000–22 000 mol. wt. classes of proteins from both normal and opaque lens; however, the higher mol. wt. class from only the opaque lens was reduced to smaller components. The lowest mol. wt. class accounted for over 50% of the total water-insoluble protein in both types of lens. [³H]leucine was incorporated into the protein classes roughly proportionate to their relative concentrations, although there was a tendency for normal lens to incorporate more label into peak 1 and 2 proteins and for cataractous lens to incorporate more into peak 3 proteins. In general, however, the greater concentration of water-insoluble protein in this cataract appeared due to a proportionately increased formation of the same proteins present in normal lens. In contrast to human senile cataracts, the opaque rat lens did not preferentially accumulate disulfidelinked very high mol. wt. insoluble protein. However, the aged (i.e. pre-existing) higher mol. wt. insoluble proteins of the U18666A cataract appeared different from those newly synthesized, since the aged proteins were reduced under conditions not affecting those newly synthesized.     

Testosterone binding in the cytosol of bovine corneal epithelium.

In the cytosol of bovine corneal epithelium [³H]testosterone binding was investigated by three independent methods: ammonium sulphate precipitation, equilibrium dialysis and electrophoresis in polyacrylamide gel. A high affinity (association constants K_as = 0·91 ? 1·5 × 10¹⁰ l. mol^?1), low capacity (number of binding sites N = 3·1 ? 7·8 fmol. mg^?1 of protein) binding specific for testosterone and dihydrotestosterone was found. Other steroid hormones exerted only a slight or negligible competitive binding to [³H]testosterone binding sites of the protein, the molecular weight of which was estimated to be about 90 000.     

Lens membranes vi. Some characteristics of the EDTA-extractable protein (EEP) from bovine lens fiber membranes.

After urea-treatment of calf lens fiber membranes, a well defined extrinsic membrane protein fraction (EEP) can be extracted by EDTA-solution. It contains polypeptides of mol. wt. 32K and 35K (SDS-gel electrophoresis). Thin-layer gel filtration shows that without detergent no oligomers are formed. It is easily lost by its adsorption characteristics, however, it can be purified from contaminating α crystallin by gel filtration. The 35K fraction comprises components with isoelectric points at 4·6–4·7 (double band) and between 6·2 and 7·2. The 32K fraction shows components at 4·6–4·7 and 6·2 as was found by two-dimensional gel electrophoresis. The method of isolation, the isoelectric focusing pattern, and the amino acid and mol. wt. composition distinguish EEP clearly from the soluble crystallins. EEP comprises two distinct antigenic components with isoelectric points between 6·2 and 7·2. This was revealed by immunoelectrofocusing of EEP vs. anti-EEP antiserum from rabbit. Combining the results of two-dimensional electrophoresis and immunoelectrofocusing it was found that the 32K and 35K polypeptide have different antigenic properties. Traces of crystallins are difficult to remove. However, by means of specific anti-crystallin antisera it has been shown that EEP has a determinant in common with γ crystallin.     

Stimulation of the synthesis of specific glycoproteins in corneal epithelium by vitamin A.

The uptake of labeled glucosamine into a specific, purified glycopeptide of corneal epithelium was stimulated twofold by incubating whole corneas of vitamin A-deficient rats with retinol. This glycopeptide was found to be a moiety of a glycoprotein isolated from the incubation medium. The synthesis of this glycoprotein from incubated vitamin A-deficient corneas was stimulated to normal levels with as little as 2 × 10^?7m added retinol in 3 hr; stimulation was 60% of maximum at 2 × 10^?9m-retinol. No such stimulation of synthesis was observed when normal corneas were incubated with retinol. The glycoprotein stimulated by the added retinol was not a glycosaminoglycan and took up no labeled sulfate; its origin is not conjunctival. It is probably derived from corneal epithelium.     

SDS-gradient polyacrylamide gel electrophoresis of individual ocular mucus samples from patients with normal and diseased conjunctiva

Individual mucus samples were collected from normal individuals and from patients with ocular cicatricial pemphigoid (CP), Stevens-Johnson syndrome (SJS), and various types of conjunctival inflammation (rosacea, meibomianitis, atopy, keratoconjunctivitis sicca, etc.). The mucus samples were dissolved in sample buffer containing 8M urea, 2% SDS and 5% 2-mercaptoethanol and were electrophoresed on gradient 2-16% polyacrylamide gels. Four glycoproteins with molecular weights greater than 200,000 daltons were consistently observed in both individuals with normal conjunctiva and patients with CP, SJS, and other diseases exhibiting conjunctival inflammation. The amounts of each glycoprotein appeared to vary from one individual to another; however, the presence or absence of specific glycoproteins could not be correlated with the different ocular diseases. The techniques described for mucus analysis offer advantages over previously published techniques since improved resolution of the mucous glycoproteins can be achieved by electrophoresis on 2-16% gradient gels, and individual samples can be analyzed. Our results suggest that substantial amounts of ocular mucous glycoprotein are present in the eyes of patients with CP and SJS, diseases which have been previously described as mucin-deficient dry eye syndromes.     

Protein composition of bovine lens cortical fiber cell membranes.

A plasma membrane-enriched fraction was obtained after treatment of the water-insoluble residue of the adult bovine lens cortex with 8 m-urea for the solubilization of albuminoid (i.e. intracellular matrix). Morphological characterization of the urea-insoluble plasma membrane-rich fraction by electron microscopy showed it to consist of membrane fragments in the form of clear vesicles of various sizes. No obvious damage to the integrity of the membranes could be detected as a result of this treatment.Electrophoretic analysis of the urea-insoluble plasma membrane-rich fraction in 5·13% polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS) showed it to consist of seven major polypeptide components of estimated molecular weights (I) 200 000, (III) 68 000, (VI) 43 000, (VIII) 35 000, (IX) 27 500, (X) 22 500, and (XI) 20 000 daltons. Component III (mol. wt. 68 000) was the major PAS-positive component and probably represents the major glycoprotein fraction of the lens membranes. Components VIII, IX, X, and XI had mobilities identical to the mobilities of the soluble crystallins in the 1% SDS electrophoresis system. The predominant polypeptide component of the membrane-rich fraction, component IX (estimated mol. wt. 27 500), comprised nearly half of the protein recovered from this fraction as determined by densitometric scanning of the gels. Electrophoretic comparison with the soluble lens fraction revealed that this component had a mobility identical to that of the predominant polypeptide chain (B_P) of beta-crystallin in the 1% SDS electrophoresis system. The next most abundant component of the membrane-rich fraction, band X, comprised 24% of the protein recovered from this fraction and had a mobility identical to that of alpha-crystallin in the 1% SDS electrophoresis system. Whether the polypeptide components of bands VI–XI in the membrane-rich fraction represent homomonomers (crystallins or membrane components) or heteromonomers (crystallins and membrane components) of similar molecular size (i.e. mobility) remains to be determined.     

Fractionation and partial characterization of macromolecular components from human ocular mucus

Crude human ocular mucus was extracted with 0·154 m-NaCl to separate soluble protein components from mucus. Small amounts of lipoglycoprotein of high molecular weight, as well as twelve plasma proteins, were detected in the soluble extract by gel filtration and immunodiffusion studies. After the NaCl extraction, the remaining mucus residue was further extracted with 6 m-urea-0·2 m-Tris-phosphoric acid buffer. From this portion of soluble extract, a relatively larger amount of lipoglycoprotein of high molecular weight, as well as a lower molecular weight fraction containing eight detectable plasma proteins, were both isolated by gel filtration. The glycoprotein moieties of the lipoglycoproteins of high molecular weight had similar chemical composition. Both contained approximately 40–43% protein and 57–60% carbohydrate, giving a carbohydrate-protein ratio of 1·30 to 1·48. Fucose, galactose, N-acetylhexosamine and N-acetylneuraminic acid comprised about 423–516 residues per 1000 amino acid residues, while serine and threonine constituted about 285–299. All analyses indicated mucin-like character in the lipoglycoproteins of high molecular weight.Plasma proteins constituted approximately three-fifths of the macromolecular components in ocular mucus. These proteins also appeared to be in complexes with lipids, but to a much lesser extent than the high molecular weight fractions. The relevance of present findings to the structure and composition of precorneal tear film is discussed.     

The effect of N‐acetylcysteine on the microscopic fluid dynamics of rat tears

Background: The classical view of the tear film is of a 10‐micron film of aqueous tears, sandwiched between thin layers of lipid and mucus. This has been challenged recently by the revelation that the tear film may be considerably thicker than 10 microns and that dissolved mucus and protein may play a much more important role than simply promoting tear adherence. In particular, the primary role of mucus may be to form a structured aqueous gel that adheres closely to the corneal surface and evens out its irregularities, thus providing a high‐quality optical surface. Methods: We have used the robust tear film of the rat eye as an animal model to investigate the contribution of mucus and low‐molecular‐weight (LMW) proteins to tear film structure. The ocular surface was first exposed to saline, which washed away the tear film. Single drops of a tear/saline mixture, treated with various concentrations of the thiol‐reducing agent N‐acetylcysteine (NAC), were placed on the ocular surface and the resulting fluid behaviour was recorded with video‐microscopy. Results: At five per cent concentration, NAC appeared to degrade the gap‐filling and anti‐evaporative qualities of the tears, features that give the rat tear film its robust characteristics. Lower concentrations had no significant effect. Discussion: In a previous publication, we showed that five per cent NAC alters the profile of LMW proteins in rat tears. The present observations suggest that the robust wetting properties of rat tears depend critically on their mucus and/or LMW protein content and possibly are related to the formation of an aqueous/mucous gel.     

Isolation and characterization of BCP 54, the major soluble protein of bovine cornea

The most abundant soluble protein of bovine cornea, which we have named “BCP 54” (Bovine Corneal Protein, molecular weight 54000 daltons), has been isolated and biochemically characterized. This protein constitutes approximately 30% of the total soluble protein of whole cornea. BCP 54 is present in both epithelium and stroma; however, its presence in endothelium has not been demonstrated conclusively. Immunochemical analysis indicates that BCP 54 is of corneal, but not of serum, origin.Polyacrylamide gel electrophoresis in SDS of either unreduced or reduced BCP 54 yields a single protein band with a molecular weight of 54000 daltons. By gel filtration the molecular weight of the native molecule was also shown to be about 54000 daltons. Isoelectric focusing under denaturing conditions yields four bands clustered between pH 6·6 and pH 6·9. Thus BCP 54 appears to be a protein composed of a single polypeptide chain, but with some degree of microheterogeneity. The amino acid composition of BCP 54 demonstrates that it is not a collagenous or actin-like protein; furthermore, it is not a glycoprotein. The abundance and distribution of BCP 54 suggest that this protein fulfills an important, but as yet unknown, function in the cornea.     

Corneal ulceration and the serum antiproteases. II. Complexes of corneal collagenases and alpha-macroglobulins.

Previous work has demonstrated that whole rabbit or human serum and the human α₂-macroglobulin (Hα₂-m) inhibit corneal collagenases. In the present study, the ability of rabbit and human serum to cause rabbit corneal collagenase, mol. wt 45 000, to elute in high molecular weight fractions from sieving columns is taken as presumptive evidence for the formation of collagenase-serum protein complexes. The recovery of increased collagenase activity by thiocyanate treatment of serum effluent fractions containing the human α₂-macroglobulin indicates that it is the α₂-m in human serum that complexes rabbit corneal collagenase. This conclusion is supported by the demonstration that purified Hα₂-m transports the rabbit corneal collagenase through a molecular sieve. Moreover, the chromatography of day one culture media from ulcerating rabbit corneas has demonstrated the presence of a significant amount of the total collagenase activity in the high molecular weight (850 000 – 1 000 000) fractions in which rabbit α-macroglobulin (Rα₁-m) was also demonstrated. These observations support the hypothesis that the α-macroglobulins play an important role in the regulation of corneal collagenase activity.Crossed-gel immunoelectrophoretic methods have shown that rabbit and human corneal collagenase preparations perturb the patterns of their respective α-macroglobulins. The perturbed patterns are taken as evidence for the formation of collagenase-α-macroglobulin complexes. The application of crossed-gel methods to the tears of human ulcer patients shows the utility of such methods for examining the status of α₂-m in tears.     

Age and cataract-related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens.

Heavy molecular weight (HMW) proteins were detected in normal and cataractous mouse lenses. The HMW aggregates increased with the age of the lens in normal mouse. Alpha and β-crystallins were detected by immunodiffusion in the HMW fractions from normal and Nakano mice. No γ-crystallin could be detected in these aggregates by immunodiffusion; however, a slight amount of this crystallin was detected using the radioimmunoassay. The polypeptide composition of the HMW proteins was different in the Nakano mouse from the normal. By SDS polyacrylamide gel electrophoresis, a polypeptide of 27 000 mol. wt. was evident in the Nakano HMW material that was not present in the normal HMW protein, but a 15 000 mol. wt. band was absent in the Nakano.Two other differences were seen with the Nakano lens. First, the water insoluble lens protein was extremely high. By 90 days, about two thirds of the protein was insoluble in these lenses. Secondly, the sharp drop in γ-crystallin at the time of complete opacification of the lens was in part a result of the leakage of this protein into the anterior chamber of these mice. By radioimmunoassay, the level of γ-crystallin in the Nakano aqueous humor at the time of the cataract was greater than 100 ng per microliter. These data demonstrate that crystallins are converted to the insoluble proteins and some diffuse out of the lens during cataract formation.     

A morphometric study of the Müller cell in rod and cone retinas with and without retinal vessels

A comparative morphometric study of the Müller cells, their nuclei, and mitochondria was made in the following animals: Rattus norwegicus (albino rat) which has a vascular rod retina, Gecko eublipharis macularis (nocturnal gecko) which has an avascular rod retina, Eutamios sciurus (Korean ground squirrel) which has a vascular cone retina, and Natrix natrix (grass snake) which has an avascular cone retina.The study was restricted to a zone 30° wide, extending 30–60° from the centre, and each retinal layer in this zone was examined separately.The rat retina proved to contain 377 000 Müller cells/mm³, each having a volume of 260 μm³ and a surface of 2759 μm². The Müller cell cytoplasm made up 8·8% of the total retinal volume, and the mean surface: volume ratio was 114·4 × 10⁸ μm²/mm³ Müller cell. Mitochondria average 78·2 × 10⁶/mm³ retina and were situated mainly in the vitreal part of the retina. There were 16·4 microvilli/μm² at the outer limiting membrane.In the gecko retina there were 154 000 Müller cells/mm³ retina, each having a volume of 1338 μm³ and a surface of 20 325 μm². The cytoplasmic volume percentage was 19·8%, and the mean surface: volume ratio was 156·9 × 10⁸ μm²/mm³ Müller cell. There was an average of 44·5 × 10⁶ mitochondria/mm³ retina, situated exclusively in the scleral part of the retina. At the outer limiting membrane there were 43·7 microvilli/μm².In the gound squirrel there were 328 000 Müller cells/mm³ retina, each having a volume of 378 μm³ and a surface of 2378 μm². The cytoplasmic volume percentage was 10·5% and the mean surface: volume ratio 67·8 × 10⁸ μm²/mm³ Müller cell. The mean mitochondrial content was 29·9 × 10⁶ mm³ retina, with a fairly even distribution in all layers. At the outer limiting membrane there were 49·2 rather short microvilli/μm².In the grass snake retina there were 373 000 Müller cells/mm³ retina, each having a volume of 209 μm³ and a surface of 2038 μm². The cytoplasmic volume percentage made up 5·6% and the mean surface: volume ratio was 106·5 × 10⁸ μm²/mm³ Müller cell. There was an average of 43·6 × 10⁶ mitochondria/mm³ retina, approximately equally distributed over the entire retina. At the outer limiting membrane there were 27·3 microvilli/μm².     

Mucus fishing syndrome: case report and new treatment option.

BACKGROUND: Mucus fishing syndrome (MFS) is a cascading cyclic condition characterized by continuous extraction of mucous strands from the eye. It is usually initiated by ocular irritation. In response to irritation, ocular surface cells produce excess mucus. A "snow balling" cycle begins when the patient extracts ("fishes") excess mucus from the ocular surface, thereby causing further irritation and a more-profound mucous discharge. To date, treatment includes eliminating the initiating element and educating the patient not to touch the eye when extracting the excess mucus, CASE REPORT: Presented is a case of mucus fishing syndrome initiated by dry eye. The patient's diagnosis, MFS, was identified by persistent mucous discharge, his admittance and demonstration of digitally extracting mucus from the ocular surface, and a characteristic rose bengal staining pattern. The conventional treatment initiated by using artificial tears for the dry eye condition and educating the patient not to touch the ocular surface did not provide relief from the excess mucous discharge. Therefore, a new approach to treatment was pursued. In order to break the cycle, a mucolytic agent and an antihistamine-mast cell stabilizer were prescribed, until the ocular surface healed. After treatment, the patient reported alleviation of symptoms and demonstrated improvement in ocular surface integrity by a profound reduction in rose bengal staining. CONCLUSION: Mucus fishing syndrome is challenging to resolve with conventional treatment because it requires a certain level of psychological tolerance and perseverance from the patient. By eliminating the present mucus and diminishing mucous production pharmacologically, the practitioner is able to remove the stimulus for digital extraction and thus accelerate ocular surface healing. We present a proposed new treatment option for patients who are refractory to conventional treatments.     

Molecular weight and sequence complexity of delta-crystallin mRNA.

δ-Crystallin mRNA, isolated from lens fibers of 15-day-old embryonic chicks, has a mol. wt of 680 000 as determined by its migration in formamide polyacrylamide gels. This value, which corresponds to about 2200 nucleotides, is consistent with the observed sedimentation value of approximately 17 S. Radioactive δ-crystallin cDNA has been synthesized in vitro by avian myeloblastosis virus RNA-dependent DNA polymerase, and the kinetics of the reaction between the [³H]cDNA and excess δ-crystallin mRNA have been analyzed to determine the sequence complexity of the mRNA. The results indicate that δ-crystallin mRNA is composed primarily of a single species with a sequence complexity of 2200–2500 nucleotides. This suggests that the frequently observed heterogeneity of δ-crystallin may be due to post-translational modifications of a single subunit.     

Effects of temperature, concentration and carboxymethylation on interactions of calf lens crystallins.

Concentration and temperature dependent interactions of calf cortical crystallins were studied by means of gel filtration and disc gel electrophoretic analyses. Low temperature (8°C) gel filtration of either low or high levels of cortical crystallin extracts (5–59 A₂₈₀ units) yields elution profiles that are indistinguishable in the distributions of α-, β_H-, β_L- and γ-crystallins. Gel filtration of low levels of extracts between 22 and 44°C causes a gradual but ultimately complete temperature-dependent disappearance of the region of β_H-crystallins and to a lesser degree in the γ-crystallin peak. Chromatography of high levels of extracts at 44°C results in the reappearance of a high mol. wt. β-crystallin peak with electrophoretic property and polypeptide composition different from those of β_H-crystallin isolated at the low temperature of 8°C. Preincubation of high levels of extracts at 44°C causes some small but irreversible changes in the elution profile obtained at 8°C. Taken together these results suggest that the presence of high mol. wt. β-crystallin is the result of largely reversible equilibria between β-crystallins and other lenticular components.The reaction of calf cortical extracts with increasing amounts of iodoacetic acid results in a progressive loss in the β_H-crystallin region of the 8°C filtration profile. Results from amino acid analyses and sodium dodecylsulfate (SDS) gel electrophoreses of the respective α-, β_L- and γ-crystallin fractions indicate that the SH moieties of the cysteine residues are essential to the formation of high mol. wt. β-crystallins.     

Immunological study of proteins and mucosubstance in saline soluble human ocular mucus

Proteins and mucosubstance of the saline extract of human ocular mucus were studied by immunological analysis. A minor study was made with human tears for comparison. Immunoelectrophoresis of proteins from these two sources consistently revealed similar characteristic gel patterns. Proteins were found as the major constituents of both samples. However, more mucosubstance was present in the saline extract of human ocular mucus than in tears. Seventeen proteins were identified in the mucus extract. Albumin, IgA, and lactoferrin appeared to be the three major proteins, while lysozyme, lactoferrin, tear prealbumin, and ocular mucoisolate were tear and ocular mucus specific. Although saline soluble mucoisolate is complex in structure, it seemed to resist electrical dissociation, producing only one major precipitation line along with a line of IgA during immunoelectrophoresis. The ocular mucoisolate accounted for about 12% of the saline extractable proteins of human ocular mucus.     

Evidence for two sodium pumps in the crystalline lens of the rabbit eye.

The effect of ouabain on the ATPase activity, the electrical potential, ⁸⁶Rb uptake, and sodium content in the crystalline lens of the rabbit were studied. The ouabain-sensitive ATPase activity of the lens epithelium was compared to that of segments of lens cortex. The lens fibres were found to possess one-third of the total ouabain-sensitive ATPase activity of the lens. Dose-response studies of ATPase inhibition by ouabain gave a K_I of about 1·4 × 10^?6m for the epithelium and about 3·3 × 10^?5m for the anterior cortex. Although ouabain sensitive ATPase was present in the posterior cortex, ouabain had no effect on ⁸⁶Rb uptake across the posterior lens surface.Application of ouabain to the anterior surface of the lens resulted in an 8 mV decrease in the absolute value of the lens potential, a reduction of ⁸⁶Rb uptake to about one-third of its value in unpoisoned lenses, and a significant increase in sodium content after 2 hr of incubation. Dose-response studies of these effects suggested that the $\text{Na}\text{K}$ ATPase of the anterior cortex, but not that of the epithelium, makes an electrogenic contribution to the measured lens potential. On the other hand, inhibition of the epithelial ATPase effects a greater reduction in ⁸⁶Rb uptake than inhibition of the cortical ATPase. The maintenance of lens sodium content cannot be ascribed solely to the activity of the sodium pump in the lens epithelium.The results of these studies suggest that two types of $\text{Na}\text{K}$ ATPase with different anatomical localizations contribute to the maintenance of cation balance in the rabbit lens.     

Energetic requirements of active transepithelial Na and Cl transport in the isolated bullfrog cornea.

In the isolated bullfrog cornea, the energetic requirements were estimated of anaerobic active transepithelial Na and Cl transport (i.e. $\text{Δ}\text{Na}\text{Δ}\text{ATP}$ and $\text{Δ}\text{Cl}\text{Δ}\text{ATP}$). These estimates were done by measuring the corresponding effects of various stimulators and inhibitors of active transepithelial Na and Cl transport on anaerobic lactate efflux and the short-circuit current (s.c.c.). In NaCl Ringer's solution, 10^?5m-amphotericin B stimulated the calculated rate of active transepithelial Na transport by between 0·86 and 1·12 μEq/hr.cm². The corresponding increase in lactate efflux was 0·29 μmol/hr.cm². Assuming that glucose is the sole substrate for anaerobic glycolysis, the energetic requirement as estimated from these stimulatory effects on active transepithelial Na transport is between 3·0 and 3·9. The energetic requirement of active transepithelial Cl transport was studied by considering the corresponding effects on lactate efflux and the Cl-originated s.c.c. of various known selective inhibitors and stimulators of the Cl-originated s.c.c. The Cl energetic requirement could not be estimated from either the inhibitory effects of 10^?5m-bumetanide or 3 × 10^?4m-furosemide since both of these drugs only inhibited the Cl-originated s.c.c. without affecting lactate efflux. Only 10^?3m-ouabain had a slight inhibitory effect on lactate efflux. The selective stimulants of active transepithelial Cl transport, 10^?4m-epinephrine and 2 × 10^?3m-theophylline had large stimulatory effects on the s.c.c. but did not affect lactate efflux. Only the stimulant 10^?5m-A23187 (calcium ionophore), which had the largest stimulatory effect on the Cl-originated s.c.c, significantly stimulated the lactate efflux. From the inhibitory effects of 10^?3m-ouabain on the A23187 stimulated s.c.c. and lactate efflux, the energetic requirement of active transepithelial Cl transport is equivalent to a movement of 4·0 mol of Cl per mol of ATP.     

Fundus autofluorescence in patients with retinal pigment epithelial (RPE) tears: an in-vivo evaluation of RPE resurfacing

Aim

Investigate RPE resurfacing by changes in fundus autofluorescence (AF) in patients with retinal pigment epithelial (RPE) tears secondary to age-related macular degeneration (AMD).

Methods

A retrospective case series of patients presenting with RPE tears from 1 March 2008 to 1 April 2011. The pattern and area of AF signal distribution in RPE tears were evaluated. The change in the size of the area of debrided RPE over the follow-up period was used as the main outcome measure. A reduction in this area was termed “RPE resurfacing”, and an enlargement termed “progression of RPE cell loss”.

Results

Thirteen patients (14 eyes) with RPE tears (mean age 82 years) were included in this study. The mean baseline area of reduced AF signal was 4.1 mm² (range 0.33–14.9, median 0.29). “Resurfacing” of the RPE occurred in ten eyes and “progression of RPE cell loss” in four eyes after a median follow-up of 11.5 months (range, 1–39). The mean area of healing was 2.0 mm², and progression was 1.78 mm².

Conclusion

A consistent AF pattern was observed in patients with RPE tears. RPE resurfacing over the area of the RPE tear occurred, to a varying degree, in the majority of the cases.