Possible reactions of 1,2-naphthaquinone in the eye

1. Reactions of 1,2-naphthaquinone with amino acids, glutathione and proteins of the lens have been studied in connexion with investigations of naphthalene-induced cataract. 2. Cysteine reacts probably through its amino group with 1,2-naphthaquinone to form either purple or brown compounds with characteristic absorption spectra. 3. Glutathione reacts with 1,2-naphthaquinone through its thiol group. 4. Spectroscopic evidence suggests that 1,2-naphthaquinone reacts with the amino group of amino acids. This reaction may take place in the aqueous humour. 5. The proteins of lens react with 1,2-naphthaquinone to form brown compounds. 6. There is loss of protein thiol in this reaction and the products are less easily digestible by pancreatin than normal lens proteins. 7. The compound of α-crystallin and 1,2-naphthaquinone is soluble at neutrality, but the compounds of β-crystallins and of γ-crystallins are largely insoluble. 8. The brown reaction products of glutathione or cysteine with 1,2-naphthaquinone catalyse the oxidation of ascorbic acid in the same way as 1,2-naphthaquinone itself. 9. These results are discussed in relation to naphthalene-induced cataract.     

Taurine and other free amino acids in the retina,vitreous, lens,irisciliary body,and cornea of the rat eye

Levels of free amino acids were determined quantitatively in whole ocular tissues of the rat eye with aid of a sensitive amino acid analyzer. The tissues studied were the retina, vitreous, lens, iris-ciliary body, and cornea. The retina and lens contained a more concentrated free amino acid pool than other tissues. The neuroactive amino acids taurine, GABA, glutamic acid, aspartic acid, and glycine were clearly enriched in the retina. Taurine was the most abundant amino acid in all five tissue studied, and its high concentration in non-neural tissues, especially the lens, suggests that it must have other functions as well as neurotransmitter ones in the rat eye.     

Vitreous body collagen. Evidence for a dual origin from the neural retina and hyalocytes

Two different cells types have been shown to synthesize embryonic chick vitreous collagen (vitrosin) at different stages of development. Identification of vitrosin was established by labeling the embryos in ovo [3H]proline at stages 23 and 28 and separating the extracted vitreous collagen alpha-chains by carboxymethylcellulose chromatography. The labeled collagen consisted predominately of alpha 1 chains, indicating a molecule in the form of a trimer of identical chains designated (alpha 1)3. The molecular weight of the labeled chains measured approximately 95,000 daltons by molecular sieve chromatography, and contained 41% of their imino acid as 4- hydroxyproline. To establish which eye tissues synthesize vitrosine, the collagens produced in organ culture by the isolated neural retina, lens and vitreous body from stages 26-27, 29-30, and 40 were examined. At the two earlier stages, only the neural retina synthesized large quantities of (alpha 1)3 collagen whereas the lens and the cells within the vitreous body itself synthesized relatively small amounts of collagen characterized by an alpha 1:alpha 2 ratio of about 2:1. At stage 40, however, the cells of the vitreous body itself synthesized the greatest quantities of collagen, which now was predominantly an (alpha 1)3 type molecule. Stage 40 neural retina and lens synthesized lesser amounts of collagen with an alpha 1:alpha 2 ratio of 2 to 3:1. Chick vitrosin thus appears to be synthesized by the neural retina in early embryonic stages, whereas the major contribution derives from cells within the vitreous body in later development.     

Regulation of hydrogen peroxide in eye humors effect of 3-amino-1H-1,2,4-triazole on catalase and glutathione peroxidase of rabbit eye

Activities of catalase (H₂O₂ : H₂O₂ oxidoreductase, EC 1.11.1.6) and GSH peroxidase (GSH : H₂O₂ oxidoreductase, EC 1.11.1.9) have been measured in iris, ciliary body, retina, corneal epithelium, corneal endothelium, lens capsule-epithelium and decapsulated lens. 3-Amino-1H-1,2,4-triazole is a specific inhibitor of catalase and a potent cataractogenic agent. We observed marked inhibition of catalase activity in these tissues 1–6 h after the administration of a single intravenous dose of 1 g 3-aminotriazole per kg body weight in rabbit. This was associated with a 2–3-fold increase in the H₂O₂ concentrations of aqueous humor and vitreous humor. The increased peroxide concentrations were restored to the physiological levels as the catalase activity of eye tissues gradually returned to normal with time after injection. Under the conditions, GSH peroxidase activity of the afore-mentioned eye tissues was unaltered, GSH and protein sulfhydryl of lens were not changed, and ascorbic acid of aqueous humor and vitreous humor was not significantly altered. Based on these findings our conclusion is that catalase of eye tissues regulates the endogenous H₂O₂ in eye humors to the physiological level. We speculate that H₂O₂ may be the triggering factor in cataract induced by 3-aminotriazole.     

Relationships between Presence of the Eye Cup and Maintenance of Lens-Forming Capacity in Larval Xenopus laevis

The lentectomized eye of larval Xenopus laevis can regenerate a lens by a process of lens-transdifferentiation of the cornea and pericorneal epidermis. These tissues can form the lens only when they become in direct communication with the environment of the vitreous chamber (neural retina) indicating that the eye cup plays a fundamental role in this process.
In this work the role of the eye cup in the maintainance of the lens-forming capacity of the cornea and pericorneal epidermis was studied by allowing these tissues to cover the enucleated orbit for different periods, and then implanting them into the vitreous chamber of the contralateral eye. Under these experimental conditions the maintainance of the lens-forming capacity of the cornea and pericorneal epidermis showed no significant correlation with the time from enucleation to implantation.     

The nature and origin of the glycosaminoglycans of the embryonic chick vitreous body

Glycosaminoglycans of the embryonic chicken vitreous were characterized and then were used as markers to establish which tissues synthesize the vitreous humor during development. The glycosaminoglycans are predominantly chondroitin sulfates by several criteria. They are resistant to streptomyces hyaluronidase, an enzyme which degrades only hyaluronate, and are digested by testicular hyaluronidase and chondroitinase AC, enzymes which degrade hyaluronate plus chondroitin 4- and 6-sulfates. On electrophoresis on cellulose acetate in 0.15 M phosphate buffer, pH 6.7, the vitreous glycosaminoglycans migrate slightly slower than authentic chondroitin sulfate, but, in 0.1 N HCl, they migrate very close to chondroitin sulfate standards. Finally, the disaccharides produced by digestion of these radioactively labeled glycosaminoglycans with chondroitinases AC and ABC were identified as Δdi-4S and Δdi-6S, as expected for chondroitin 4- and 6-sulfate. By using incorporation of radioactive precursors into chondroitin sulfates in vitro, we than determined which tissues synthesize the vitreous humor in the developing eye. Late in development, on Day 12–13, the isolated vitreous is very active in chondroitin sulfate synthesis, while the neural retina, the lens, and the pecten are less active and produce a high proportion of enzyme-resistant GAG. The eye tissues isolated from embryos labeled in ovo retain similar amounts and types of glycosaminoglycans, indicating that cells within the vitreous synthesize the vitreous humor glycosaminoglycans at this time. Earlier in development, from Days 6 to 8, the isolated vitreous incorporates very low levels of radioactivity into GAG, but the neural retina incorporates high levels of radioactivity into chondroitin sulfate. When the embryos are labeled in ovo and the same tissues are isolated following incorporation, the vitreous retains more radioactive chondroitin sulfate than does the neural retina. Thus, the vitreous humour glycosaminoglycan is initially synthesized by the neural retina and is secreted into the vitreous space.     

Desferal-Mn(III) in the therapy of diquat-induced cataract in rabbit.

In rabbit lenses subjected to oxidative stress, induced by 1 mM diquat in vitro, there were 7- to 10-fold increases (p less than 0.001) in malondialdehyde, conjugated dienes, and carbonyl dienes, indicating extensive peroxidation of cellular membrane lipids, and approximately a 60% decrease in reduced glutathione. In the presence of 0.1-5 mM Desferal-Mn(III) these changes were diminished by 50-70%. In an experimental group of 12 rabbits having diquat-induced cataract, Desferal-Mn(III) (5% w/v) applied topically as a 50-microliters eye drop four times per day and a single intraperitoneal dose of 64 mg/kg body wt daily for 5 weeks (including pretreatment for 1 week) retarded the progression of lens opacities, whereas, in a control group of 6 rabbits treated with the vehicle (0.15 M NaCl) cataract progressed to an advanced grade. Treatment with Desferal-Mn(III) also significantly diminished production of O2.- and OH. in the lens, aqueous humor, and vitreous humor, and of H2O2 in the aqueous humor and vitreous humor. It also suppressed lipid peroxidation and oxidation of protein-SH of the lens and restored lenticular glutathione and ascorbate to normal levels.     

Transdifferentiation of ocular tissues in larval Xenopus laevis

Transdifferentiation phenomena offer a useful opportunity to study experimentally the mechanisms on which cell phenotypic stability depends. The capacities of vertebrate eye tissues to reprogram cell differentiation are well known in avian and mammalian embryos, and in larval and adult newt. From research into the capacity of anuran eye tissues to reprogram differentiation into a new pathway, considerable data have accumulated concerning the transdifferentiative capacities of eye tissues in larval Xenopus laevis. This work reviews the data concerning the transdifferentiative phenomena of eye tissues in that species and, based on these, aims to establish the extent of our knowledge about the mechanism controlling these processes. In larval Xenopus laevis the outer cornea can regenerate a lens by a lens-transdifferentiation process triggered and substained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye phenotypic stability of the outer cornea is guaranteed by the presence of the inner cornea and lens, which prevent the spread of retinal factor(s). The stimulus for lens transdifferentiation of the outer cornea can be supplied by other tissues as well, but this capacity is not widely distributed. The iris and retinal pigmented epithelium can transdifferentiate into neural retina if isolated from the surrounding tissues and implanted in the vitreous chamber. As for lens transdifferentiation of the outer cornea, retinal transdifferentiation of the iris can be stimulated by certain nonocular tissues as well.     

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. ^1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. ^1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.     

Homeostasis in the vertebrate lens: mechanisms of solute exchange

The eye lens is avascular, deriving nutrients from the aqueous and vitreous humours. It is, however, unclear which mechanisms mediate the transfer of solutes between these humours and the lens' fibre cells (FCs). In this review, we integrate the published data with the previously unpublished ultrastructural, dye loading and magnetic resonance imaging results. The picture emerging is that solute transfer between the humours and the fibre mass is determined by four processes: (i) paracellular transport of ions, water and small molecules along the intercellular spaces between epithelial and FCs, driven by Na(+)-leak conductance; (ii) membrane transport of such solutes from the intercellular spaces into the fibre cytoplasm by specific carriers and transporters; (iii) gap-junctional coupling mediating solute flux between superficial and deeper fibres, Na(+)/K(+)-ATPase-driven efflux of waste products in the equator, and electrical coupling of fibres; and (iv) transcellular transfer via caveoli and coated vesicles for the uptake of macromolecules and cholesterol. There is evidence that the Na(+)-driven influx of solutes occurs via paracellular and membrane transport and the Na(+)/K(+)-ATPase-driven efflux of waste products via gap junctions. This micro-circulation is likely restricted to the superficial cortex and nearly absent beyond the zone of organelle loss, forming a solute exchange barrier in the lens.     

Free Radical Enhancer Xenobiotic is an Inducer of Cataract in Rabbit

Free radical enhancers, diquat, paraquat, plumbagin and juglone were used to study the oxy radical-induced damage to the rabbit lens in vitro and in vivo. Each compound caused a 6–8 fold increase in malondialdehyde (MDA) and a 30–55% decrease in reduced glutathione of the lens in vim. These peroxidative and oxidative changes were potentiated in the presence of 100% 0., abolished by N, and prevented by desferal-Mn (III) (DF-Mn) or liposomal superoxide dismutase (LSOD) indicating the involvement of O₂^?.

Diquat injected intravitreally as a single dose (300nmole in 30μl of isotonic saline) in the right eye of a 5-wk-old Dutch belted rabbit, induced early cataract after 24–72h. The lens of the contralateral control eye injected with isotonic saline had no change. In the right eye, O₂,^? and OH -productions were significantly (P < 0.01) higher; O₂-, was about 16 fold higher in the aqueous humor and vitreous humor, and 5 fold in the lens and retina, and OH. was 35 fold higher in the aqueous humor, 2 fold in vitreous humor and 5 fold in the lens and retina as compared to the respective tissues of the control eye. Enhanced lipid peroxidation in the lens was apparent from the higher levels of MDA and formation of aminophospholipid-MDA Schiff-base conjugates.

We propose that cyclic oxidation-reduction of xenobiotics coupled to the endogenous redox systems in the eye, could generate oxy radicals in excessive amounts, triggering cataractogcnesis.     

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.^1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.^1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.⁵ This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.     

Antioxidant and anticataractogenic effects of topical captopril in diquat-induced cataract in rabbits.

1-[(2s)-3-Mercapto-2-methylpropionyl]-L-proline (captopril), an antihypertensive and free radical scavenger, protected the rabbit lens from peroxidative and oxidative damage induced by 1 mM diquat in vitro. To evaluate the anticataract efficacy of captopril, an experimental group of five rabbits was treated with topical captopril (1% in 0.15 M NaCl, w/v), and 50 microliters was instilled onto both eyes four times a day for a total of 8 weeks. Following the same procedure, the eyes of five rabbits were treated with topical 0.15 M NaCl as a control for captopril treatment. At the end of the first week of treatment, a single intravitreal dose of 120 nmole diquat in 30 microliters of 0.15 M NaCl was injected into the right eye of each rabbit of both the groups. As a control for intravitreal diquat injection, the left eye of all the rabbits were injected with the diluent, 30 microliters per eye. The intravitreal diquat or its diluent injection was only for one time. From slit-lamp biomicroscopic observation of the diquat-injected right eyes, the anticataract effect of captopril in the treatment group was indicated by the finding that in four of five rabbits the cataract did not advance; whereas in four of five rabbits treated with the diluent the cataract progressed to grade 3. The lenses in the diluent-injected control left eyes of the rabbits treated with the captopril or diluent were normal. However, since the number of animals used for the in vivo studies was few, further confirmation of the anticataract effect of captopril is necessary. In diquat-injected right eyes of animals treated with captopril, the integrated rate of O2- production was about 50% less (p less than .001) in the aqueous humor, vitreous humor, and lens, compared with O2-, 33.49 +/- 2.26 microM (mean +/- SEM) in the aqueous humor, 17.12 +/- 0.75 microM in the vitreous humor, and 31.44 +/- 1.29 nmole/g wet weight in the lens of the diquat-injected right eyes treated with the diluent. Similar significant (p less than .01) differences in the production of .OH and H2O2 in eye tissues were also observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

New sesquiterpenoids of Bombax malabaricum

Hemigossypol-6-methyl ether, reported to be present in the root bark of Bombax malabaricum, has been shown to be isohemigossypol-1-methyl ether. Isohemigossypol-1,2-dimethyl ether, 8-formyl-7-hydroxy-5-isopropyl-2-methoxy-3-methyl-1,4-naphthaquinone, 7-hydroxycadalene and an unidentified phenolic compound have also been isolated. Long range couplings in the ¹H NMR spectrum of isohemigossypol-1-methyl ether have been established by decoupling experiments.     

The metabolism of 3-methylcholanthrene and some related compounds by rat-liver homogenates

1. A chromatographic investigation of the products of the metabolism of 3-methylcholanthrene by rat-liver homogenates showed the formation of compounds with the properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene and 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. A glutathione conjugate that is probably S-(11,12-dihydro-12-hydroxy-3-methyl-11-cholanthrenyl)glutathione was also detected. 3-Methylcholanthrene-1- and -2-one and -1,2-quinone were also present, but these products may have arisen by the chemical oxidation of the corresponding hydroxy compounds. 2. Other metabolic products were tentatively identified as 9- and 10-hydroxy-3-methylcholanthrene, 4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene and 3-hydroxymethylcholanthrene. 3. 1- and 2-Hydroxy-3-methylcholanthrene were converted by homogenates into the related ketones and into products with the properties of cis- and trans-1,2-dihydroxy-3-methylcholanthrene: 3-methylcholanthren-1- and -2-one were converted into their related hydroxy compounds and into the isomeric 1,2-dihydroxy compounds. The isomeric 1,2-dihydroxy compounds were each partly converted into the other isomer by these homogenates. All the above substrates also yielded products that appeared to be derivatives of 3-hydroxymethylcholanthrene. 4. 3-Methylcholanthrylene was converted by rat-liver homogenates into products with the properties of trans-1,2-dihydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene and 3-methylcholanthren-2-one. A small amount of the cis-1,2-dihydroxy compound was also formed, together with a glutathione conjugate that is possibly S-(2-hydroxy-3-methyl-1-cholanthrenyl)glutathione or its positional isomer. 5. An unidentified product was detected in the metabolism of 3-methylcholanthrene, the monohydroxy compounds, the ketones and the dihydroxy compounds, the formation of which appeared to involve metabolism at the 1,2-bond. 6. 11,12-Epoxy-11,12-dihydro-3-methylcholanthrene was converted by rat-liver homogenates into products with the properties of 11-hydroxy-3-methylcholanthrene (or, less likely, the 12-isomer), 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and the glutathione conjugate described above. Products with the properties of these compounds were formed when the epoxide was allowed to react with glutathione in an aqueous medium. 7. Mouse-liver homogenate converted 3-methylcholanthrene into products with the chromatographic properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, 3-methylcholanthrene-1- and -2-one and -1,2-quinone and the unidentified hydroxy-3-methylcholanthrenes. 8. The syntheses of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 3-methylcholanthren-2-one, 2-hydroxy-3-methylcholanthrene, 3-methylcholanthrylene, 11,12-epoxy-11,12-dihydro-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene are described.     

Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage.

1. Activities of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H2O2 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-1H-1,2,4-triazole (3-aminotriazole) or NaN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H2O2. 4. In rabbit having early cataract (vacuolar stage) induced by feeding-3-aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-Fold increase in H2O2 of aqueous humor and vitreous humor. We conclude that catalase of eye affords protection to the lens from H2O2 and it also protects superoxide dismutase of lens from inactivation by H2O2. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2.-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the hydroxyl radical, under pathological conditions when H2O2 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.     

Lens Subluxation after Plasmin and SF6 Injections in Rabbit Eyes

Purpose

To investigate the rate of lens subluxation following plasmin and/or SF₆ injections in eyes, and whether a subsequent elevated level of vascular endothelial growth factor (VEGF) and vitreous tap would aggravate subluxation.

Methods

Four groups of rabbits were used. Group 1 received an intravitreal injection (IVI) of plasmin and SF₆ in the right eye; group 2 received an IVI of plasmin in the right eye; group 3 received an IVI of SF₆ in the right eye; and group 4 received an IVI of balanced salt solution in the right eye. After treatment, IVIs of VEGF were given and vitreous tap was performed three times, followed by clinical observation of lens subluxation and scanning electronic microscope evaluation of the zonular fibers.

Results

After IVIs of plasmin and SF₆, and VEGF and vitreous tap had been performed one to three times, lens subluxation was noted in 0%, 43%, 71%, 71%, and 86% of the eyes in group 1. After IVIs of plasmin, VEGF, and vitreous tap had been performed one to three times, lens subluxation was noted in 11%, 22%, 44%, 44%, and 67% of the eyes in group 2. The eyes in group 3 and 4 did not show signs of lens subluxation after VEGF IVIs and vitreous tap. Histology confirmed zonular fiber damage in the eyes treated with plasmin.

Conclusions

The incidence of lens subluxation increased following plasmin injections in the eyes, and this was aggravated by the subsequent high VEGF level in the eyes and vitreous tapping. Zonular fibers were disrupted following plasmin treatment. These effects should be kept in mind when using plasmin enzymes in patients with vitreoretinal abnormalities.     

Ultrastructure of the eye of the amber snail Succinea putris (L.) (Gastropoda, Stylommatophora)]

The structure and some aspects of the development of the eye of Succinea putris were studied with the aid of the electron microscope. The eye is of the closed vesicle type and is composed of retina, cornea, vitreous body, lens and optic nerve. Three different types of cell are to be found in the retina: (1) the small elongated pigment cell with an avoid nucleus, many pigment granulae and short microvilli at the apical end of the cell; (2) the sensory cell type I with a large irregular nucleus, long microvilli, which extend to under the surface of the lens, a large number of light-cored vesicles, 700 A in diameter and the axon; (3) the elongated slender sensory cell type II with many dense cored vesicles, several pigment granulae in the distal region of the cell and short irregular microvilli at the apical end of the cell. This type is few in number. Two results of the study of the embryonic eye are described: the cornea cells differ from those in the adult eye in the nucleus-cytoplasm relation and the optic nerve is smaller than in the adult eye.