摘要:

Purpose. To learn the influence of the ciliary ganglion on the postnatal growth of eyes with unimpaired visual input and of eyes beneath an image diffusing goggle.Methods. Newborn chicks received unilateral ciliary ganglion-ectomy or unilateral sham operation and were reared either with or without a goggle ipsilateral to the surgical procedure. Ocular refractions and ultrasound measurements were made on anesthetized chicks; eyes enucleated postmortem were measured in axial and equatorial dimensions with calipers and studied histologically.Results. Excessive growth of open eyes in the equatorial dimensions followed ciliary ganglionectomy and became more pronounced as the chicks grew older. There was only a modest increase in axial growth. Ganglionectomy also induced relative hyperopia; lens thinning contributed to this effect and likely was a direct result of disrupted parasympathetic input to the ciliary muscle. Ganglionectomy also slightly increased the thickness of the choroid in the posterior pole but not in more peripheral locations.Conclusion. We conclude that the ciliary ganglion exerts an inhibitory influence on the postnatal growth of open eyes; the main effect is in the equatorial dimension of the vitreous cavity, with a smaller effect on axial length. Ciliary ganglionectomy exerted minimal influence on the development of experimental myopia, known to be induced by the goggle regimen. The amount of equatorial expansion in goggle-induced myopia was greater than after ganglionectomy alone, indicating that other factors besides the ciliary ganglion can influence the equatorial dimension of the vitreous cavity.

ISSN:0271-3683    

DOI:10.3109/02713689609000756

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. Following the solubilization of protein from patient worn soft contact lenses and subsequent analysis via SDS-PAGE, an unidentified 30 kDa protein deposit was commonly observed. The mysterious deposit was found to accumulate on a variety of soft contact lens material.Methods. Acuvue®, Cibasoft®, Excelens®and Newvue®soft contact lenses were worn by three asymptomatic patients using both daily-wear and extended wear regimens. To characterize the unknown deposit, human tear samples and lens eluted protein were subjected to SDS-PAGE, immunoblotting, enzymatic assays and protein sequencing.Results. Results show that the 30 kDa protein deposit is the homologous dimer of tear lysozyme. Polymerized lysozyme was found on each of the three lens materials within one h of wear. However, the dimer was not present in the normal tear film.Conclusions. Therefore, this dimerization phenomenon is the result of an aggregation and interaction of lysozyme with various soft contact lens polymers.

ISSN:0271-3683    

DOI:10.3109/02713689609000757

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. Outer segments of the photoreceptor rods that are phagocytized by the retinal pigment epithelial (RPE) cells contain a high proportion of polyunsaturated fatty acids (PUFA). PUFA are susceptible to lipid peroxidation. We hypothesized that the resulting peroxides could injure RPE cells leading to retinal degeneration. Accordingly, we compared the effects of linoleic acid (LA) and its hydroperoxide (LHP) on the growth and morphology of RPE cells using laser scanning microscopy and transmission microscopy.Methods. We counted the number of RPE cells after incubation for 24 and 48 hrs with concentrations of LA or LHP of 0.035, 0.175, and 0.35 mM. To observe the actin filaments, cultured RPE cells were stained with rhodamine phalloidin. The cells were prefixed with 2% glutaraldehyde and postfixed in 1% osmium tetroxide. Specimens were embedded in Epon 812 after dehydration, and the ultrathin sections were doubly stained with 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy.Results. Exposure to LA or LHP produced dose-dependent damage to RPE cells with a significantly greater effects of LHP than LA. After incubation for 24 hrs with 0.35 mM LA, the number of vacuoles in RPE cells exceeded that observed in control RPE cells by 365 nm laser microscopy. Exposure to 0.35 mM LHP for 24 hrs produced a pycnotic nucleus, with diffuse and granular autofluorescences observed in and around it. Exposure of RPE cells to 0.35 mM LA for 24 hrs showed that the LA incorporated into the lysosomes was digested and released extra-cellularly from lysosomes via exocytotic vesicles. However, such exposure to LHP damaged the RPE cells, including the membranes in the pinocytotic vesicles. The packed membranes resembled myelin.Conclusions. While the LA incorporated into the lysosomes was released extracellularly, LHP persisted in the RPE cells, being observed as autofluorescent lipofuscin-like materials. LHP was cytotoxic, and caused damage to the membranes of pinocytotic vesicles and lysosomes.

ISSN:0271-3683    

DOI:10.3109/02713689609000758

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. A human retinal cell line, KGLDMSM, developed by SV-40T antigen gene transfection, is stable in culture for a long period, unlike the primary cells. The cell line shows some degree of morphological differentiation with limited extension of stublike neurites upon transfer to defined medium. In our effort to explore genes implicated in neuritic extension and neuronal differentiation seen in response to cAMP and TPA, we have analyzed time dependent induction of a variety of proto-oncogenes: c-myc, H-ras, c-ras, and c-fos.Methods. Cells were adapted to grow in defined media and exposed to differentiation inducing agents cAMP, TPA, Retinoic Acid, and sodium butyrata. Cells were assessed for phenotypic changes and altered expression of proto-oncogenes as evaluated by Northern Blot analysis and immunocytochemistry.Results. Exposure of the cells to cAMP and TPA induced dramatic changes, with 100% of the cells extending neuritic processes. However, other differentiation inducing agents such as retinoic acid and sodium butyrata failed to elicit any response. We report that agents that promote neuritic extension also induce expression of c-fos. Transcriptional activation of c-fos in response to cAMP (30 min) and TPA (lhr) is also accompanied by expression of fos gene product as evaluated by using fos antibody. No fos expression was seen in uninduced cells.Conclusion. In retinal cell line KGLDMSM, agents that enhance neuronal differentiation (cAMP, TPA) also induce c-fos expression. Expression of c-fos may be a necessary prerequisite in neuronal differentiation and the established retinal cell line offers an excellent cell model for dissecting the molecular events underlying neuronal differentiation.

ISSN:0271-3683    

DOI:10.3109/02713689609000759

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. Phagocytosis of rod outer segments (ROS) is an important function of retinal pigment epithelial (RPE) cells. Since the details of the process are not fully known, we studied effects of cytokines produced by RPE and photoreceptor cells on phagocytosis of ROS by rat RPE cells.Methods. RPE cells were isolated and cultivated from two strains of rats: Sprague-Dawley (SD) rats with normal phagocytosis and Royal College of Surgeons (RCS) rats, which have genetic deficiencies in ROS phagocytosis. A double immunofluorescence staining technique was used to study the effectsin vitroof several cytokines on phagocytosis of ROS.Results. We found that transforming growth factor beta-1 (TGF-β1) had dose-dependent effects on RPE cells of both strains of rat: at a concentration of 10 ng/ml, TGF-β1 significantly (p<0.01) reduced total ROS (to 74% of control in SD rats and to 51% of control in RCS rats), reduced bound ROS (to 56% of control in SD rats and to 48% in RCS rats), and increased the ratio of ingested ROS to total ROS (to 140% in SD rats but not significantly in RCS rats). Treatment of medium with anti-TGF-β1 antibody before incubation of RPE cells of SD rats with TGF-β1 decreased the magnitude of these effects. The cytokine acidic fibroblast growth factor (aFGF, 10 ng/ml) affected RPE cells of SD rats only, decreasing ROS ingested to 56% of control and the ratio of ingested ROS to total ROS to 64% of control. We also examined effects of basic fibroblast growth factor and insulin-like growth factor. None of the cytokines tested increased ingestion of ROS by RPE cells of RCS rats.Conclusions. Our results suggest that TGF-β1 and aFGF have roles in regulating ROS phagocytosis by normal and dystrophic RPE cells in the rat.

ISSN:0271-3683    

DOI:10.3109/02713689609000760

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. To apply a high performance liquid chromatographic radiotracer method to test a variety of L-cysteine prodrugs and one dipeptide prodrug for their ability to synthesize glutathione in cultured rat lenses.Methods. Rat lenses were incubated for 48 h in a medium containing [14C(U)]-glycine and prodrugs. Following homoge-nization and derivatization, lens extracts were analyzed to determine the extent of biosynthetic incorporation of this labeled amino acid into [14C]-glutathione using high performance liquid chromatography with radioisotope and ultraviolet absorption detection. All of the thiazolidine prodrugs contained masked sulfhydryl groups to stabilize them against air oxidation. L-buthionine-(S,R)-sulfoximine—an inhibitor of the first step in glutathione biosynthesis-was present in media containing the dipeptide prodrug.Results. In all cases, a large [14C]-labeled peak eluted just prior to [14C]-glutathione. This peak had some characteristics of the mixed disulfide of glutathione and L-cysteine, viz., L-cysteine/ glutathione disulfide, but requires further investigation in order to be positively identified. Of the eleven L-cysteine prodrugs investigated, the most effective was 2(R,S)-methylthiazolidine-4(R)-carboxylic acid, which increased the rate of [14C]-glutathi-one biosynthesis 35% over that of the controls. A number of other L-cysteine prodrugs were somewhat effective, increasing glutathione synthesis 5-30% over the controls, while several L-cysteine prodrugs were totally ineffective. The only dipeptide prodrug investigated, viz.,γ-L-glutamyl-L-cysteine ethyl ester, increased the biosynthesis of [&14C]-glutathione 18% over control. Biosynthetic rates based on ultraviolet absorption of the deriv-atized glutathione demonstrated a similar pattern, the compounds most effective in synthesizing [14C]-glutathione generally yielding the highest ultraviolet glutathione concentrations and the ineffective compounds showing the lowest concentrations.Conclusions. 2(R,S)-methylthiazolidine-4(R)-carboxylic acid, 2(R,S)-n-propylthiazolidine-4(R)-carboxylic acid and N-acetyl-L-cysteine were the only compounds that were statistically significant in yielding higher levels of both ultraviolet and radioactive glutathione as compared to their respective controls. Thus, these prodrugs have very promising anti-cataract potential.

ISSN:0271-3683    

DOI:10.3109/02713689609000761

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. The aims of this study were to determine in the human lens water soluble-high molecular weight (WS-HMW)-proteins: (a) the levels of degraded polypeptides (crystallin fragments), and (b) thein vivocleavage sites in the parent crystallins to produce the major fragments.Methods. The WS-HMW proteins (Mr>15×106daltons) were isolated as a void volume peak from homogenates of lenses of donors of different ages using Agarose A 15m gel-filtration chromatography. The degraded polypeptides (Mr<18 kDa), present in the WS-HMW proteins, were separated by a preparative SDS-PAGE method and quantified as a percent of total WS-HMW proteins. In addition, the parent crystallins of the major polypeptides were identified by the Western blot method using antibodies raised either to the whole crystallin molecule or to desired regions at N- and C-terminals or middle of individual crystallins. The partial N-terminal sequences of purified individual polypeptides were determined to identity the cleavage sites in parent crystallins.Results. The levels of degraded polypeptides as percent of the total WS-HMW proteins increased with aging, i.e. about 5% in lenses of 16 to 19 year-old-donors compared to 27% in the 60-80 year-old-donors. As many as thirteen polypeptide species with Mr's between 3 to 17 kDa were separated from WS-HMW proteins by a preparative SDS-PAGE method. The Western blot analyses showed that the polypeptides originated fromα-,β-andγ-crystallins and the cleavage sites varied in different regions of crystallins as identified by partial N-terminal sequence analyses.Conclusions. The data showed an age-related increase in levels of degraded polypeptides in the WS-HMW proteins and the polypeptides were derived fromα-,β- andγ-crystallins.

ISSN:0271-3683    

DOI:10.3109/02713689609000762

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. Ocular cicatricial pemphigoid (OCP) is a chronic autoimmune cicatrizing disease which affects the conjunctiva and other squamous epithelium, resulting in a scarring process. A similar process, limited only to the conjunctiva, observed in some patients using eye drops for the treatment of glaucoma, is called pseudo-ocular cicatricial pemphigoid (P-OCP). Im-munofluorescence studies demonstrate deposition of immuno-globulins and complement components in the basement membrane zone (BMZ) of the conjunctiva and an anti-basement membrane zone antibody in the serum of patients. A striking association between OCP and MHC class II gene DQBl*0301 has been observed. The purpose of this study was to determine some of the differences in the binding of OCP and P-OCP sera to different lysate in an immunoblot assay, in an attempt to partially characterize the OCP and P-OCP antigens. Furthermore, we wanted to determine if the MHC class II gene association of P-OCP is similar to that of OCP.Methods. We studied sera from 11 patients with active ocular cicatricial pemphigoid and seven patients with pseudo-ocular cicatricial pemphigoid and controls. Indirect immunofluorescence (IIF) studies were done using monkey esophagus and salt split normal human skin as substrate. A sensitive immunoblot assay (IBA) was developed using normal human epidermis, dermis and conjunctiva as substrate. Typing for MHC class II genes was performed on eight pseudo-ocular cicatricial pemphigoid patients by dot-blot analysis and compared to 38 matched controls.Results. Weak staining of the basement membrane zone was observed in nine of ten ocular cicatricial pemphigoid sera and five of seven pseudo-ocular cicatricial pemphigoid sera in the IIF assay using monkey esophagus. Using salt split human skin as substrate, ten of eleven ocular cicatricial pemphigoid sera demonstrated low titer weak binding to the epidermal side of the split. No consistent pattern of staining was seen with pseudo-ocular cicatricial pemphigoid sera. Ten of the 11 ocular cicatricial pemphigoid sera demonstrated binding to 230, 205, 160 and 85 kDa proteins in the IBA using normal human epidermis and conjunctiva lysates. When the lysates were first reacted with BP sera and then immunoblotted with ocular cicatricial pemphigoid sera, the 230, 160, and 86 kDa bands disappeared, and only the 205 kDa band persisted. The sera of five of seven pseudo-ocular cicatricial pemphigoid patients bound to 290, 230, 205, 180, 97, and 85 kDa proteins in the epidermis and conjunctiva. However, the 230, 205, 180, and 85 kDa proteins are depleted when the lysates are first reacted with BP and ocular cicatricial pemphigoid sera. In the dermal lysate, the pseudo-ocular cicatricial pemphigoid sera recognize 400, 290, 150 and 45 kDa proteins. None of these are absorbed by BP, ocular cicatricial pemphigoid or pemphigus vulgaris or epidermolysis bullosa acquisita sera. The 290 kDa proteins identified in the dermis and epidermis are distinct from each other. No binding was seen with control sera with the 3 lysates. Statistically, dot-blot analysis did not demonstrate a significant increase in the frequency of the MHC DQB1*O3O1 gene.Conclusions. Patients with ocular cicatricial pemphigoid and pseudo-ocular cicatricial pemphigoid produce several autoanti-bodies. However, there are similarities and differences between them. The MHC class II genes associated with pseudo-ocular cicatricial pemphigoid are different from those with ocular cicatricial pemphigoid. This provides a new model system to study the immune abnormalities in idiopathic and drug-related organ specific autoimmunity.

ISSN:0271-3683    

DOI:10.3109/02713689609000763

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. The goal of this study was to compare the ultrastruc-ture of the oldest cells in opaque and transparent human lenses.Methods. Age-related nuclear cataracts, late-onset diabetic nuclear cataracts and normal aged lenses were examined by transmission electron microscopy. Cross-sectional profiles of fiber cells in the embryonic, fetal and juvenile nuclear regions were obtained to facilitate direct comparisons between lens regions and between sample groups. Image analysis was performed to determine cross-sectional areas of fiber cells in each region.Results. The average cross-sectional area increased approximately sixfold from the outer to the inner nuclear regions in all lenses measured. In each nuclear region, fiber cells displayed a characteristic size, shape, arrangement and type of interdig-itations which were consistently seen in all the lenses examined. Some lenses had more complex interdigitations than others. Gap junctions were identified as pentalamellar structures having 16 nm width and appeared identical throughout the nuclei of both normal and cataractous lenses. The cytoplasm of all lenses was smooth and free of large density variations. However, the cytoplasm of some cataractous lenses appeared more granular in texture than noncataractous lenses. Cellular degeneration, debris or large cellular defects were not seen in the cores of cataractous lens nuclei.Conclusions. These results indicate that only minor ultrastruc-tural differences exist between the oldest fiber cells in normal and cataractous lenses, and that the presence of extensive cellular damage and disruptions is not necessary for the generation of nuclear opacities in aged lenses. Our observations suggest that light scattering sufficient for vision impairment may involve structural alterations much smaller than previously proposed.

ISSN:0271-3683    

DOI:10.3109/02713689609000764

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. To investigate the time course of effects of intravenous administration of a calcium antagonist, nicardipine, on the choroidal circulation of rabbit eyes using a laser speckle tissue circulation analyzer.Method. The rabbit fundus was illuminated by a diode laser spot and its image speckle was detected by an image sensor. The difference between the average of the speckle intensity (Imean) and the speckle intensity for successive scannings was calculated, and the ratio of Imeanto this difference was defined as normalized blur (NB); a quantitative index of tissue blood velocity. The average NB over the field measured (0.620.62 mm in the choroid) was calculated to give Nbav. Under general anesthesia, 0.4 ml/kg of 0.01% nicardipine hydrochloride dissolved in physiological saline was injected intravenously in a group of albino rabbits (control group) for measurement in the choroid (nicardipine groups). To serve as control, 0.4 ml/kg of physiological saline was injected in other groups of albino rabbits (control groups). Nbavwas recorded at 1-min intervals for the first 5 min and at 5-min intervals for the next 85 min. During the experiment mean femoral arterial blood pressure (FABPm), pulse rate (PR), arterial blood pH, Pco2and Po2body temperature (BT) and intraocular pressure (IOP) were also monitored. Before and 45 min after nicardipine administration, the choroidal blood flow measurement using the microsphere technique and the Nbavmeasurement were carried out in the same eye in another group of rabbits.Results. Only the rabbits which did not show any significant change in the PR, pH, Pco2, Po2and BT during the experiment were accepted. FABPmin the nicardipine group dropped to the minimum at 1 min postadministration and this level remained significantly lower than that in the controlgroup up to 15 min post-injection, while te 10P did not show any significant change. The Nbavin the nicardipine group showed a significant increase after the FABPmreturn to the baseline, which was maintained throughout the experiment. The averaged increase between 30 and 90 minutes after administration was 27±1% (mean±S.E.M., n = 10). Relative increase in choroidal blood flow determined by the microsphere technique showed a good correlation (r = 0.90, P<0.001, n = 12) with those determined by Nbavafter nicardipine administration.Conclusions. The present result indicates that the time course of drug effects on the choroidal circulation can be noninvasively and sensitively followed by Nbavmeasurements. Further, it was shown that nicardipine may have considerable potential for the treatment of ocular diseases associated with insufficient choroidal blood flow and that nicardipine's effects here observed deserve to be further studied in humans.

ISSN:0271-3683    

DOI:10.3109/02713689609000765

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor