摘要:

Purpose. Experimental autoimmune uveoretinitis (EAU) is an invaluable animal model for studying inflammatory eye disease in humans. Indocyanine green (ICG) is a fluorescent dye that can be used to image both retinal and choroidal vessels. This study was performed to examine the retinal and choroidal vascular abnormalities of a rat model of EAU using ICG and fluo-rescein as the contrast media and to assess the suitability of this model for studying ICG angiographic abnormalities in inflammatory eye disease in humans.Methods. Twenty-six male black-hooded Lister rats were inoculated with bovine retinal S-antigen plus adjuvant with or withoutBordetella pertussisantigen. Fluorescein and ICG angio-grams were performed at different stages of clinical disease with a scanning laser ophthalmoscope.Results. EAU was a more severe and primarily choroidal disease in rats givenBordetella pertussis, but no animals showed evidence of dye leakage from large choroidal vessels. There was frank leakage of indocyanine green from retinal vessels. Leakage of both fluorescein and ICG from retinal vessels largely correlated with disease activity. Retinal pigment epithelial lesions either corresponded to areas of hypofluorescence on the ICG angiogram alone or were represented by areas of ICG hyperfluo-rescence that had overlying areas of fluorescein leakage from retinal capillaries.Conclusions. This study has demonstrated the vascular abnormalities of this model of EAU using ICG and fluorescein as the contrast media. The suitability of this model for studying ICG angiographic abnormalities in inflammatory eye disease in humans is encouraging.

ISSN:0271-3683    

DOI:10.3109/02713689608995149

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. Hyaluronic acid (HA) is the predominant glycosami-noglycan (GAG) of the human vitreous. Interaction of this HA with vitreous collagen is important for maintaining gel structure. The mechanism of HA homeostasis in the vitreous is incompletely understood. The aim of this study was to determine whether hyaluronidase, an endoglycosidase that degrades HA, was present in human vitreous.Methods. Vitreous samples were collected from post-mortem eye bank specimens and from non-hemorrhagic, non-inflamed biopsy specimens. Vitreous hyaluronidase was purified by a series of column chromatographic steps, and its activity was measured by an ELISA-like assay and by substrate gel electro-phoresis through an HA-impregnated gel. The purified hyaluronidase was also analyzed by SDS-polyacrylamide gel elec-trophoresis (SDS-PAGE) and by Western blotting.Results. Hyaluronidase activity was detected in vitreous samples from both post-mortem and biopsy specimens. The enzyme was most active at acid pH, but demonstrated significant activity at neutral pH. The partially purified enzyme migrated as a 59 kDa protein on SDS-PAGE, and a single band on Western blots.Conclusions. Hyaluronidase is present in the human vitreous. Thus, hyaluronidase may be involved in HA catabolism in the vitreous and may play a role in determining its gel structure.

ISSN:0271-3683    

DOI:10.3109/02713689608995150

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. The present study was conducted to develop and characterize a functional primary culture of pigmented rabbit conjunctival epithelial cells on permeable support exhibiting tight barrier properties.Methods. Conjunctival epithelial cells were isolated by 0.2% protease treatment, cultured at 0.5–1.8×106cells/cm2onto collagen-treated Transwell filters, and were maintained either in the presence of 1 % fetal bovine serum throughout or serum-free media from day 3 onwards. Transepithelial potential difference (PD) and transepithelial electrical resistance (TEER) were measured and equivalent short-circuit current (Ieq= PD/ TEER) estimated.Results. There appears to be a critical plating density of 1.5×106cells/cm2for functional development of tight epithelial cell cultures. The culture conditions as noted above did not affect either the time when peak bioelectric parameters were attained (days 8–10) or the magnitude of these parameters at a plating density of 1.5×106cells/cm2. Specifically, cells grown in a serum-free media showed a peak TEER of 1.9±0.2 kÒ.cm2, a PD of 14.2±1.6 mV (apical side negative), and an Ieqof 8.0±0.4μA/cm2(mean±SEM, n = 45). Electron microscopy of serum-weaned cultures revealed a multilayered epithelium with numerous microvilli on the outermost layer of cells, while sporadic positive Periodic Acid Schiff (PAS) staining under light microscopy suggested the presence of mucin-secretory goblet cells.Conclusions. A functional, tight, epithelial barrier of the pigmented rabbit conjunctiva on a permeable support has been developed, which may be useful for mechanistic studies of ion and drug transport at the cellular level.

ISSN:0271-3683    

DOI:10.3109/02713689608995151

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. To evaluate the permeability characteristics of primary cultured rabbit conjunctival epithelial cell (RCEC) layers to low molecular weight drugs of varying lipophilicity.Methods.3H-mannitol; hydrophilic sotalol and atenolol; moderately lipophilic metoprolol, timolol, propranolol; and highly lipophilic betaxolol were used as model compounds.Results. The conjunctival apparent permeability coefficient (Papp) of mannitol (I×10−7cm/s) was 2.4 times lower than that of the most hydrophilicβ-blocker, sotalol (Papp= 2.4×107cm/s). Differences in the degree of tightness of the epithelial cell layers brought about a 30-fold difference in the transport of atenolol in favor of the leaky cell layers, while not affecting the transport of the lipophilic drug, propranolol. Within the log partition coefficient (PC) range of -0.62 (sotalol) and 3.44 (betaxolol), there was a hundred-fold difference in the Papp. A sigmoidal curve was used to depict the influence of lipophilicity on solute permeation across conjunctival epithelial cell layers. An effective half-maximal Pappwas observed at a log PC value of 1.2.Conclusions. These findings on the lipophilicity effect on drug transport are generally similar to those reported for the isolated rabbit conjunctiva, suggesting the utility of cultured rabbit conjunctival epithelial cell layers as anin vitromodel for evaluating drug transport.

ISSN:0271-3683    

DOI:10.3109/02713689608995152

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. To investigate the metabolic profile of the rabbit lens using high resolution1H NMR spectroscopy including two-dimensional shift correlated (COSY) technique.Methods. Perchloric acid extracts of the rabbit lens were analysed with a Bruker AM-500 spectrometer and the metabolites were assigned in the spectra. Some of these were also quantified.Results. More than 20 metabolites were detected in the perchloric acid extract of a single lens, including amino acids, nucleotides and other related compounds. Of particular importance is the ability to detect and identify glutathione, myo-inositol, scyllo-inositol and taurine.Conclusions. The study demonstrated the potential of1H NMR spectroscopy for monitoring the metabolic profile of the lens in normal and pathologic conditions.

ISSN:0271-3683    

DOI:10.3109/02713689608995153

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. Solutions of the bovine lens proteinγB (orγII) crystallin at neutral pH in the absence of reducing agents, undergo a slow, partial conversion to a new protein species,γIIH. This species is an aggregate composed of an intermolecular, disulfide-crosslinked dimer (≈32% of total protein by weight) and loosely associated dimers (≈66%).γIIHhas a phase separation temperature (Tph), at least 40d`C higher than that of nativeγII crystallin at any given protein concentration. In this paper we demonstrate that pantethine, a derivative of coenzyme A, inhibits the formation ofγIIH.Methods.γII crystallin solutions were incubated at pH 7.1 and room temperature with increasing amounts of pantethine. The Tphof the solutions was monitored as a function of incubation time. Corresponding to each Tphmeasurement, aliquots of each solution were analyzed by cation-exchange HPLC to determine the amount ofγIIHformed.Results. Incubation ofγII crystallin with increasing amounts of pantethine lowers Tphand suppresses the formation ofγIIH. With pantethine to protein mole ratios of 0.66, 1 and 2, the TphofγII crystallin is lowered from 8d`C in the native protein, to 2d`C, -3d`C and -4.3d`C respectively, at a protein concentration of≈200 mg/ml. The amount ofγIIHaccumulated decreases from∼25% in the native protein to 10%, 1% and 0% respectively in these pantethine-trcatcd protein solutions. For complete suppression of the rise in Tphand inhibition ofγIIHformation, a 2:1 mole ratio of pantethine to protein is required.Conclusions. We suggest that pantethine reacts with two cys-leine residues ofγII crystallin by forming a mixed disulfide, and effectively suppresses protein aggregation and lowers Tph. This is due to the strong polar character of pantethine which reduces the net attractive interactions between the protein molecules.

ISSN:0271-3683    

DOI:10.3109/02713689608995154

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. To investigate the activity profile of cyclic nucleotide phosphodiesterase (PDE) isoenzymes and the effects of isoen-zyme selective inhibitors in the superior and inferior lacrimal glands, Harderian gland, and zygomatic gland of the rabbit.Methods. Protein fractions extracted from crude homogenates on an anion exchange column were examined for PDE activity using an HPLC method for detecting nucleotides.Results. The superior and inferior lacrimal glands had identical PDE activity profiles. PDE 1 was the major type of activity and there was also a minor PDE III peak of activity. The main activity detected in the lipid secreting Harderian gland was PDE II and for the mucus secreting zygomatic gland PDE III. All glands contained PDE IV activity. The kinetics of the peak enzyme activities were examined and found similar, but not identical to the kinetics for PDE activities obtained from other tissues. Inhibitors of specific PDE classes and the general PDE inhibitor, IBMX, were tested on the peak enzyme activities. Activities designated by their substrate specificity or co-factor modification were most strongly inhibited by the corresponding class selective inhibitor. For example, PDE I activity in the lacrimal gland was most strongly inhibited by nicardipine. All activities were inhibited by IBMX.Conclusions. The superior and inferior lacrimal glands of the rabbit have the same PDE profile and this differs from the PDE subtypes detected in the mucus secreting zygomatic gland and the lipid secreting Harderian gland.

ISSN:0271-3683    

DOI:10.3109/02713689608995155

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. To characterize the effects of medium Ca2+levels on rabbit lens electrical properties. Early studies with wholly submerged lenses had shown that Ca2+removal from the bath resulted in an increased Rb+efflux, a consequence of an increased Na+permeability and lens depolarization.Methods. Lenses were bathed within Ussing-type chambers under short-circuited conditions, an arrangement in which the translens short-circuit current (lsc) is carried across the posterior lens surface mainly by an influx of Na+, and across the anterior face largely by a K+efflux.Results. Under the present conditions in which the effects of Ca2+were characterized unilaterally, the above established effects could only be ascribed to the posterior surface. When Ca2+removal was limited to the anterior face, the Iscincreased from 11.87±1.17 to 17.04±1.52μA/cm2(means±SE's, n = 18; an accompanying translens resistance (Rt) decrease of 0.23±0.049 KOM-cm2was also recorded). Conversely, increasing the control, anterior-bath [Ca2+] from 1.8 to 3.6 mM reduced the K+efflux-dependent Iscfrom 10.54±1.09 to 8.93±1.02 (n = 10, with an Rtincrease of 0.11±0.013). These changes were reversible, Na+-independent, and fully inhibited by the presence of K+channel blockers (quinidine or Ba2+). Inhibitions of the Ca2+effects were also obtained with strontium, a Ca2+surrogate. The Iscwas less responsive to changes in the Ca2+content of the posterior bath. Removal of the cation caused a gradual 1.65±0.72μA/cm2increase (n = 9, with an Rtdecrease of 0.090±0.021 KOM-cm2). In the absence of posterior Na+, Ca2+withdrawal resulted in highly variable responses, with some specimens exhibiting salient current increases, suggesting that an outwardly directed, posterior efflux of an anion could also have been affected. During the course of this study it was consistently observed that the removal of Na+from the anterior bath led to an Iscdecrease of 2.62±0.22μA/cm2(n = 32, with an Rtincrease of 0.35±0.029 KOM-cm2). This change occurred in both the presence of ouabain and the absence of Ca2+, suggesting that it did not result from an inhibition of the Na+-K+pump current nor from a reversal in putative Na+/Ca2+exchange activity. Small Iscincreases upon anterior Na+withdrawal (1.68±0.17, n = 7), consistent with Na+efflux from the lens, could only be observed with K+channels inhibited with Ba2+. Also congruent with the observations of a relatively limited anterior Na+permeability, was the finding that the induction of nonspecific cation channels with amphotericin B reduced the Iscby allowing Na+from the anterior bath to enter the lens. Thus, changes in lens Isc: can differentiate changes in K+permeability across the native anterior epithelium from changes in Na+permeability.Conclusions. Overall, these results suggest that lens Ca2+-mobilizing agents (e.g. acetylcholine) could trigger the inhibition of epithelial K+conductance(s) by the direct action of Ca2+on K+channels.

ISSN:0271-3683    

DOI:10.3109/02713689608995156

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. Corneal injury stimulates the formation of both pros-taglandins (PG) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), the major lipoxygenase metabolite. The purpose of this study was to investigate the metabolism of arachi-donic acid (AA) in a model of corneal graft rejection.Methods. Corneal tissue from Dutch belted rabbits was transplanted to vascularized corneas of New Zealand white rabbits. Rejected corneas were removed at the endstage of allograft failure. The allograft, the host corneal rim, the contralateral control corneal rim of equal size and normal Dutch belted cornea from the same site as the allograft were incubated with 0.25μCi [3H]AA and the released eicosanoids were analyzed by high-performance liquid chromatography.Results. The host corneal rims, adjacent to the failed allografts, produced up to five times as much 12(S)-hydroxyeicosatet-raenoic acid (12(S)-HETE) as contralateral control corneal rims. Additionally, prostaglandin E2(PGE2) formation in the host rims increased 100% above controls, and 12(S)-HETE and PGE2synthesis in the rejected corneal graft also increased. 12(R)-HETrE, an endogenous corneal angiogenic factor, was not detected in rejected corneas.Conclusions. The results point to the importance of selective AA pathways as the source of key inflammatory components found in rejected allografts.

ISSN:0271-3683    

DOI:10.3109/02713689608995157

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

ISSN:0271-3683    

DOI:10.3109/02713689608995158

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor