摘要:

PurposeIn this electron microscopical study, we compared effects of chemical fixation versus cryofixation on the ultrastructure of acute rod outer segment alterations in the rat retina.MethodsThe alterations were induced by toxic levels of diffuse white light. Albino rats were exposed to 2000 lux for 30 min. Samples from one eye of each animal were fixed by high pressure freezing and samples from the other eye were fixed by standard glutaraldehyde procedures.ResultsLight exposed retina showed major differences in their rod outer segments, inner segments and photoreceptor synaptic regions in chemical fixation. In particular gross vesiculations of outer segment membranes were produced in light exposed experiments. In contrast, in cryo-fixed samples such prominent changes were not observed in outer segment membranes. There was, however, occasional formation of small vesicles and a reduction of the cilium diameter in response to light damage. In the dark adapted control group the morphology of chemically fixed and cryo-fixed photoreceptors was similar.ConclusionsWe conclude, that cryo-fixed samples better represent the living state of the retina, because high pressure freezing is a purely physical method and acts much faster than chemical fixation. Curr. Eye Res. 15: 807-814, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017621

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

PurposeLiposomes and collagen corneal shields (CCS) have been used as ophthalmic drug delivery devices. With regard to a possibly combined application, we studied the effects of surface charge and bilayer fluidity of liposomes on their uptake and release by CCS.Methods12-hours-CCS were soaked in large unilamellar liposomes, which had been labelled with 4,5-carboxyfluorescein (CF) and N-(lissamine rhodamine B sulfonyl)-diacylphosphatidylethanolamine (PE-RhB) in the aqueous space and in the liposome bilayer, respectively. Released fluorophores were determined fluorometrically in the elution buffer at intervals from 1 to 240 min after immersion.ResultsThe CF concentration in the CCS soaked in a CF solution was two to seven times higher than immersion in the liposome suspensions. Among those, the negatively charged, cholesterol-containing preparation led to the highest CF concentration in the CCS. The PE-RhB concentration was highest after soaking the CCS in neutral, cholesterol-free liposomes. All types of liposomes were found inside the CCS by freeze fracture electron microscopy. The release kinetics data indicate a first order release. More than 90% of CF was released by the CCS within the first 30 min. This was equal after soaking the CCS in the CF solution or in liposomes. With DOPC-liposomes, the maximal release was already attained after 10 min. In general, the differences in the release kinetics of both hydrophilic and lipophilic markers, obtained by the various liposome types were small.ConclusionsOur results indicate that surface charge and bilayer fluidity are of minor importance for the interaction with collagen corneal shields. However, since the release kinetics of a liposomeencapsulated hydrophilic or lipophilic substance are similar to the release of a non-encapsulated drug, the combination of liposomes with collagen shields may be useful mainly with respect to the encapsulation of drugs which do not penetrate the ocular surface as well as to prolong corneal contact time of the liposomes. Curr. Eye Res. 15: 815–823, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017622

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

PurposeWhile transformation of epithelial cells to a motile form is the first step in wound healing of the corneal epithelium, the migratory mechanism in these cells is not fully understood. We studied the expression of proto-oncogene mRNAs: c-fos; c-jun; fos B; jun B; jun D in injured corneal epithelium usingin situhybridization. Moreover, we examined immunolocalization of c-Fos and cJun protein products to elucidate the transcriptional activation prior to the onset of migration in corneal epithelium.MethodsAn epithelial defect was made on one cornea of 60 Wistar rats. The affected eye was enucleated immediately (within 5 min) or was allowed to heal for 15, 30, 60, 90, 120 and 180 min. Frozen sections were processed forin situhybridization with c-fos, c-jun, fos B, jun B and jun D mRNAs or were stained with anti-c-fos and anti-c-jun antibodies.ResultsFifteen min after the epithelial ablation, weak signals for c-fos and c-jun mRNAs were detected in the corneal epithelium surrounding the wound. These signals reached a peak 30 to 60 min after ablation, but were no longer evident at 120 min. Immunoreactivities for these proteins were also detected in the same area at 60 to 120 min after the epithelial ablation. Fos B mRNA was detected in the same region at 30 min after the ablation, and reached its peak after 30 to 60 min, but was no longer evident at 120 min. Jun B mRNA was detected in the epithelium around the defect 60 min after the ablation, later than the other proto-oncogenes, and reached its peak after 90 min. The message for jun D was detected in normal epithelium, and was not affected by wounding.ConclusionsThese findings indicate that transcriptional activation of epithelial cells is initiated in the early phase after epithelial ablation, before the cells start to migrate, and that these proto-oncogene products may play important roles in wound healing in corneal epithelium. The time lag of the peak of expression of these proto-oncogenes might suggest the different roles for each proto-oncogene in this process. Curr. Eye Res. 15: 824–832, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017623

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

PurposeViral-mediated gene transfer to retina, as well as to other tissues, is evolving rapidly. We have evaluated the potential of a retroviral vector with an internal opsin promoter fragment to direct gene expression to retinal photoreceptor cells.MethodsTwo recombinant retroviral vectors were prepared; in each vector, a 1.4 kb fragment of the mouse opsin promoter was placed downstream from the neoRgene in the Moloney murine leukemia virus-based vector G1Na. The opsin promoter fragment was linked either to the cDNA for mouse rod photo-receptor phosphodiesterase (PDE)β-subunit or to the bacterial lacZ reporter gene. These vectors were tested for their ability to direct gene expression after transduction of 3T3 and Y79 cells, or of dissociated retinal cell cultures or retinal explants from neonatal mice.ResultsAs expected, PDEβ-subunit andβ-galactosidase mRNAs were expressed only at low levels in 3T3 fibroblasts and Y79 retinoblastoma cells. Northern blot analysis indicated that expression was derived from the viral long terminal repeat (LTR) promoter. Infection of primary retinal cell cultures or explants from neonatal mice with BAG retrovirus, in whichβ-galactosidase is driven by the viral LTR, resulted in expression in many cell types, while the opsin-lacZ vector mediated the expression of the lacZ reporter gene specifically in photoreceptor cells.ConclusionsThe internal opsin promoter fragment appears capable of selectively directing gene expression to photoreceptor cells after retroviral-mediated gene transfer. These findings serve as a basis for future studies using the opsin promoter-RPDE retroviral vector to rescue photoreceptor cells in therdmutant mouse, in which theβ-PDE gene is mutated resulting in degeneration of photoreceptor cells during the early postnatal period. Curr. Eye Res. 15 : 833–844, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017624

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

PurposeTo analyze the activities of catalase, glutathione peroxidase and superoxide dismutase, three enzymes involved in the detoxification of reactive oxygen species in organ-cultured Rhesus monkey lenses.MethodsLenses freshly obtained from Rhesus monkeys were incubated at 37°C for 2 h and assessed for lens integrity. Lenses were then oxidatively stressed by exposure to a bolus of hydrogen peroxide. The three enzyme activities were assayed 2, 4 and 24 h after exposure to the peroxide challenge.ResultsFreshly dissected lenses placed in organ culture exhibited a 20% decrease in catalase activity within 2 h. During the course of a 24 h incubation, catalase activity continued to decrease to a level 58% below that of freshly dissected monkey lenses. In contrast, the activity levels of both glutathione peroxidase and superoxide dismutase increased dramatically within the first 2 h of organ culture, with superoxide dismutase being most affected. Although glutathione peroxidase activity declined with incubation time, its level at the end of 24 h was still 36% greater than that of the fresh lenses. Superoxide dismutase activity remained elevated throughout the 24 h incubation period. The addition of a bolus of 0.25mM H2O2to monkey lenses in culture had no effect on catalase activity. Two h after the peroxide insult, glutathione peroxidase activity decreased in comparison to control levels while the activity of superoxide dismutase increased by 43%. After 24 h, superoxide dismutase activity returned to values equivalent to the controls. In lenses challenged with 0.50mM H2O2, catalase and glutathione peroxidase activities decreased at 2 h, while superoxide dismutase activity increased 67% above control levels. At subsequent timepoints, catalase activity increased and reached control levels. In contrast, glutathione peroxidase activity continued to decrease with time eventually reaching fresh lens levels. Superoxide dismutase activity levels remained elevated and were equivalent to control values at 24 h.ConclusionsThe data indicate that placement of monkey lenses into an organ culture system represents an environmental change sufficient to cause a response in antioxidant enzyme levels. The addition of H2O2to this environment caused only superoxide dismutase to be stimulated above control lens levels. Curr. Eye Res. 15: 845–851, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017625

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

PurposeTo quantify the corneal diffusion and metabolism of tritiated 12(R)-hydroxyeicosatetraenoic acid (12(R)HETE) in thein vitro-mounted rabbit cornea to determine if this compound or one of its metabolites can diffuse across the stroma to the corneal endothelium.MethodsThe studies were performed in a Lucite block perfusion chamber by placing tritiated 12(R)HETE on the tear side of the cornea under the following conditions: (A) cornea completely intact (endothelium and epithelium present); (B) cornea with epithelium removed; and (C) cornea with both epithelium and endothelium removed. Radioactivity of 12(R)HETE and metabolites were measured in the different corneal layers and in the corneal perfusates using scintillation spectroscopy. 12(R)-hydroxyeicosatetraenoic and its metabolites were then quantified in the tissue perfusates using high performance liquid chromatography (HPLC) analysis.Results12(R)HETE is rapidly taken up and metabolized by the intact cornea to a number of more polar compounds including a metabolite which has spectral and retention time characteristics of 8(R)-hydroxyhexadecatrienoic acid (8(R)HHDTrE). Both 12(R)HETE and 8(R)HHDTrE can diffuse through the stroma to the endothelium. Corneas having the epithelium removed also allow diffusion of 12(R)HETE across the stroma; however, there is significantly less metabolism. When both the epithelium and endothelium are removed, 12(R)HETE is capable of diffusing across the stroma; however, there is little metabolism, which suggests that the majority of 12(R)HETE metabolism occurs in the epithelium and, to a lesser degree, in the endothelium and stroma.Conclusions12(R)HETE and its metabolites are capable of diffusing from the epithelium through the cornea where they may adversely affect the endothelium. Curr. Eye Res. 15: 852–859, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017626

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

PurposeTo compare the susceptibility of crystallins from various animal species to formation of light scattering elements after proteolysis by calpain II enzyme (EC 3.4.22.17).MethodsLens, total soluble proteins from: 12-day and 4-week old rat, fetal and adult bovine, 16-day embryonic and 10-week chicken, and young human cortex and nucleus were proteolyzed by either endogenous lens calpain or addition of purified calpain II for 24 h followed by incubation for up to 11 days. Absorbance of light at 405 nm estimated light scattering by crystallins; SDS-PAGE and 2D-electrophoresis assessed proteolysis on the crystallins.ResultsMost rapid light scattering occurred with total soluble proteins from young rat lens, either after adding purified calpain or by activating endogenous lens calpain with calcium. (Only rat lens showed activation of endogenous calpain II.)β-crystallin polypeptides from rat, bovine, human, and to a more limited extent, chick lens were partially proteolyzed by addition of purified calpain II. In spite of this proteolysis, total soluble proteins from chicken, bovine, and human lenses showed no obvious light scattering by action of calpain. Crystallins from older rat lens showed approximately 50% of the light scattering displayed by crystallins from younger rats after 3 days, but only when purified calpain was added.ConclusionsOur results indicate an unusually high susceptibility of crystallin polypeptides from young rat lens to formation of light scattering elements after limited proteolysis. Thus, young rat lens provides a unique opportunity to investigate how properties of crystallins influence the development of light scattering found in cataract. Curr. Eye Res. 15: 860–868, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017627

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

PurposeThe mechanism by which prostaglandin(PG)F2αincreases uvcoscleral outflow and lowers intraocular pressure in primates is not known. In cultured human ciliary muscle cells, PGF2α, induces the expression of the protooncogene c-fos which is known to induce the transcription of genes such as matrix metalloproteinase-1 (MMP-1) and MMP-3 in other cell systems. As these enzymes are initially secreted as proenzymes, the present study was undertaken to determine if PG treatment induces ciliary muscle cells to secrete either proMMP-1 or proMMP-3.MethodsHuman ciliary smooth muscle cells were grown to confluence in monolayer cell cultures and then treated with PGF2α, 17-phenyltrinor-PGF2α, or 11-deoxy-PGE1. Medium harvested at various times after treatment was assayed for proMMP-1 and proMMP-3 content using sandwich ELISAs.ResultsThree days after adding 10 nM PGF2α, proMMP-1 and pro-MMP-3, concentrations in the culture medium were increased by 254±33% (mean±SE) and 128±13%, respectively. Compared with vehicle controls, 24 h treatment with 200 nM PGF2α, 17-phenyltrinor-PGF2α, or PGE1, increased proMMP-1 by 116±29%, 169±26%, and 273±16%, respectively. In parallel experiments, proMMP-3 was increased by 99±18%, 82±24%, and 214±16%, respectively.ConclusionsThese results suggest that induction of MMPsin situfollowing topical PG treatment may degrade ciliary muscle extracellular matrix and possibly contribute to increased uveoscleral outflow, as well. Curr. Eye Res. 15: 869–875, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017628

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

PurposeIL-15 and IL-15 receptor expression was measured in retinal pigment epithelial (RPE) cells to support a possible role of IL-15 in ocular inflammatory and immune responses.MethodsReverse transcription-coupled polymerase chain reaction (RT-PCR) and Northern blot analysis of IL-15 mRNA in previously characterized non-transformed and simian virus (SV)-40 transformed human fetal RPE cells were carried out. Biological activities of IL-15 produced by the RPE cells were assayed by co-culture with IL-15 responsive cells. Expression of the IL-15 receptor (IL-15R)α, IL-2Rβandγchains were examined by RT-PCR.ResultsBoth non-transformed and SV-40 transformed human fetal RPE cells express IL-15, a T cell growth factor which has similar biological activities to IL-2, and the expression of IL-15 is enhanced by interferon-γ(IFN-γ) or tumor necrosis factor-α(TNF-α) stimulation. In addition, transcripts for all three IL-15 receptor components (IL-15Rα, IL-2Rβand IL-2Rγ) were detected in these cells.ConclusionsRPE cells produce IL-15, which may play an important role in ocular immune and inflammatory responses by stimulating infiltrated T cells and RPE cells via paracrine and autocrine loops, respectively. Curr. Eye Res. 15: 876–882, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017629

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

ISSN:0271-3683    

DOI:10.3109/02713689609017630

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor