RESUMO
Although organic solvents have been associated with CNS toxicity, neurotoxicity testing is rarely a regulatory requirement. We propose a strategy to assess the potential neurotoxicity of organic solvents and predict solvent air concentrations that will not likely produce neurotoxicity in exposed individuals. The strategy integrated an in vitro neurotoxicity, an in vitro blood-brain barrier (BBB), and an in silico toxicokinetic (TK) model. We illustrated the concept with propylene glycol methyl ether (PGME), widely used in industrial and consumer products. The positive control was ethylene glycol methyl ether (EGME) and negative control propylene glycol butyl ether (PGBE), a supposedly non-neurotoxic glycol ether. PGME, PGBE, and EGME had high passive permeation across the BBB (permeability coefficients (Pe) 11.0 × 10-3, 9.0 × 10-3, and 6.0 × 10-3 cm/min, respectively). PGBE was the most potent in in vitro repeated neurotoxicity assays. EGME's main metabolite, methoxyacetic acid (MAA) may be responsible for the neurotoxic effects reported in humans. No-observed adverse effect concentrations (NOAECs) for the neuronal biomarker were for PGME, PGBE, and EGME 10.2, 0.07, and 79.2 mM, respectively. All tested substances elicited a concentration-dependent increase in pro-inflammatory cytokine expressions. The TK model was used for in vitro-to-in vivo extrapolation from PGME NOAEC to corresponding air concentrations (684 ppm). In conclusion, we were able to predict air concentrations that would not likely result in neurotoxicity using our strategy. We confirmed that the Swiss PGME occupational exposure limit (100 ppm) will not likely produce immediate adverse effects on brain cells. However, we cannot exclude possible long-term neurodegenerative effects because inflammation was observed in vitro. Our simple TK model can be parameterized for other glycol ethers and used in parallel with in vitro data for systematically screening for neurotoxicity. If further developed, this approach could be adapted to predict brain neurotoxicity from exposure to organic solvents.
Assuntos
Éter , Propilenoglicóis , Humanos , Toxicocinética , Propilenoglicóis/metabolismo , Propilenoglicóis/toxicidade , Éteres/toxicidade , Etilenoglicóis/toxicidade , Etilenoglicóis/metabolismo , SolventesRESUMO
A growing number of companies within the cheese-making industry are now using high-protein (e.g., 4-5%) milks to increase cheese yield. Previous studies have suggested that cheeses made from high-protein (both casein and whey protein; WP) milks may ripen more slowly; one suggested explanation is inhibition of residual rennet activity due to elevated WP levels. We explored the use of microfiltration (MF) to concentrate milk for cheese-making, as that would allow us to concentrate the casein while varying the WP content. Our objective was to determine if reducing the level of WP in concentrated cheese milk had any impact on cheese characteristics, including ripening, texture, and nutritional profile. Three types of 5% casein standardized and pasteurized cheese milks were prepared that had various casein:true protein (CN:TP) ratios: (a) control with CN:TP 83:100, (b) 35% WP reduced, 89:100 CN:TP, and (c) 70% WP reduced, 95:100 CN:TP. Standardized milks were preacidified to pH 6.2 with dilute lactic acid during cheese-making. Composition, proteolysis, textural, rheological, and sensory properties of cheeses were monitored over a 9-mo ripening period. The lactose, total solids, total protein, and WP contents in the 5% casein concentrated milks were reduced with increasing levels of WP removal. All milks had similar casein and total calcium levels. Cheeses had similar compositions, but, as expected, lower WP levels were observed in the cheeses where WP depletion by MF was performed on the cheese milks. Cheese yield and nitrogen recoveries were highest in cheese made with the 95:100 CN:TP milk. These enhanced recoveries were due to the higher fraction of nitrogen being casein-based solids. Microfiltration depletion of WP did not affect pH, sensory attributes, or insoluble calcium content of cheese. Proteolysis (the amount of pH 4.6 soluble nitrogen) was lower in control cheeses compared with WP-reduced cheeses. During ripening, the hardness values and the temperature of the crossover point, an indicator of the melting point of the cheese, were higher in the control cheese. It was thus likely that the higher residual WP content in the control cheese inhibited proteolysis during ripening, and the lower breakdown rate resulted in its higher hardness and melting point. There were no major differences in the concentrations of key nutrients with this WP depletion method. Cheese milk concentration by MF provides the benefit of more typical ripening rates.
Assuntos
Queijo , Animais , Queijo/análise , Leite/química , Proteínas do Soro do Leite/análise , Caseínas/análise , Proteólise , Cálcio/análise , Manipulação de Alimentos/métodos , Nitrogênio/análise , Concentração de Íons de HidrogênioRESUMO
Bisphenol A (BPA) in vitro skin permeation studies have shown inconsistent results, which could be due to experimental conditions. We studied the impact of in vitro parameters on BPA skin permeation using flow-through diffusion cells with ex-vivo human skin (12 donors, 3-12 replicates). We varied skin status (viable or frozen skin) and thickness (200, 400, 800 µm), BPA concentrations (18, 250 mg/l) and vehicle volumes (10, 100 and 1000 µl/cm2). These conditions led to a wide range of BPA absorption (2%-24% after 24 h exposure), peak permeation rates (J = 0.02-1.31 µg/cm2/h), and permeability coefficients (Kp = 1.6-5.2 × 10-3 cm/h). This is the first time steady state conditions were reached for BPA aqueous solutions in vitro (1000 µl/cm2 applied at concentration 250 mg/l). A reduction of the skin thickness from 800 and 400 µm to 200 µm led to a 3-fold increase of J (P < 0.05). A reduction of the vehicle volume from 1000 to 100 led to a 2-fold decrease in J (P > 0.05). Previously frozen skin led to a 3-fold increase in J compared to viable skin (P < 0.001). We found that results from published studies were consistent when adjusting J according to experimental parameters. We propose appropriate J values for different exposure scenarios to calculate BPA internal exposures for use in risk assessment.
Assuntos
Compostos Benzidrílicos/farmacologia , Fenóis/farmacologia , Absorção Cutânea , Pele/metabolismo , Administração Cutânea , Humanos , Técnicas In Vitro , Pele/anatomia & histologiaRESUMO
Some European dairies use low concentration factor microfiltration (MF) in their cheese plants. Removal of whey protein (WP) from milk before cheesemaking using microfiltration without concentration provides the opportunity to produce a value-added by-product, milk-derived whey. However, few studies have focused on the effects on cheese properties caused by the depletion of WP from cheese milk. Most studies have concentrated cheese milk using MF in addition to depletion of WP. In our approach, cheese milk was not concentrated during WP depletion using MF. We wanted to quantify residual WP levels in cheese made from MF milk and to explore whether WP depletion from milk would influence functionality, nutritional profile, and cheese quality during ripening. Casein (CN) contents for all milks were kept at â¼2.5%, to eliminate the confounding factor of concentration of CN, which was observed in some previous MF studies. Cheese milks had similar ratios of CN to fat. Three standardized milks were produced with various CN:true protein (TP) ratios: (a) control with a CN:TP ratio of 83:100, (b) 35% WP depletion, 89:100 CN:TP, and (c) 70% WP depletion, 95:100 CN:TP. Cheddar cheeses were made from MF milk with various WP depletion levels and aged for 9 mo, and their functionality was evaluated during ripening. We found no major differences in cheese composition or pH values between samples. Cheese yield, solids recovery, and nitrogen recovery were slightly higher in the 95:100 CN:TP cheeses compared with the control. These enhanced recoveries reflect that MF-treated milk started with a higher fraction of CN-based protein solids, rather than WP solids. The standardized milk from the 95:100 CN:TP treatment also had a slightly higher fat content compared with the control, likely helping to increase cheese yield. Rheological properties of cheeses during heating were similar between treatments. Hardness initially decreased with age for all cheeses due to proteolysis or solubilization, or both, of calcium phosphate. Maximum loss tangent (LT), an index of cheese meltability, was slightly lower for the control cheese until 30 d of ripening, but after 30 d, all treatments exhibited similar maximum LT values. The temperature where LT = 1 (crossover temperature), an index of softening point during heating, was slightly lower for MF cheese compared with the control cheeses during ripening. Microfiltration treatment had no significant influence on proteolysis. Sensory properties were similar between the cheeses, except for bitterness. Bitterness intensity was slightly lower in the MF cheeses than in the control cheeses and increased in all cheeses during ripening. We detected no major differences in the concentrations of key nutrients or vitamins between the various cheeses. Depletion of WP in cheese milk by MF did not negatively affect cheese quality, or its nutritional profile, and resulted in similar cheesemaking yields.
Assuntos
Queijo/análise , Leite/química , Proteínas do Soro do Leite/análise , Animais , Caseínas/análise , Queijo/normas , Filtração , Manipulação de Alimentos , Nitrogênio/análise , Reologia , Paladar , TemperaturaRESUMO
At the 8th conference of Occupational and Environmental Exposure of the Skin to Chemicals (OEESC) (16-18 September 2019) in Dublin, Ireland, several researchers performing skin permeation assays convened to discuss in vitro skin permeability experiments. We, along with other colleagues, all of us hands-on skin permeation researchers, present here the results from our discussions on the available OECD guidelines. The discussions were especially focused on three OECD skin absorption documents, including a recent revision of one: i) OECD Guidance Document 28 (GD28) for the conduct of skin absorption studies (OECD, 2004), ii) Test Guideline 428 (TGD428) for measuring skin absorption of chemical in vitro (OECD, 2004), and iii) OECD Guidance Notes 156 (GN156) on dermal absorption issued in 2011 (OECD, 2011). GN156 (OECD, 2019) is currently under review but not finalized. A mutual concern was that these guidance documents do not comprehensively address methodological issues or the performance of the test, which might be partially due to the years needed to finalize and update OECD documents with new skin research evidence. Here, we summarize the numerous factors that can influence skin permeation and its measurement, and where guidance on several of these are omitted and often not discussed in published articles. We propose several improvements of these guidelines, which would contribute in harmonizing future in vitro skin permeation experiments.
Assuntos
Congressos como Assunto/normas , Exposição Ambiental/normas , Fidelidade a Diretrizes/normas , Exposição Ocupacional/normas , Organização para a Cooperação e Desenvolvimento Econômico/normas , Absorção Cutânea/efeitos dos fármacos , Exposição Ambiental/prevenção & controle , Substâncias Perigosas/metabolismo , Substâncias Perigosas/toxicidade , Humanos , Irlanda , Exposição Ocupacional/prevenção & controle , Absorção Cutânea/fisiologiaRESUMO
Renin immunoreactivity was localized at the light and electron microscopic level in two elasmobranch fish species, the Atlantic stingray, Dasyatis sabina, and river ray, Potamotrygon humerosa. At the light microscopic level, the peroxidase-anti-peroxidase method showed a positive immunoreactivity in modified smooth muscle cells in kidney afferent arterioles as well as in arterioles of several organs: rectal gland, inter-renal gland, conus arteriosus, and gill. Electron microscopic renin-positive immunogold localization was confined to the contents of membrane bound granules in the modified smooth muscle cells of these arterioles. The presence of renin-containing granules in the modified smooth muscle, "granular cells," of the renal glomerular afferent arteriole of these two stingray species adds support to earlier studies which showed the structural components of a complete juxtaglomerular apparatus and some of the biochemical and molecular components of a renin-angiotensin system (RAS) as found in teleost fish, reptiles, birds, and mammals. A notable result, however, was the renin-positive immunoreaction in the arteriolar wall of all other organs studied here. The presence of this "diffuse renin system" in the connective tissue of various organs suggests that in these two stingray species in addition to local organ-specific functions, the RAS may act as a systemic mechanism to regulate blood pressure and blood flow in the body.
Assuntos
Elasmobrânquios/metabolismo , Miócitos de Músculo Liso/metabolismo , Renina/metabolismo , Animais , Arteríolas/anatomia & histologia , Arteríolas/metabolismo , Arteríolas/ultraestrutura , Elasmobrânquios/anatomia & histologia , Mucosa Gástrica/metabolismo , Imuno-Histoquímica , Rim/anatomia & histologia , Rim/metabolismo , Microscopia Eletrônica , Miócitos de Músculo Liso/ultraestrutura , Reto/metabolismo , Reto/ultraestrutura , Estômago/anatomia & histologiaRESUMO
The mechanisms responsible for the progression of nonalcoholic fatty liver disease (NAFLD) to more severe liver injury are still poorly understood. Data from animal models suggest that oxidative stress contributes to steatohepatitis and an increase of lipid peroxidation has been documented in human NAFLD. By measuring the titers of circulating antibodies against lipid peroxidation products as markers of oxidative stress we have observed that NAFLD patients have titers of these antibodies significantly higher than in controls. Moreover, the titers of lipid peroxidation-related antibodies are associated with a 3-fold increase in the risk of developing advanced fibrosis/cirrhosis. Although the mechanisms causing oxidative stress in NAFLD have not been elucidated, these results support the involvement of lipid peroxidation in the processes leading to liver fibrosis associated with NAFLD.
Assuntos
Fígado Gorduroso/metabolismo , Estresse Oxidativo/fisiologia , Anticorpos/sangue , Progressão da Doença , Fígado Gorduroso/complicações , Fígado Gorduroso/imunologia , Humanos , Peroxidação de Lipídeos/imunologia , Peroxidação de Lipídeos/fisiologia , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismoRESUMO
AIMS: Factors responsible for the progression of non-alcoholic fatty liver disease (NAFLD) to more severe liver injury are poorly understood. In the present study, we investigated the association between immune reactions triggered by oxidative stress and stage of NAFLD. METHODS: Titres of IgG against human serum albumin adducted with malondialdehyde (MDA-HSA) or arachidonic acid hydroperoxide (AAHP) and against oxidised cardiolipin (Ox-CL) were measured in 167 NAFLD patients with steatosis only (n = 79), steatohepatitis (n = 74), or steatosis plus cirrhosis (n = 14), and in 59 age and sex matched controls. RESULTS: Circulating IgG against lipid peroxidation products was significantly higher (p<0.001) in NAFLD patients than in controls. Oxidative stress dependent immune responses were not associated with obesity, type 2 diabetes, or with serum cholesterol, ferritin, or aminotransferase levels. Titres of lipid peroxidation related antibodies were also independent of the extent of steatosis and were similarly distributed in patients with and without necroinflammation. In contrast, the same antibodies were significantly increased in patients with advanced fibrosis or cirrhosis. Logistic regression analysis confirmed that anti-MDA antibodies were independently associated with progression of NALFD and that NAFLD patients with titres of anti-MDA-HSA antibodies above the control threshold value had a threefold (relative risk 2.82 (95% confidence interval 1.35-5.90); p = 0.007) higher risk of having advanced fibrosis/cirrhosis than patients whose antibody titres were within the control range. CONCLUSIONS: These results indicate that the presence of immune reactions triggered by oxidative stress can be an independent predictor of progression of NAFLD to advanced fibrosis.
Assuntos
Autoanticorpos/sangue , Fígado Gorduroso/imunologia , Peroxidação de Lipídeos/imunologia , Cirrose Hepática/imunologia , Adulto , Idoso , Biomarcadores/sangue , Progressão da Doença , Fígado Gorduroso/complicações , Feminino , Humanos , Imunoglobulina G/sangue , Cirrose Hepática/etiologia , Modelos Logísticos , Masculino , Malondialdeído/imunologia , Pessoa de Meia-Idade , Estresse Oxidativo/imunologia , Albumina Sérica/imunologiaRESUMO
Short-chain fatty acids (SCFA) in particular butyrate are regarded as an energy source acting in beneficial, protective manner on the colonic mucosa. Previous investigations showed that the colonic mucosa bathed in Ussing chamber with a solution lacking butyrate induced massive apoptosis of epithelial cells. The apoptotic material (bodies and cells) was shed at the mucosa surface. In the present study we aimed to investigate the effects caused in vivo on the colonic mucosa by the absence of butyrate. For this purpose the colon of guinea pigs was perfused in situ with solutions either containing or lacking butyrate. The results show that within 2h of perfusion without butyrate a large amount of epithelial cells underwent apoptosis as in the in vitro experiments. However, apoptotic material instead to be extruded at the epithelial surface accumulates into the intercellular spaces from which it becomes removed by an unusual high number of macrophages. These, engorged with phagocytozed material, lie assembled in a layer below the epithelium. Similar alterations have not been observed after perfusion in the presence of butyrate. The results suggest that this SCFA may protect the colonic mucosa in that it prevents apoptosis. The alterations occurring during 2h of its absence allow to assume that a protracted butyrate deprivation may lead to a breakdown of the integrity of the mucosa thus influencing differently the activity of the macrophages.
Assuntos
Apoptose/fisiologia , Butiratos/metabolismo , Colo/metabolismo , Colo/patologia , Células Epiteliais/patologia , Ácidos Graxos Voláteis/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Animais , Apoptose/efeitos dos fármacos , Butiratos/farmacologia , Colo/fisiopatologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Ácidos Graxos Voláteis/farmacologia , Cobaias , Mucosa Intestinal/fisiopatologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Macrófagos/ultraestrutura , Masculino , Microscopia EletrônicaRESUMO
The structure of the membrana limitans interna (MLI) in the region of the macula has been investigated by electron microscopy in (a) 2 enucleated human adult eyes and (b) 38 surgically removed samples associated with an epiretinal membrane (ERM). In the enucleated eye, the glia cells were vitrad bordered either by the lamina rara or, directly, by the lamina densa. Both extended into a coarse network whereby the lamina densa, through repeated branches and anastomoses, delimited large meshes, the lamina rara formed their contents. High magnification revealed that both meshes and contents of this network were composed by a further, finer network. It is suggested that strips and small openings of the finer network are homologous to the cords and intercordal spaces, respectively, which have been indicated as the common, basic structures of most of the basement membranes. The MLI excised with an ERM had the same structure. In some of the ERM associated with a macular hole, myofibroblasts prevailed among the cells. They showed indented nucleus, stress fibers abuting on the plasma membrane or in apparent continuity with bundles of extracellular filaments (microtendons), gap junctions. The cells lay on or were surrounded by a discontinuous basement membrane.
Assuntos
Membrana Basal/patologia , Membrana Basal/ultraestrutura , Membrana Epirretiniana/patologia , Neuroglia/patologia , Neuroglia/ultraestrutura , Retina/patologia , Retina/ultraestrutura , Perfurações Retinianas/patologia , Idoso , Membrana Basal/fisiopatologia , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Membrana Epirretiniana/fisiopatologia , Feminino , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Humanos , Masculino , Microfibrilas/patologia , Microfibrilas/ultraestrutura , Microscopia Eletrônica , Pessoa de Meia-Idade , Retina/fisiopatologia , Perfurações Retinianas/fisiopatologiaRESUMO
The organization of the aggregates occurring in the stroma: (1) of the murine and human cornea after incubation in an ATP acidic solution; (2) of surgically excised epiretinal membranes (ERM); and (3) of the trabecular meshwork of monkey eyes was investigated morphologically and immunocytochemically on thin section electron microscopy. Morphology. The aggregates in the cornea appeared as cross-banded fibrils. The bands were uniformly electron dense (single banded form); they were separated from each other by interbands consisting of a bundle of filaments emerging in cross section as small areas of randomly assembled dot-like structures. In the ERM, most of the aggregates stood out as heteromorphic cross-banded bodies showing dense bands with electron denser borders (double banded form) and interbands composed of longitudinally oriented, parallel sheets or laminae of amorphous material enclosing thin, similarly oriented filaments. These extended, thinner and double in number (since interlacing with similar components of the opposite sheet), into the pale central zone of the dense band. The aggregates of the trabecular meshwork were heteromorphic, had uniformly dense bands (single banded form as in the cornea), but their interbands displayed longitudinal sheets (as the ERM aggregates). Immunocytochemistry revealed type VI collagen in the three eye aggregates with gold particles preferentially localized at the interbands. The specificity of the antibodies used was tested by Western blot analysis of type VI collagen samples extracted from human placenta and on homogenates of human cornea. In conclusion, the results indicate that the tetramers of type VI collagen may aggregate differently into structures with distinct supramolecular arrangements. These are illustrated in schematic drawings.
Assuntos
Colágeno/metabolismo , Olho/metabolismo , Idoso , Animais , Olho/patologia , Feminino , Humanos , Masculino , Camundongos , Microscopia Imunoeletrônica/métodos , Pessoa de Meia-Idade , Coelhos , Retina/metabolismo , Retina/patologia , Inclusão do Tecido , Malha Trabecular/metabolismo , Malha Trabecular/patologiaRESUMO
Epoxy resins provide optimal tissue morphology at both the light and the electron microscopic level and therefore enable correlative studies on semithin and thin sections from the same tissue block. Here we report on an approach to retain these advantages for immunolabeling studies by adapting and combining well-known techniques, i.e., surface etching with sodium ethoxide and heat-mediated antigen retrieval. We propose a simple procedure for immunostaining semithin and thin epoxy resin sections. To check its applicability, well characterized, commercially available antibodies (against E-cadherin, alpha-catenin, and beta-catenin) were used on sections of human small intestine. By light microscopy, the immunostaining efficiency was compared on cryo-, paraffin, and epoxy semithin sections processed in parallel. The most detailed results were obtained on semithin sections, where the labeling precisely delineated the lateral plasma membrane of the enterocytes. At the electron microscopic level the procedure did not damage the structures and allowed an efficient, reproducible immunogold labeling extending homogeneously over exceptionally wide tissue areas. The three antibodies specifically labeled the zonula adherens of the junctional complex between epithelial cells and, in agreement with light microscopic observations, the lateral plasma membrane.
Assuntos
Resinas Epóxi , Microscopia/métodos , Inclusão em Plástico , Transativadores , Caderinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Humanos , Imuno-Histoquímica , Jejuno/metabolismo , Luz , Microscopia Imunoeletrônica/métodos , alfa Catenina , beta CateninaRESUMO
The brush cells (BC) are the second most frequent cellular component of the epithelium of the mouse gallbladder. They have a topographical distribution, being present in large numbers toward the neck and in the fundic regions of the organ and are scattered in the body. Serial section studies demonstrate that BC have a characteristic shape consisting of a narrow apical portion, bulky body and basal cytoplasmic projections. BC are located obliquely among the principal cells. Scanning electron microscopy demonstrates that the microvilli forming the prominent brush border, after which the cell was named, have a triangular arrangement. Due to their size and stiffness, the microvilli of BC have more similarity with stereocilia of sensory cells than with conventional microvilli. Furthermore freeze-fracture replicas demonstrate that, like stereocilia, the P face of the microvilli plasma membrane of BC is smoother than the E face but several intramembranous particles form small aggregates on the microvillus tip of both P and E faces. Numerous intramembranous particles are scattered on the lateral plasma membrane. An unusual, spatially organized cytoskeleton characterizes the apical cytoplasm of BC. The use of the appropriate fixative reveals that it consists of bundles of actin filaments originating from the axis of the apical microvilli and stretching continuously up to the supranuclear region of the cell. Microtubuli, also assembled in bundles, flank in alternating manner the actin filaments over their whole course. Due to the strong parallel arrangement of both cytoskeletal structures, the apical cytoplasm of the BC assumes a typical stiffness, observable in both thin sections and freeze-fracture replicas. A variable number of vesicles of different size are aligned between the bundles of actin filaments and microtubuli; their shape is highly influenced by the fixative used. Intraluminal injection of horseradish peroxidase demonstrates that these vesicles are not resorptive as they are not filled by the tracer. The BC possess a large number of lateral microvilli. These, whether single or in pairs, are rigid cytoplasmic protrusions that leave the lateral surface of the cell in all directions and penetrate deeply into the cytoplasm of the adjacent principal cells. The bundle of actin filaments emanating from each lateral microvillus extends at different angles into the cytoplasm. A conspicuous amount of bundles of 10 nm filaments is intertwined around the nucleus and extends toward the desmosomes of the lateral plasma membrane and into the basal cellular body. Arguments are considered in support of the view that interactions between the plasma membrane with its differentiations on the one hand and the cytoskeleton elements on the other hand, play a key role in the function of BC as a receptor (sensory) cell.
Assuntos
Vesícula Biliar/ultraestrutura , Animais , Núcleo Celular/ultraestrutura , Tamanho Celular/fisiologia , Citoplasma/ultraestrutura , Camundongos , Microvilosidades/ultraestruturaRESUMO
UNLABELLED: The recommended treatment for full-thickness macular holes is removal of the posterior hyaloid and sometimes the epiretinal membrane from the retina during vitrectomy in order to release the assumed intravitreous traction. We have employed a technique involving the additional removal of the membrana limitans interna (MLI) from the retina in the vicinity of the macular hole. We report on our clinical results and ultrastructural findings. MATERIALS AND METHODS: Between December 1995 and July 1996, we performed vitrectomies on 39 eyes of 37 patients with full-thickness macular hole. After removal of the attached posterior hyaloid, a specially developed forceps was used to remove a circular area of the MLI approximately three to four disc diameters in size. At the conclusion of the operation, 20% C3F8 gas was injected and the patient instructed to stay in a prone position for 8 days. RESULTS: Intraoperatively, "rhexis" of the MLI only rarely produced bleeding or recognizable retinal edema. Complete closure of the hole was observed postoperatively in 36 of the 39 eyes (92%). A visual improvement of at least two lines was achieved in 77% of eyes with successful closure. Pigment irregularities or edematous changes could not be detected either clinically or by fluorescein angiography in any of the 39 eyes. Electron microscopy was performed on 23 of the membranes. The salient feature was the MLI. Canals leading from the inner to the outer surface of the MLI contained Müller cell processes with clear signs of necrosis or degeneration. On the vitreous side, the MLI usually exhibited myofibroblasts. CONCLUSIONS: The MLI was successfully removed in all 39 eyes with a full-thickness macular hole. This procedure led to very good anatomic and functional results. It remains for future studies to determine the pathogenic significance of the necrotic processes detected by electron microscopy in the MLI canals.
Assuntos
Membrana Epirretiniana/cirurgia , Hialina , Perfurações Retinianas/cirurgia , Vitrectomia/métodos , Adulto , Idoso , Membrana Epirretiniana/patologia , Feminino , Humanos , Hialina/ultraestrutura , Macula Lutea/patologia , Macula Lutea/cirurgia , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Perfurações Retinianas/patologia , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: Butyrate stimulates proliferation and suppresses differentiation in normal colonic epithelial cells. Because the involved intracellular signaling mechanisms are unclear, this study investigated certain molecular effects of butyrate. METHODS: Tissue sheets from guinea pig proximal colon were incubated in Ussing chambers in the presence and absence of butyrate. Colonic tissues were examined by scanning and transmission electron microscopy, DNA laddering, Western blots, and immunohistochemistry. RESULTS: After incubation of the colonic mucosa for 150 minutes without butyrate, morphological studies showed massive apoptosis of colonocytes. Simultaneously, these colonocytes exhibited a significant oligonucleosomal DNA fragmentation. In contrast, addition of physiological concentrations of butyrate (10 mmol/L) to colonic sheets showed no detectable DNA fragmentation within 150 minutes. Western blot analysis showed little if any difference in the level of Bcl-2 expression in colonocytes incubated with or without butyrate up to 150 minutes. In contrast, expression of Bax proteins continuously increased after 45 minutes without butyrate and reached a fivefold induction after 150 minutes compared with cells incubated in the presence of butyrate. Moreover, immunohistochemistry using an anti-Bax antibody system showed enhanced labeling of the epithelial colonocytes in the absence of butyrate. CONCLUSIONS: Removal of butyrate induces increased expression of Bax proteins paralleled by rapid apoptosis of colonocytes in vitro.
Assuntos
Apoptose , Butiratos/farmacologia , Colo/patologia , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/análise , Animais , Butiratos/análise , Ácido Butírico , Divisão Celular/efeitos dos fármacos , Colo/química , Colo/ultraestrutura , Fragmentação do DNA , Cobaias , Masculino , Proteína X Associada a bcl-2RESUMO
Butyrate exerts a trophic effect on the colonocytes and plays a protective role in ulcerative colitis. In the present study, we investigated the effect of butyrate withdrawal on the colonic mucosa of the guinea-pig. The samples were mounted in Ussing chambers and bathed for 45, 60, 90 and 150 min with standard Ringer solution with or without sodium butyrate. Light and electron microscopy for morphology, electrophysiological methods for testing tissue function, histochemistry using the TUNEL method for localization of apoptotic cells and flow cytometry for cell cycle analysis were applied. Morphological observations revealed that butyrate deprivation caused a time-dependent hypoplasia and a rapid triggering of massive apoptosis as substantiated by the TUNEL assay. The epithelium, however, did not show discontinuities at any time. Electrophysiological data confirmed that no leakage of the epithelium had occurred. In the control specimens, the mucosa underwent a moderate reduction in height; apoptotic epithelial cells were infrequently observed. Cell cycle analysis of colonocytes isolated from the mucosa deprived of butyrate revealed a decrease in the percentage of cells occupying each phase of the cycle, especially the G0/G1 phase. Thus, in the absence of butyrate, apoptosis was enhanced and cell renewal reduced. The trophic protective action exerted by butyrate in both physiological and pathological conditions could derive from its capacity to modulate survival and death of colonocytes.
Assuntos
Apoptose/efeitos dos fármacos , Butiratos/farmacologia , Ciclo Celular/efeitos dos fármacos , Colo/citologia , Mucosa Intestinal/efeitos dos fármacos , Animais , Biotina , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Colo/efeitos dos fármacos , Dano ao DNA , Nucleotídeos de Desoxiuracil , Cultura em Câmaras de Difusão , Eletrofisiologia , Citometria de Fluxo , Fase G1/efeitos dos fármacos , Cobaias , Mucosa Intestinal/citologia , Mucosa Intestinal/ultraestrutura , Masculino , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Coloração e RotulagemRESUMO
The effect of externally applied mechanical stress was investigated by thin section electron microscopy of the microvessels in the unaffected palmar fascia in the carpal tunnel syndrome and in patients with Dupuytren's contracture before and after application of a continuous elongation device. In the unaffected palmar fascia the microfilaments of the endothelial cells were connected to a few adherens junctions and focal contacts; stress fibres were absent. In the cord of Dupuytren's disease the microfilaments were increased in quantity. The length ratios of the connections with the lateral and basal cell membrane were significantly higher than in the control group and increased to an even greater extent in the continuously extended fascia. Stress fibres appeared in the endothelial cells of postcapillary venules in the nonextended cord and in the endothelium of both arterioles and venules after extension elongation. the numerous intermediate filaments and the rare microtubules remained unchanged in the endothelial cells of all palmar fasciae analysed. In the endothelial cells of the microvessels the mechanical stress applied from outside mainly affected the contractile component of the cytoskeleton.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Síndrome do Túnel Carpal/patologia , Contratura de Dupuytren/patologia , Endotélio Vascular/patologia , Fáscia/irrigação sanguínea , Mãos/irrigação sanguínea , Idoso , Feminino , Humanos , Masculino , Microcirculação , Microscopia Eletrônica , Pessoa de Meia-Idade , Estresse MecânicoRESUMO
It is current opinion that the intercellular spaces of the stria vascularis represent a closed compartment isolated from the endolymph by the tight junctions of the marginal cells and from the perilymph by the junctional complexes of the basel cells. However, it has not yet been investigated whether these two barriers meet at the stria margins toward Reissner's membrane and the spiral prominence. Possible candidates for this sealing could be junctions between the spindle-shaped cells. In the present study freeze-fracture replicas of guinea pig specimens fixed in the presence of filipin were used in order to investigate the junctions of the spindle-shaped cells and to localize the cholesterol in their plasma membrane. Replicas reveal that, below the belt-like apical zonula occludens, the basolateral plasma membranes of the spindle-shaped cells adjacent to each other and to the basal cells are joined over their entire extension by a large number of junctional strands intermingled with numerous filipin-cholesterol-complexes. Gap junctions are present in the meshes formed by these junctional strands. Thus, the plasma membrane of the spindle-shaped cells shows morphological and cytochemical characteristics which indicate that they are the anatomical components completing the barrier isolating the intrastrial compartment from the surrounding fluids.
Assuntos
Endolinfa/fisiologia , Junções Intercelulares/fisiologia , Perilinfa/fisiologia , Estria Vascular/fisiologia , Animais , Tamanho Celular/fisiologia , Colesterol/metabolismo , Filipina/química , Técnica de Fratura por Congelamento , Cobaias , Junções Intercelulares/ultraestrutura , Masculino , Microscopia Eletrônica de Varredura , Estria Vascular/citologia , Estria Vascular/ultraestruturaRESUMO
Virtually all vertebrates acidify their gastric contents to a pH between 0.8 and 2.0. In mammals, acid secretion is mediated by a K-stimulated proton-translocating adenosine triphosphatase (H,K-ATPase), which establishes a million-fold gradient of protons across the apical membrane of the gastric parietal cell. The earliest phylogenetic appearance of gastric acid secretion is in cartilaginous fish, and we sought to verify in this class (Chondrichthyes) the presence and distribution of H,K-ATPase in gastric epithelial cells. An antibody against a synthetic peptide based on the C-terminus of pig H,K-ATPase alpha-subunit was localized in the gastric glands of the Atlantic stingray Dasyatis sabina. The C-terminal antibody stained all cells with tubulovesicles and the apical membrane domain of mucous neck cells. In proximal stomach, gastric glands showed the strongest immunoreactivity in cells close to the isthmus; in the distal stomach, strongest immunoreactivity was found in cells at the base of the glands. Oxyntic cells were more intensely immunoreactive than oxynticopeptic cells. This antibody labeled a single band of M(r) 100,600 on immunoblots of D. sabina gastric microsomes. These results show the earliest phylogenetic occurrence of a gastric ATPase in putative acid-secreting cells and suggest that this enzyme shares structural features with mammalian H,K-ATPase.