RESUMO
Microfluidic synthesis techniques can offer improvement over batch syntheses which are currently used for radiopharmaceutical production. These improvements are, for example, better mixing of reactants, more efficient energy transfer, less radiolysis, faster reaction optimization, and overall improved reaction control. However, scale-up challenges hinder the routine clinical use, so the main advantage is currently the ability to optimize reactions rapidly and with low reactant consumption. Translating those results to clinical systems could be done based on calculations, if kinetic constants and diffusion coefficients were known. This study describes a microfluidic system with which it was possible to determine the kinetic association rate constants for the formation of [177Lu]Lu-DOTA-TATE under conditions currently used for clinical production. The kinetic rate constants showed a temperature dependence that followed the Arrhenius equation, allowing the determination of Arrhenius parameters for a Lu-DOTA conjugate (A = 1.24 ± 0.05 × 1019 M-1 s-1, EA = 109.5 ± 0.1 × 103 J mol-1) for the first time. The required reaction time for the formation of [177Lu]Lu-DOTA-TATE (99% yield) at 80 °C was 44 s in a microfluidic channel (100 µm). Simulations done with COMSOL Multiphysics® indicated that processing clinical amounts (3 mL reaction solution) in less than 12 min is possible in a micro- or milli-fluidic system, if the diameter of the reaction channel is increased to over 500 µm. These results show that a continuous, microfluidic system can become a viable alternative to the conventional, batch-wise radiolabelling technique.
RESUMO
The hepatotropism and intrahepatic distribution of adenoviral vectors may be species dependent. Hepatocyte transduction was evaluated in three rabbit strains after transfer with E1E3E4-deleted adenoviral vectors containing a hepatocyte specific alpha1-antitrypsin promoter-driven expression cassette (AdAT4). Intravenous administration of 4 x 10(12) particles/kg of AdAT4 induced human apo A-I levels above 40 mg/dl in Dutch Belt, but below 1 mg/dl in New Zealand White and Fauve de Bourgogne rabbits. Diameters of sinusoidal fenestrae were significantly (P=0.0014) larger in Dutch Belt (124+/-3.4 nm) than in New Zealand White (108+/-1.3 nm) and Fauve de Bourgogne (105+/-2.6 nm) rabbits, suggesting that a smaller size constitutes a barrier for hepatocyte transduction. Indeed, intraportal transfer preceded by intraportal injection of sodium decanoate, which increases the diameter of sinusoidal fenestrae to 123+/-3.4 nm (P<0.01) in New Zealand White rabbits, increased human apo A-I levels 32- and 120-fold in New Zealand White and Fauve de Bourgogne rabbits, respectively, but did not affect expression in Dutch Belt rabbits. In conclusion, size of sinusoidal fenestrae appears to be a critical determinant of hepatocyte transduction after adenoviral transfer.
Assuntos
Adenoviridae/genética , Apolipoproteína A-I/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Fígado/ultraestrutura , Transdução Genética/métodos , Animais , Apolipoproteína A-I/metabolismo , Hepatócitos/metabolismo , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Coelhos , Especificidade da Espécie , TransgenesRESUMO
Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO-labelled colon carcinoma cells (CC531s) in 100 microm thick liver sections. In vitro and in vivo experiments revealed that DiO-labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO-labelled CC531s cells could be traced all over the tissue depth and anti-metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine-phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO-labelled cells in tissue sections.
Assuntos
Carbocianinas/metabolismo , Neoplasias Hepáticas/patologia , Microscopia Confocal/métodos , Animais , Corantes Fluorescentes/metabolismo , Técnicas In Vitro , Lasers , Masculino , Ratos , Coloração e Rotulagem , Células Tumorais CultivadasRESUMO
Atomic force microscopy (AFM) has been used to image a wide variety of cells and has proven to be successful in cellular imaging, by comparing results obtained by AFM with SEM or TEM. The aim of the present study was to investigate further the conditions for AFM imaging of living cells and compare the results with those obtained by SEM. We chose to image skin fibroblast and liver sinusoidal endothelial cells of two different sources, because these cells have been well described and characterized in earlier studies. AFM imaging of living cells mainly reveals submembranous structures, which could not be observed by SEM. This concerns the visualization of the overall cytoskeletal architecture and organelles, without the necessity of any preparative steps. The AFM study of living cells allows a time lapse study of dynamic changes of the actin cytoskeleton under the influence of the cytoskeleton-disturbing drug cytochalasin B in cells that can be followed individually during the process. However, softer samples, such as the fenestrated parts of living rat liver sinusoidal endothelial cells in culture could not be visualized. Apparently, these cell parts are disrupted due to tip-sample interaction in contact mode. To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was applied, resulting in images of higher quality. Still, endothelial fenestrae could not be visualized. In contrast, contact imaging of immortomouse liver sinusoidal endothelial cells, which are devoid of fenestrae, could easily be performed and revealed a detailed filamentous cytoskeleton.
Assuntos
Fígado/citologia , Microscopia de Força Atômica/métodos , Pele/citologia , Animais , Células Cultivadas , Citoesqueleto/ultraestrutura , Endotélio/citologia , Endotélio/ultraestrutura , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Fígado/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura , Ratos , Pele/ultraestrutura , Estresse MecânicoRESUMO
The atomic force microscope (AFM) is a powerful tool to investigate surface and submembranous structures of living cells under physiological conditions at high resolution. These properties enabled us to study the interaction between live hepatic natural killer (NK) cells, also called pit cells, and colon carcinoma cells in vitro by AFM. In addition, the staining for filamentous actin and DNA was performed and served as a reference, because actin and nuclear observations at the light microscopic level during the cytotoxic interaction between these two cell types have been presented earlier. In this study, we collected evidence that conjugation of hepatic NK cells with CC531s colon carcinoma cells results in a decreased binding of CC531s cells to the substratum as probed with the AFM in contact mode as early as 10 min after cell contact (n = 11). To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was performed on hepatic NK/CC531s cell conjugates, resulting in identical observations (n = 3). In contrast, the first cytotoxic signs, as determined with the nuclear staining dye Hoechst 33342, could be observed 3 h after the start of the co-culture. This study illustrates that the AFM can be used to probe early cytotoxic effects of effector to target cell contact in nearby physiological conditions. Other routine cytotoxicity tests detect the first cytotoxic effects after 1.5-3 h co-incubation at the earliest.
Assuntos
Neoplasias do Colo/imunologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Fígado/imunologia , Animais , Células Matadoras Naturais/ultraestrutura , Masculino , Microscopia de Força Atômica , Microscopia de Fluorescência , Ratos , Ratos WistarRESUMO
Hepatic endothelial fenestrae are dynamic structures that act as a sieving barrier to control the extensive exchange of material between the blood and the liver parenchyma. Alterations in the number or diameter of fenestrae by drugs, hormones, toxins, and diseases can produce serious perturbations in liver function. Previous studies have shown that disassembly of actin by cytochalasin B or latrunculin A caused a remarkable increase in the number of fenestrae and established the importance of the actin cytoskeleton in the numerical dynamics of fenestrae. So far, however, no mechanism or structure has been described to explain the increase in the number of fenestrae. Using the new actin inhibitor misakinolide, we observed a new structure that appears to serve as a fenestrae-forming center in hepatic endothelial cells.
Assuntos
Actinas/antagonistas & inibidores , Citoesqueleto/ultraestrutura , Endotélio Vascular/ultraestrutura , Fígado/citologia , Macrolídeos/farmacologia , Animais , Citoesqueleto/efeitos dos fármacos , Endotélio Vascular/fisiologia , Fígado/irrigação sanguínea , Masculino , Ratos , Ratos WistarRESUMO
Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried-coated, wet-fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde-fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented. AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip-sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high-resolution real-time studies of dynamic changes in submembranous structures.
Assuntos
Adenocarcinoma/ultraestrutura , Neoplasias do Colo/ultraestrutura , Fibroblastos/ultraestrutura , Macrófagos/ultraestrutura , Microscopia de Força Atômica/métodos , Microscopia Eletrônica/métodos , Animais , Células Cultivadas , Fibroblastos/fisiologia , Fígado/citologia , Fígado/ultraestrutura , Macrófagos/fisiologia , Ratos , Ratos Wistar , Pele/citologia , Pele/ultraestrutura , Células Tumorais CultivadasRESUMO
Critical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS-dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole-mount cells for TEM and AFM.
Assuntos
Fixadores , Técnicas de Preparação Histocitológica , Fígado/ultraestrutura , Compostos de Organossilício , Animais , Células Cultivadas , Endotélio/ultraestrutura , Estudos de Avaliação como Assunto , Microscopia de Força Atômica/métodos , Microscopia Eletrônica/métodos , Microscopia Eletrônica de Varredura/métodos , RatosRESUMO
Pit cells are liver-specific natural killer (NK) cells and belong to the group of sinusoidal cells, together with Kupffer, endothelial, and fat-storing cells. Pit cells are lymphoid cells containing specific granules, classifying them also as large granular lymphocytes (LGL). They probably originate from the bone marrow, circulate in the blood, and marginate in the liver, where they develop into pit cells by lowering their density and increasing the number of granules, which decrease in size. Pit cells remain in the liver about 2 weeks and are dependent on Kupffer cells. Pit cells also proliferate locally, when stimulated with interleukin-2, biological response modifiers, or other agents. Pit cells adhere to tumor target cells during killing. They possess a high level of natural cytotoxicity against a variety of tumor cell lines, which is comparable to the cytotoxicity level of lymphokine-activated killer (LAK) cells. Tumor cell killing is synergistically enhanced when pit cells attack tumor cells together with Kupffer cells. Further investigations are needed to clarify the mechanisms of pit cell cytotoxicity and the role of these cells in killing virus-infected cells, such as during viral hepatitis.
Assuntos
Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Células de Kupffer/imunologia , Fígado/citologia , Animais , Apoptose , Moléculas de Adesão Celular/metabolismo , Técnicas de Cultura de Células/métodos , Movimento Celular , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/prevenção & controle , Hematopoese , Humanos , Imunofenotipagem , Células Matadoras Ativadas por Linfocina/imunologia , Fígado/imunologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/prevenção & controle , Ratos , Especificidade da Espécie , Viroses/imunologiaRESUMO
This report describes the effect of the actin-disrupting marine toxin latrunculin A on the cytoskeleton and fenestrae of liver endothelial cells (LECs). Fluorescence microscopy and whole mount-transmission electron microscopic preparations of isolated, purified, and cultured LECs showed that latrunculin A, which sequesters actin monomers and depolymerizes actin filaments, caused profound changes in microfilament organization in LECs. Scanning electron microscopic preparations showed that latrunculin A almost doubles the number of fenestrae within 10 minutes, whereas the diameter is only slightly reduced. All new fenestrae possess the earlier described fenestrae-associated cytoskeleton ring. Cytochalasin B, which disrupts the network of actin filaments, principally by capping the fast growing end of actin filaments, produced comparable effects with regard to actin organization and the number and size of fenestrae. After 1 hour of treatment, an equal maximum number of fenestrae was observed for both agents. The effect of latrunculin A was obtained at concentrations about 100 times lower than cytochalasin B. Thus, two agents that alter the state of actin organization in LECs, albeit by different mechanisms, cause the doubling of the number of fenestrae within 10 to 30 minutes. This indicates that the state of assembly of the actin cytoskeleton is important in the numerical dynamics of LEC fenestrae and that the actin cytoskeleton of LECs is probably the main mechanical regulator for sieving between the sinusoidal blood and the parenchymal cells. Latrunculin A represents a new agent in the study of the de novo formation of fenestrae.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citocalasina B/farmacologia , Endotélio Vascular/efeitos dos fármacos , Circulação Hepática/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Células Cultivadas , Endotélio Vascular/citologia , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Poríferos , Ratos , TiazolidinasRESUMO
Endothelial fenestrae control the exchange of fluids, solutes and particles between the sinusoidal lumen and the microvillous surface of the parenchymal cells. Fenestrae have a critical dimension in the order of 150-200 nm, making it necessary to use microscopes with a resolution better than the light microscope. Comparative whole-mount preparations of isolated, purified and cultured rat liver sinusoidal endothelial cells (LEC) were studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Examination of detergent-extracted LEC by SEM and TEM shows an integral cytoskeleton: sieve plates are delineated by a sieve plate-associated cytoskeleton ring and fenestrae by a fenestrae-associated cytoskeleton ring. By using microtubule altering agents we could demonstrate: (1) the architectural role of microtubules in arranging fenestrae, (2) the existence of a population of microtubules resistant against low temperature and colchicine, (3) the ability of LEC to shift the microtubule assembly-disassembly steady state under various conditions, (4) and the necessity of an intact microtubular cytoskeleton to support the increase in the number of fenestrae after cytochalasin B. Topographical examinations of AFM images revealed that sieve plates are delineated by elevated borders, probably projections of the underlying tubular cytoskeleton.
Assuntos
Endotélio/ultraestrutura , Fígado/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Animais , Células Cultivadas , Colchicina/farmacologia , Citocalasina B/farmacologia , Endotélio/citologia , Técnicas de Preparação Histocitológica , Masculino , Microtúbulos/efeitos dos fármacos , Paclitaxel/farmacologia , Ratos , Ratos WistarRESUMO
The hepatic sinusoid harbors 4 different cells: endothelial cells (100, 101), Kupffer cells (96, 102, 103), fat-storing cells (34, 51, 93), and pit cells (14, 107, 108). Each cell type has its own specific morphology and functions, and no transitional stages exist between the cells. These cells have the potential to proliferate locally, either in normal or in special conditions, that is, experiments or disease. Sinusoidal cells from a functional unit together with the parenchymal cells. Isolation protocols exist for all sinusoidal cells. Endothelial cells filter the fluids, exchanged between the sinusoid and the space of Disse through fenestrae (100), which measure 175 nm in diameter and are grouped in sieve plates. Fenestrae occupy 6-8% of the surface (106). No intact basal lamina is present under these cells (100). Various factors change the number and diameter of fenestrae [pressure, alcohol, serotonin, and nicotin; for a review, see Fraser et al (32)]. These changes mainly affect the passage of lipoproteins, which contain cholesterol and vitamin A among other components. Fat-storing cells are pericytes, located in the space of Disse, with long, contractile processes, which probably influence liver (sinusoidal) blood flow. Fat-storing cells possess characteristic fat droplets, which contain a large part of the body's depot of vitamin A (91, 93). These cells play a major role in the synthesis of extracellular matrix (ECM) (34, 39-41). Strongly reduced levels of vitamin A occur in alcoholic livers developing fibrosis (56). Vitamin A deficiency transforms fat-storing cells into myofibroblast-like cells with enhanced ECM production (38). Kupffer cells accumulate in periportal areas. They specifically endocytose endotoxin (70), which activates these macrophages. Lipopolysaccharide, together with interferon gamma, belongs to the most potent activators of Kupffer cells (28). As a result of activation, these cells secrete oxygen radicals, tumor necrosis factor, interleukin 1, interleukin 6, and a series of eicosanoids (28) and become cytotoxic against tumor cells [e.g., colon carcinoma cells (19, 22, 48)]. Toxic secretory products can cause necrosis of the liver parenchyma, which constitutes a crucial factor in liver transplantation (55). Pit cells possess characteristic azurophylic granules and display a high level of spontaneous cytolytic activity against various tumor cells, identifying themselves as natural killer cells (10). The number and cytotoxicity of pit cells can be considerably enhanced with biological response modifiers, such as Zymosan or interleukin 2 (8). Pit cell proliferation occurs within the liver, but recent evidence indicates that blood large granular lymphocytes develop into pit cells in 2 steps involving high- and low-density pit cells (88). Kupffer cells control the motility, adherence, viability, and cytotoxicity of pit cells (89), whereas cytotoxicity against tumor cells is synergistically enhanced (80, 81).
Assuntos
Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Fígado/irrigação sanguínea , Fígado/fisiologia , Animais , Endotélio Vascular/ultraestrutura , Humanos , Fígado/ultraestruturaRESUMO
Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparations techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference. AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (-15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).
Assuntos
Endotélio/ultraestrutura , Fígado/citologia , Animais , Células Cultivadas , Centrifugação com Gradiente de Concentração , Glutaral , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Ratos , Fixação de Tecidos/métodosRESUMO
Fenestrae control the exchange of fluids, dissolved compounds and small particles between the blood and the space of Disse, and are primarily limited at one side by parenchymal cells. We recently described a simple and rapid method for the isolation, purification and cultivation of rat liver sinusoidal endothelial cells. With regard to the purity and morphology of liver endothelial cells, a detailed microscopic study was performed. Purity and viability after selective adherence was 74 and 95%, respectively. Liver endothelial cell purity was further enhanced to about 95% during adherence and spreading on collagen after 8 h of culture. Liver endothelial cells isolated by this method provide a viable cell population, enabling the study of structure and function of these cells in vitro. We investigated the cytoskeleton associated with fenestrae and sieve plates of liver endothelial cells. Cultured cells were slightly fixed and treated with cytoskeleton extraction buffer containing 0.1% Triton. Whole mounts of extracted liver endothelial cells were prepared for scanning and transmission electron microscopy. Extracted liver endothelial cells show an integral, intricate cytoskeleton. Sieve plates and fenestrae are clearly delineated by cytoskeleton elements. Fenestrae are surrounded by a filamentous, fenestrae-associated cytoskeleton ring with an average filament thickness of 16 nm. Additionally, sieve plates are surrounded and delineated by microtubuli, which form a network together with additional branching cytoskeletal elements. Microtubuli are sometimes found delineating linear arrangements of fenestrae. In conclusion, liver endothelial cells possess a cytoskeleton, that defines and supports sieve plates and fenestrae. Fenestrae-associated cytoskeleton rings are involved in determining the size of fenestrae. The fenestrae-associated cytoskeleton therefore probably controls the important hepatic function of endothelial filtration.
Assuntos
Endotélio Vascular/ultraestrutura , Fígado/irrigação sanguínea , Animais , Técnicas de Cultura de Células/métodos , Separação Celular , Citoesqueleto/ultraestrutura , Masculino , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Ratos , Ratos WistarRESUMO
This article describes the cytoskeleton associated with fenestrae and sieve plates of rat liver sinusoidal endothelial cells. Fenestrae control the exchange between the blood and parenchymal cells. We present evidence indicating that several agents that change the fenestrae and sieve plates also cause changes in the cytoskeleton. Cultured liver endothelial cells (LECs) were slightly fixed and treated with cytoskeleton extraction buffer. Detergent-extracted whole mounts of cultured cells were prepared for either scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Extracted cells show an integral intricate cytoskeleton; sieve plates and fenestrae are delineated by cytoskeleton elements. Fenestrae are surrounded by a filamentous, fenestrae-associated cytoskeleton with a mean filament thickness of 16 nm. Sieve plates are surrounded and delineated by microtubuli, which form a network together with additional branching cytoskeletal elements. The addition of ethanol to cultured cells enlarged the diameter for these fenestrae-associated cytoskeleton rings by 5%, whereas serotonin treatment reduced the diameter by 20%. These observations indicate that the fenestrae-associated cytoskeleton probably changes the size of fenestrae after different treatments. After treatment with cytochalasin B the number of fenestrae increased. However, cytochalasin B did not change the structure of the fenestrae-associated cytoskeleton ring, but disperses the microtubuli. In conclusion, LECs have a cytoskeleton that defines and supports sieve plates and fenestrae. Fenestrae-associated cytoskeleton is a dynamic structure and plays a role in maintaining and regulating the size of fenestrae after different treatments. Therefore, the fenestrae-associated cytoskeleton controls the important hepatic function of endothelial filtration.
Assuntos
Citoesqueleto/ultraestrutura , Fígado/ultraestrutura , Animais , Células Cultivadas , Citocalasina B/farmacologia , Citoesqueleto/efeitos dos fármacos , Endotélio/citologia , Endotélio/ultraestrutura , Etanol/farmacologia , Fígado/citologia , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Ratos , Serotonina/farmacologiaRESUMO
BACKGROUND: This paper describes a simple, low cost, and rapid method for the isolation, purification, and cultivation of rat liver sinusoidal endothelial cells (LEC). With regard to the purity, morphology, and responsiveness of the LEC, a detailed electron microscopy study was performed. In addition, we developed a method of automatic detection and analysis of the endothelial fenestrations using digitized scanning electron microscope images, allowing us to collect large sets of data. EXPERIMENTAL DESIGN: LEC were isolated by collagenase perfusion of the liver, isopycnic sedimentation in a two-step Percoll gradient, and selective adherence. The purification and cultivation of LEC was evaluated by light and electron microscopy. The addition of ethanol to LEC cultures showed responsiveness of LEC. RESULTS: Purity and viability of LEC after selective adherence was 73.7 +/- 5.8% and > or = 95%, respectively. LEC purity was further enhanced during adherence and spreading on collagen (type I, III). After 8 hours of culture, LEC monolayers were contaminated with less than 5% of other cells. After treatment with ethanol for 90 minutes, the diameters of LEC fenestrae increased approximately 10%. CONCLUSIONS: LEC isolated by this method provide a vital and responsive cell population enabling the study of structure and function of these cells in vitro. This method, in combination with a computer-assisted, on-line image analysis allows the acquisition of large numbers of measurements on these cells with high accuracy and with a minimum of bias.
Assuntos
Separação Celular/métodos , Técnicas de Cultura/métodos , Fígado/citologia , Animais , Células Cultivadas , Centrifugação com Gradiente de Concentração/métodos , Colagenases , Endotélio/citologia , Endotélio/ultraestrutura , Fígado/ultraestrutura , Masculino , Microscopia Eletrônica , Perfusão , Ratos , Ratos WistarRESUMO
Collagen type I and procollagen type III were localized at the ultrastructural level on ultrathin frozen sections of rat liver by the protein A-gold technique using affinity-purified primary antibodies. Both collagen type I and procollagen type III were localized on nearly all solitary and bundled fibrils in the space of Disse. Simultaneous localization of collagen type I and procollagen type III by a double-labeling procedure using protein A-gold probes of different sizes unequivocally demonstrated the presence of both collagens in the same fibrils. Measurement of the diameter of large numbers of collagen fibrils in the space of Disse of the rat liver showed a unimodal distribution of the fibril diameters around an average value of 62.4 nm (S.D. = 12.8 nm), and 91% of the collagen bundles contained less than 30 fibrils. Additional measurements on epoxy resin-embedded material of five biopsy specimens of normal human liver showed a comparable unimodal distribution of the fibril diameters around an average value of 57.2 nm (S.D. = 9.6 nm), and 74% of the bundles contained less than 60 fibrils. The latter observation demonstrates that human liver contains broader interstitial collagen bundles than rat liver. From these results, we conclude that the space of Disse of normal rat and human liver contains a uniform population of striated interstitial collagen fibrils. In the rat liver, these fibrils contain both collagen type I and procollagen type III. Therefore the concept that procollagen type III is predominantly localized in small diameter fibrils or bundles, whereas collagen type I is preferentially localized in thick ones, does not hold.
Assuntos
Colágeno/metabolismo , Fígado/metabolismo , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Colágeno/imunologia , Humanos , Imuno-Histoquímica , Fígado/ultraestrutura , Masculino , Ratos , Ratos Endogâmicos , Valores de ReferênciaRESUMO
The cellular and subcellular distribution of biologically tritiated Salmonella abortus equi lipopolysaccharide (LPS) was studied at different time intervals after intravenous injection in rats. At 1 min after injection of LPS via the portal vein label was present over Kupffer cell phagosomes. Between 30 min and 7 days after injection, silver grains were mainly associated with phagosomes and lysosomes and occasionally with the membrane of Kupffer cells. A few parenchymal cells were labeled at 5 min in their mitochondria, cell membrane and the periphery of the cell. Radioactivity was also present in the rough endoplasmic reticulum (from 15 min), fat droplets and the nucleus (from 3 h) up to 7 days. Sinusoidal endothelial and fat-storing cells were never labeled. In conclusion, both Kupffer cells and parenchymal cells play a role in the uptake of LPS by the liver. The uptake and processing of endotoxin is rapid, since label is found early after administration and radioactivity is detected in the bile within 1 h. This radioactivity represents non-detoxified LPS, since it is lethal for galactosamine-sensitised mice after extraction with hot phenol/water. However, in the presence of bile salts, the LPS is non-lethal and not capable of clotting the limulus amebocyte lysate. LPS injection causes bile flow reduction within 45 min.
Assuntos
Ácidos e Sais Biliares/fisiologia , Lipopolissacarídeos/metabolismo , Fígado/metabolismo , Lisossomos/ultraestrutura , Fagossomos/ultraestrutura , Animais , Ácidos e Sais Biliares/farmacologia , Transporte Biológico , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/toxicidade , Fígado/patologia , Fígado/ultraestrutura , Lisossomos/metabolismo , Microscopia Eletrônica , Fagossomos/metabolismo , Ratos , Salmonella/imunologiaRESUMO
In normal rat liver, Kupffer cells were unequivocally identified using peroxidase cytochemistry by light microscopy in semithin plastic sections. The Kupffer cell population was found to constitute 31% of the sinusoidal cells and by morphometry and serial sectioning, a mean absolute number of 14 to 20 X 10(6) Kupffer cells per g liver was calculated. The mean distribution of Kupffer cells in the liver lobules was 43% in the periportal, 28% in the midzonal and 29% in the central area of the lobule. Administration of latex particles labeled only 64% of all Kupffer cells, and in particular centrally located cells, showed a lower activity of latex uptake, even at overloading doses. Furthermore, the latter cells were of smaller size than periportal Kupffer cell profiles. The mean number and distribution of latex-labeled Kupffer cells did not change over a period of 3 months, indicating a long lifetime for these resident macrophages. This slow population turnover was supported by the observed small mitotic index, 0.06% after a 6 hr arrest by vinblastine, and by the small [3H]thymidine labeling index which did not change over a period of 3 weeks after administration of the label. It is proposed that the Kupffer cell population, under physiologic conditions, is a long-living and self-renewing population, the kinetics of which substantially differ from those of other sinusoidal cell types.