RESUMEN
Experimental autoimmune encephalomyelitis (EAE) is a mouse model of demyelinating diseases, such as multiple sclerosis (MS). MS can be accompanied by autoimmune hepatitis. In this study, nanomechanical, biorheological and histological examinations were carried out by atomic force microscopy (AFM), rheology, and immunofluorescence microscopy to investigate changes in the liver tissue of EAE mice and the effect of natalizumab, a monoclonal antibody against α4-integrin (VLA-4) cell adhesion molecule, used in MS therapy. Liver samples collected from EAE mice in three successive phases of the disease showed inflammatory changes manifested by leukocyte infiltrations and elevated levels of proinflammatory cytokine IL-1ß. Liver stiffness and viscoelasticity increased in the onset phase of EAE, decreased in the peak phase and increased again in the chronic phase to reach the highest values. These changes were not associated with inflammation parameters which increased in the peak phase and decreased to the lowest values in the chronic phase. Moreover, anti-VLA treatment, which reduced the inflammation parameters, had an ambiguous effect on stiffness and viscoelasticity: it increased them in the peak phase but decreased in the chronic phase. The observed discrepancies can result from a complex network of interactions between inflammation and fibrosis, as well as between liver cells and the extracellular matrix influencing the biomechanical properties of the liver tissue.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Anticuerpos Monoclonales , Modelos Animales de Enfermedad , Inflamación , Integrina alfa4beta1 , Hígado , Ratones , Ratones Endogámicos C57BLRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is a commonly used mouse model of multiple sclerosis, a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination leading to brain and spinal cord malfunctions. We postulate that not only biological but also biomechanical properties play an important role in impairements of CNS function. Atomic force microscopy (AFM) was applied to investigate mechanical properties of spinal cords collected from EAE mice in preonset, onset, peak, and chronic disease phases. Biomechanical changes were compared with histopathological alterations observed in the successive phases. The deformability of gray matter did not change, while rigidity of white matter increased during the onset phase, remained at the same level in the peak phase and decreased in the chronic phase. Inflammatory infiltration and laminin content accompanied the tissue rigidity increase, whereas demyelination and axonal damage showed an opposite effect. The increase in white matter rigidity can be regarded as an early signature of EAE.
Asunto(s)
Encefalomielitis Autoinmune Experimental/patología , Esclerosis Múltiple/patología , Médula Espinal/patología , Animales , Fenómenos Biomecánicos , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/fisiopatología , Médula Espinal/fisiopatologíaRESUMEN
Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE) are characterized by three main histopathological parameters: inflammation, demyelination and axonal damage. In this study, these parameters were assessed in spinal cords of mice in the successive phases of EAE by quantitative histology and immunohistochemistry. The number of inflammatory lesions, the intensity of inflammation and expression of CD45 corresponded with the severity of clinical symptoms: they increased from the onset phase to the peak phase of the disease and subsided in the chronic phase. Demyelination increased in the peak phase and did not change in the chronic phase of EAE, although axonal damage gradually increased from the onset phase to the chronic phase, suggesting compensatory hypermyelination in that phase. The markers of myelin and axonal injury: myelin basic protein (MBP) and beta amyloid precursor protein (ß-APP) showed changes (decrease and increase, respectively) of expression parallel to changes in demyelination and axonal damage. Results of this study indicate that although inflammation intensity subsides in the chronic phase of EAE, the neurodestructive processes: demyelination and axonal damage continue in that phase.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Ratones , Animales , Encefalomielitis Autoinmune Experimental/metabolismo , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Inmunohistoquímica , Médula Espinal/metabolismo , Inflamación/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are often accompanied by optic neuritis associated with neurofilament disruption. In this study, the stiffness of the optic nerve was investigated by atomic force microscopy (AFM) in mice with induced EAE in the successive phases of the disease: onset, peak, and chronic. AFM results were compared with the intensity of the main pathological processes in the optic nerve: inflammation, demyelination, and axonal loss, as well as with the density of astrocytes, assessed by quantitative histology and immunohistochemistry. Optic nerve tissue and serum levels of neurofilament light chain protein (NEFL) were also examined by immunostaining and ELISA, respectively. The stiffness of the optic nerve in EAE mice was lower than that in control and naïve animals. It increased in the onset and peak phases and sharply decreased in the chronic phase. Serum NEFL level showed similar dynamics, while tissue NEFL level decreased in the onset and peak phases, indicating a leak of NEFL from the optic nerve to body fluids. Inflammation and demyelination gradually increased to reach the maximum in the peak phase of EAE, and inflammation slightly declined in the chronic phase, while demyelination did not. The axonal loss also gradually increased and had the highest level in the chronic phase. Among these processes, demyelination and especially axonal loss most effectively decrease the stiffness of the optic nerve. NEFL level in serum can be regarded as an early indicator of EAE, as it rapidly grows in the onset phase of the disease.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Ratones , Animales , Encefalomielitis Autoinmune Experimental/patología , Esclerosis Múltiple/patología , Filamentos Intermedios/patología , Nervio Óptico/patología , Inflamación/metabolismoRESUMEN
Matrix metalloproteinases (MMPs) regulated by their tissue inhibitors (TIMPs) play a significant role in the pathogenesis of multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), as they degrade extracellular matrix including vascular basal laminae and by damaging blood-brain barrier (BBB) facilitate transmigration of immune cells into the central nervous system. MMPs are also involved in destruction of myelin sheaths, leading to axonal and neuronal loss. The aim of the present study was to assess whether natalizumab, a transmigration-inhibiting monoclonal antibody against α4ß1 integrin, influences expression of MMPs and TIMPs in the central nervous system of mice with EAE. MMP-2 and MMP-9, their respective inhibitors TIMP-2 and TIMP-1 and laminin were assessed by quantitative immunohistochemistry in the spinal cord cryosections of C57BL/6 mice with EAE in the successive phases of the disease (onset, peak and chronic). The percentage of immunopositive areas were calculated in sections encompassing the whole spinal cord cross-sectional area occupied by the gray and white matter. Results obtained in animals administered with 5 mg/kg natalizumab were compared with those collected from control mice receiving 5 mg/kg IgG. Both studied MMPs and both TIMPs were upregulated in control EAE mice. Natalizumab treatment significantly reduced expression of MMPs and increased expression of TIMPs in the peak and chronic phases of the disease. This effect was accompanied by inhibition of laminin degradation in the vascular basal laminae and reduction of inflammatory infiltration. Results of this study demonstrate that in addition to its well known anti-integrin activity counteracting transmigration of immune cells into the central nervous system, natalizumab strengthens this effect by its probably indirect influence on MMPs and TIMPs leading to protection of blood-brain barrier integrity.
Asunto(s)
Sistema Nervioso Central/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Natalizumab/farmacología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Factores Inmunológicos/farmacología , Ratones , Ratones Endogámicos C57BL , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
The microvascular architecture of developing lateral ventricle choroid plexus was investigated by corrosion casting and scanning electron microscopy in human fetuses aged 20 gestational weeks. The areas with different microvascular patterns corresponded to the particular parts of the mature plexus: anterior part, glomus, posterior part, the villous fringe and the free margin. In the posterior part, densely packed parallel arterioles and venules were surrounded by sheath-like capillary networks. Other areas contained compact capillary plexuses of the primary villi: the most prominent, protruding basket- and leaf-shaped plexuses were observed in the villous fringe, whilst less numerous and smaller plexuses occurred in the anterior part and glomus. The capillaries of the plexuses had a large diameter and sinusoidal dilations, and showed the presence of occasional short, blind sprouts indicative of angiogenesis. Short anastomoses between arterioles supplying the plexuses and venules draining them were only rarely observed. In the upper area of the choroid plexus, the superior choroidal vein was surrounded by a capillary network forming small, glomerular or rosette-shapes plexuses. The free margin of the choroid plexus was characterized by flat, multiple, arcade-like capillary loops. The general vascular architecture of the human choroid plexus at 20 gestational weeks seems to be similar to that of postnatal/mature plexus, still lacking, however, the complex vascular plexuses of the secondary villi.
Asunto(s)
Plexo Coroideo/irrigación sanguínea , Plexo Coroideo/embriología , Ventrículos Laterales/embriología , Arteriolas/embriología , Arteriolas/ultraestructura , Capilares/embriología , Capilares/ultraestructura , Plexo Coroideo/ultraestructura , Molde por Corrosión , Femenino , Edad Gestacional , Humanos , Ventrículos Laterales/irrigación sanguínea , Ventrículos Laterales/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Embarazo , Vénulas/embriología , Vénulas/ultraestructuraRESUMEN
The structural features of cells, their surfaces and the extracellular matrix were investigated in acquired aural cholesteatoma. Cholesteatomas surgically removed from 30 patients were examined by a scanning electron microscope (SEM). The predominant part of a cholesteatoma was composed of stratified squamous epithelium, showing extensive chaotic desquamation. The surface sculpture of the keratinocytes and corneocytes varied from parallel ridges, irregular microplicae and mirovilli, to flat grooves and pits and a completely smooth surface. Sheetlike lamellar structures, probably representing an intercellular lipid-forming permeability barrier, were also observed. Small crystals located in the perimatrix were observed in one case. According to the SEM observations, cholesteatoma epithelium is characterised by abnormal and uncoordinated keratinisation, with a predominance of the advanced stages of the process.
Asunto(s)
Colesteatoma del Oído Medio/patología , Epitelio/patología , Epitelio/ultraestructura , Queratinocitos/patología , Queratinocitos/ultraestructura , Adolescente , Adulto , Anciano , Extensiones de la Superficie Celular/patología , Extensiones de la Superficie Celular/ultraestructura , Colesteatoma del Oído Medio/fisiopatología , Epitelio/fisiopatología , Matriz Extracelular/patología , Matriz Extracelular/ultraestructura , Femenino , Humanos , Queratinas/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Persona de Mediana EdadRESUMEN
The topography of the arterial supply and venous drainage was visualised by corrosion casting and scanning electron microscopy in the human foetal (20 weeks) choroid plexus of the lateral ventricle. Although secondary villi were not yet present at that developmental stage, the topography of the large arteries and veins almost fully corresponded to that described in adult individuals. The only major difference observed was a lack of the typical tortuosity of the lateral branch of the anterior choroidal artery and of the superior choroidal vein, which probably develops during further expansion of the vascular system associated with the formation of secondary villi.
Asunto(s)
Arterias/anatomía & histología , Plexo Coroideo/irrigación sanguínea , Plexo Coroideo/embriología , Feto/irrigación sanguínea , Ventrículos Laterales/irrigación sanguínea , Ventrículos Laterales/embriología , Venas/anatomía & histología , Cáusticos , Humanos , Masculino , Microscopía Electrónica de Rastreo , Modelos AnatómicosRESUMEN
As compared with conventional paraffin, celloidin, and frozen sections, semithin plastic sections offer a superior quality of the light microscopic image in terms of better resolution, absence of distortion and shrinkage artifacts, and suitability for calcified tissues. Application of histochemical methods to such sections often encounters, however, serious difficulties resulting from a considerably reduced reactivity of plastic-embedded biological material. Factors involved include a poor penetration of reagents into plastic embedding media due to a steric or hydrophobic hindrance, as well as a blockade of the reactive chemical groups in the sample due to interactions with fixatives and plastics. Embedding in polar (hydrophilic) plastics, such as glycol methacrylate, permits carrying out a large number of histochemical reactions, including the demonstration of enzymatic activities, directly on sections, but is less suitable for combined light/electron microscopic studies because of an imperfect ultrastructural preservation of tissues. Embedding in nonpolar epoxy resins, particularly if combined with a double aldehyde-osmium fixation, results in a high quality ultrastructure but almost fully inhibits the histochemical reactivity of the embedded material. In order to restore this reactivity, i.e. to unmask chemical groups bound by the polymerized resin, semithin epoxy sections require the removal of the embedding matrix by alkoxides prior to the histochemical procedure. Additional steps are also often necessary: treatment of osmium-fixed sections with oxidative agents, e.g., hydrogen peroxide or periodate which reoxidize the bound osmium and remove it from tissue, and a controlled proteolytic digestion, especially useful in immunocytochemical studies, which probably cleaves the bonds between the primary aldehyde fixative, and the reactive sites. This article reviews histochemical methods which have been successfully applied to plastic-embedded material. Using polar methacrylates and/or nonpolar epoxy resins as embedding media, it has been possible to demonstrate proteins and aminoacid residues, carbohydrates, lipids, nucleic acids, biogenic amines, inorganic ions, and some enzymes, although the spectrum of methods found as suitable for plastic-embedded material is far narrower than that available for paraffin or frozen sections.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Técnicas Histológicas , Microscopía/métodos , Plásticos , Animales , Aminas Biogénicas/análisis , Carbohidratos/análisis , ADN/análisis , Enzimas/metabolismo , Histocitoquímica , Humanos , Lípidos/análisis , Pigmentos Biológicos/análisis , Proteínas/análisis , ARN/análisisRESUMEN
OBJECTIVE: Microscopic investigation of the hyaloid vascular system in 5-month-old human fetuses. METHODS: Corrosion casting and light and scanning electron microscopy. RESULTS: The hyaloid artery ramifies into a tuft of vasa hyaloidea propria, which communicates with the posterior portion of the tunica vasculosa lentis, characterized by a network of anastomosing vessels. They further pass to the lateral portion of the tunica, acquiring a nonanastomosing palisadelike array and drain into the vessels of the ciliary processes or, after bending over the edge of the developing iris, drain into the outer choriocapillaris. The tunica vasculosa lentis vessels also communicate with the pupillary membrane, a system of vascular arcades arranged in several interconnected tiers, supplied by the terminal branches of the long posterior ciliary arteries. In tunica vasculosa lentis, arterioles seem to pass directly into veins, without forming a capillary bed. CONCLUSIONS: At the investigated developmental stage, the fully developed hyaloid system enters its subsequent involution, and the vessels nourishing the vitreous have already involuted. The system is generally similar to that observed in other mammals.
Asunto(s)
Ojo/irrigación sanguínea , Ojo/ultraestructura , Arterias/embriología , Arterias/ultraestructura , Molde por Corrosión , Ojo/embriología , Femenino , Feto/anatomía & histología , Edad Gestacional , Humanos , Cristalino/irrigación sanguínea , Cristalino/embriología , Cristalino/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Cuerpo Vítreo/irrigación sanguínea , Cuerpo Vítreo/embriología , Cuerpo Vítreo/ultraestructuraRESUMEN
The vascular architecture of five advanced invasive papillary tumours of the urinary bladder was investigated using corrosion casting and scanning electron microscopy. The superficial vasculature was composed predominantly of capillary systems of two types: dense flat networks with numerous interconnections and tightly packed tortuous loops, forming multiple irregular folds that reflected the papillary morphology of the tumours. The capillaries were supplied and drained by numerous straight nonanastomosing arterioles and venules, which arose by way of multiple branching of larger vessels originating from the mucosal plexus of the bladder. Differences between the tumours in the spatial arrangement of these vessels probably reflect different growth dynamics. The intramural parts of the tumours contained a chaotic network of straight, uniform capillaries with numerous sprouts, which was very different from the superficial capillary system. It is postulated that different angiogenesis-targeted growth factors may be expressed in the phases of exophytic growth and muscularis invasion of the tumour, leading to the formation of different microvascular patterns.
Asunto(s)
Carcinoma Papilar/irrigación sanguínea , Molde por Corrosión , Microscopía Electrónica de Rastreo , Neoplasias de la Vejiga Urinaria/irrigación sanguínea , Arteriolas/patología , Capilares/patología , Carcinoma Papilar/patología , Humanos , Invasividad Neoplásica , Neoplasias de la Vejiga Urinaria/patología , Vénulas/patologíaRESUMEN
A significant reduction of catalase activity, a peroxisomal marker enzyme, occurs in human hepatic neoplasias, but no information is available on other peroxisomal proteins. We have studied by means of immunohistochemistry four specific proteins of peroxisomes (catalase and three enzymes of lipid beta-oxidation) in human hepatocellular tumors of various differentiation grades from adenoma to anaplastic carcinoma. In all tumors, except the adenomas, the tumor cells contained fewer peroxisomes than extrafocal hepatocytes and the reduction of antigenic sites in the tumor types generally correlated with the degree of tumor dedifferentiation as assessed by classical histopathological criteria. Two poorly differentiated tumors had no detectable peroxisomes at all. There were no major differences in the intensities of the immunocytochemical staining for all four studied peroxisomal antigens in different tumors, suggesting that the neoplastic transformation affects the biogenesis of the entire organelle and not merely the individual peroxisomal enzyme proteins. Some tumors exhibited a distinct peripheral distribution of peroxisomes. In cases with associated liver cirrhosis, the hepatocytes in the adjacent liver showed marked peroxisome proliferation, forming large perinuclear aggregates, occupying occasionally the entire cytoplasm. Taken together, our observations indicate that peroxisomes are significantly altered in both hepatocellular tumors and liver cirrhosis and, thus, could be responsible for some of the metabolic derangements observed in those disease processes.
Asunto(s)
Adenoma de Células Hepáticas/enzimología , Carcinoma Hepatocelular/enzimología , Catalasa/metabolismo , Cirrosis Hepática/enzimología , Neoplasias Hepáticas/enzimología , Peroxisomas/enzimología , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Acetil-CoA C-Aciltransferasa/metabolismo , Acil-CoA Oxidasa , Adenoma de Células Hepáticas/ultraestructura , Carcinoma Hepatocelular/ultraestructura , Enoil-CoA Hidratasa/metabolismo , Humanos , Inmunohistoquímica , Isomerasas/metabolismo , Metabolismo de los Lípidos , Cirrosis Hepática/patología , Neoplasias Hepáticas/ultraestructura , Complejos Multienzimáticos/metabolismo , Oxidorreductasas/metabolismo , Enzima Bifuncional PeroxisomalRESUMEN
Formation of intrachondral vessels (cartilage canals) in the proximal femoral epiphysis was studied in 13- to 22-week-old human fetuses using a corrosion casting technique and scanning electron microscopy. Several successive morphological stages of angiogenesis occurring inside the hyaline cartilage were distinguished. The process of cartilage vascularization starts with the formation of hairpin loops sent off from the perichondrial vascular network into the adjacent cartilage. A capillary glomerulus is then formed at the leading end, and the entire vascular unit grows in length, assuming a mushroom-like shape. Its further elongation is accompanied by a backward expansion of the capillary network which surrounds a pair of main vessels (arteriole and venule) like a manchette. The subsequent branching of such primary vascular units proceeds according to the same morphological patterns. The resulting tree-like vascular formations become interconnected via their lateral branches. This study clearly supports the invasion theory of cartilage canal formation.
Asunto(s)
Cartílago Articular/embriología , Epífisis/embriología , Fémur/embriología , Feto/irrigación sanguínea , Feto/ultraestructura , Vasos Sanguíneos/embriología , Vasos Sanguíneos/ultraestructura , Cartílago Articular/irrigación sanguínea , Molde por Corrosión , Desarrollo Embrionario y Fetal , Epífisis/irrigación sanguínea , Fémur/irrigación sanguínea , Humanos , Microscopía Electrónica de RastreoRESUMEN
Tissue eosinophils can be easily and specifically demonstrated in frozen sections after substrateless incubation with 3,3'-diaminobenzidine (DAB) at neutral pH. Under such conditions, other cells possessing endogenous peroxidatic activity (neutrophils, macrophages) do not stain, whereas staining of mitochondria-rich cells due to cytochrome oxidase activity can be avoided by a short prefixation of sections in glutaraldehyde-containing fixatives. The method can be useful in cases when a rapid screening of tissue eosinophils is required.
Asunto(s)
Eosinófilos/ultraestructura , 3,3'-Diaminobencidina , Animales , Tejido Conectivo/ultraestructura , Células del Tejido Conectivo , Complejo IV de Transporte de Electrones/inmunología , Complejo IV de Transporte de Electrones/metabolismo , Eosinófilos/enzimología , Congelación , Histocitoquímica , Concentración de Iones de Hidrógeno , Ratones , Mitocondrias/enzimología , Mitocondrias/metabolismo , Peroxidasas/análisis , Peroxidasas/metabolismo , Ratas , Coloración y Etiquetado , Fijación del TejidoRESUMEN
Light microscopic examination of rat and mouse tissues incubated in a medium containing 3,3'-diaminobenzidine (DAB) and catalase revealed that cells known to possess abundant mitochondria (hepatocytes, cardiomyocytes, renal proximal and distal tubular cells, parietal cells of gastric mucosa, and retinal photoreceptor cells) were stained with different intensity: from moderate (parietal cells, cardiomyocytes, renal distal tubular cells) to weak (hepatocytes, renal proximal tubular cells) or even negative (photoreceptors). When exogenous cytochrome c was added to the incubation medium, all these cells displayed quite uniform, strong staining, indicating a comparable activity of cytochrome oxidase. Since DAB is oxidized directly by cytochrome c which in turn undergoes reoxidation by cytochrome oxidase, the observed differences of staining intensity in the absence of exogenous cytochrome c are postulated to result from different content of reactive endogenous cytochrome c in mitochondria of the investigated cells.
Asunto(s)
3,3'-Diaminobencidina , Grupo Citocromo c/metabolismo , Histocitoquímica/métodos , Túbulos Renales/citología , Mitocondrias/metabolismo , Miocardio/citología , Bazo/citología , Animales , Túbulos Renales/metabolismo , Túbulos Renales/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Mitocondrias/ultraestructura , Miocardio/metabolismo , Miocardio/ultraestructura , Células Fotorreceptoras/citología , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestructura , Ratas , Ratas Endogámicas , Bazo/metabolismo , Bazo/ultraestructuraRESUMEN
Activity of cytochrome c oxidase and the level of endogenous cytochrome c were investigated light microscopically in adult rat and mouse kidney by incubating unfixed frozen sections with diaminobenzidine (DAB) in the absence or presence of exogenous cytochrome c. The results suggest that DAB staining intensity mainly reflects the local density of mitochondria and only occasionally visualizes the differences in cytochrome oxidase activity and/or endogenous cytochrome c content. Most intense reaction was observed in proximal and distal tubules both in rat and mouse. Finer differentiation of reactivity in particular nephron segments and interspecies differences between rat and mouse kidney are also described.
Asunto(s)
3,3'-Diaminobencidina/metabolismo , Grupo Citocromo c/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Riñón/metabolismo , Animales , Riñón/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas EndogámicasRESUMEN
Density of eosinophils stained by substrateless DAB reaction was measured in the successive segments and the successive layers of rat digestive tract using a semiautomatic computerized image analysis system. The results were calculated in a dual way: per area of the entire layer and per area of its connective tissue compartment, in which the eosinophils were located (extremely small number of intraepithelial and intramuscular eosinophils was not included in the analysis). The density of eosinophils in the successive segments of the digestive tract seemed to depend primarily on the time of contact between the contents and the wall of the segment, being the highest in regions of food/content retention (fundus of the stomach, cecum) and the lowest in regions of rapid passage (esophagus, pylorus, duodenum). When eosinophil density in the successive layers was compared, its gradual decrease in the successive layers (villi>lamina propria>submucosa>muscularis) was observed in almost the entire small intestine and lower values in submucosa compared to mucosa were also evident in the stomach, duodenum and small intestine. It seems that there is a decreasing gradient of chemoattractants for eosinophils dependent of the distance from the lumen and the contents of the digestive tract. However, this decreasing pattern appeared only when the density was calculated for the area of the connective tissue compartment suggesting that this method of calculation more reliably reflects the distribution of eosinophils and should be used in future quantitative studies on tissue eosinophils.
Asunto(s)
Sistema Digestivo/citología , Eosinófilos/citología , Animales , Ciego/citología , Sistema Digestivo/anatomía & histología , Duodeno/citología , Esófago/citología , Íleon/citología , Citometría de Imagen , Yeyuno/citología , Recuento de Leucocitos , Píloro/citología , Ratas , Ratas Wistar , Recto/citología , Estómago/citologíaRESUMEN
Peroxisomes of different shapes were visualized in mouse Leydig cells from primary cultures by immunofluorescent demonstration of catalase and two peroxisomal lipid beta-oxidation enzymes, acyl-CoA oxidase and 3-ketoacyl-CoA thiolase. Apart from typical spherical profiles, rod-shaped, tubular and annular peroxisomes were also observed. The proportion between spherical and elongated forms varied from cell to cell; the latter forms seemed to occur more frequently in flattened cells spread on the glass in the course and after monolayer formation. On very rare occasions the Leydig cells contained small local networks of interconnected tubular peroxisomes, probably representing areas of peroxisomal reticulum, whose existence has been so far supported by substantial biochemical, but hardly any morphological evidence.
Asunto(s)
Células Intersticiales del Testículo/ultraestructura , Microcuerpos/enzimología , Acetil-CoA C-Acetiltransferasa/metabolismo , Acil-CoA Oxidasa , Animales , Células Cultivadas/citología , Células Cultivadas/enzimología , Células Cultivadas/ultraestructura , Inmunohistoquímica , Células Intersticiales del Testículo/enzimología , Masculino , Ratones , Ratones Endogámicos BALB C , Oxidorreductasas/metabolismoRESUMEN
Density of mast cells stained by Alcian Blue at pH 0.3 was measured in the successive segments and the successive layers of rat digestive tract using a semiautomatic computerized image analysis system. The results were calculated in a dual way: per area of the entire layer and per area of its connective tissue compartment, in which the mast cells were located (extremely small number of intraepithelial and intramuscular mast cells was not included in the analysis). The distribution of mast cells in the particular layers was rather uniform with exception of the fundus of glandular stomach, where they were located at two distinct levels: in the upper quarter of the mucosa and in its lowermost zone, with the layer inbetween being practically free of mast cells. The quantitative estimation of mast cell density in the successive segments of the digestive tract yielded a generally similar profile irrespectively of the way of calculating the results, with the highest overall densities being observed in the middle segments (stomach, jejunum). Especially high densities of mast cells per area unit of the connective tissue were found in the muscularis of glandular stomach and in the lamina propria of small intestine and cecum. The analysis of mast cell density in the successive layers of the particular digestive tract segments revealed no clear pattern when the results were expressed per area unit of the whole layer. Calculation of densities per area unit of the connective tissue showed that lamina propria contained the most numerous mast cells in the entire small and large intestine, moreover, a decreasing pattern of mast cell density across the wall (lamina propria>submucosa>muscularis) was observed in the jejunum, transverse colon and rectum, suggesting that the latter way of calculation more reliably reflects mast cell distribution, which may result from the joint influence of two factors: the distance from the lumen of the digestive tract and the density of nerve terminals in its wall.
Asunto(s)
Sistema Digestivo/citología , Mastocitos/metabolismo , Azul Alcián/metabolismo , Animales , Ciego/citología , Histocitoquímica , Intestino Delgado/citología , Ratas , Ratas Wistar , Estómago/citologíaRESUMEN
The oxidation of 3,3'-diaminobenzidine [DAB] by H2O2 catalysed by 4 transition metals, Cu++, Co++, Fe+++ and Mn++, was investigated spectrophotometrically in a model system. The velocity of the reactions was higher with higher concentrations of DAB, H2O2 and a metal as well as with higher temperature. Optimal pH found for reactions catalysed by different transition metals was pH = 7.0 for Cu++ and Co++, pH - 6.0 for Fe+++ and pH - 9.0 for Mn++. A rapid spontaneous DAB oxidation by H2O2 in the absence of any transition metal was observed at pH less than 5.0.