RESUMO
AIM: Mitochondria undergo continuous changes in shape as result of complex fusion and fission processes. The physiological relevance of mitochondrial dynamics is still unclear. In the field of mitochondria bioenergetics, there is a need of tools to assess cell mitochondrial content. To develop a method to visualize mitochondrial networks in high resolution and assess mitochondrial volume. METHODS: Confocal fluorescence microscopy imaging of mitochondrial network stains in human vastus lateralis single muscle fibres and focused ion beam/ scanning electron microscopy (FIB/SEM) imaging, combined with 3D reconstruction was used as a tool to analyse mitochondrial morphology and measure mitochondrial fractional volume. RESULTS: Most type I and type II muscle fibres have tubular highly interconnected profusion mitochondria, which are thicker and more structured in type I muscle fibres (Fig. 1). In some muscle fibres, profission-isolated ellipsoid-shaped mitochondria were observed. Mitochondrial volume was significantly higher in type I muscle fibres and showed no correlation with any of the investigated molecular and biochemical mitochondrial measurements (Fig. 2). Three-dimensional reconstruction of FIB/SEM data sets shows that some subsarcolemmal mitochondria are physically interconnected with some intermyofibrillar mitochondria (Fig. 3). CONCLUSION: Two microscopy methods to visualize skeletal muscle mitochondrial networks in 3D are described and can be used as tools to investigate mitochondrial dynamics in response to life-style interventions and/or in certain pathologies. Our results question the classification of mitochondria into subsarcolemmal and intermyofibrillar pools, as they are physically interconnected.
Assuntos
Metabolismo Energético/fisiologia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Mitocôndrias , Músculo Esquelético/citologia , Adulto , Humanos , Imageamento Tridimensional/métodos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Fibras Musculares de Contração Lenta/citologia , Músculo Esquelético/metabolismoRESUMO
The myotendinous junction (MTJ) is a specialized structure in the musculotendinous system, where force is transmitted from muscle to tendon. Animal models have shown that the MTJ takes form of tendon finger-like processes merging with muscle tissue. The human MTJ is largely unknown and has never been described in three dimensions (3D). The aim of this study was to describe the ultrastructure of the human MTJ and render 3D reconstructions. Fourteen subjects (age 25 ± 3 years) with isolated injury of the anterior cruciate ligament (ACL), scheduled for reconstruction with a semitendinosus/gracilis graft were included. Semitendinosus and gracilis tendons were stripped as grafts for the ACL reconstruction. The MTJ was isolated from the grafts and prepared for transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy. It was possible to isolate recognizable MTJ tissue from all 14 patients. TEM images displayed similarities to observations in animals: Sarcolemmal evaginations observed as finger-like processes from the tendon and endomysium surrounding the muscle fibers, with myofilaments extending from the final Z-line of the muscle fiber merging with the tendon tissue. The 3D reconstruction revealed that tendon made ridge-like protrusions, which interdigitiated with groove-like indentations in the muscle cell.
Assuntos
Lesões do Ligamento Cruzado Anterior , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/ultraestrutura , Miofibrilas/ultraestrutura , Sarcolema/ultraestrutura , Tendões/ultraestrutura , Adulto , Ligamento Cruzado Anterior/cirurgia , Reconstrução do Ligamento Cruzado Anterior , Citoesqueleto/ultraestrutura , Humanos , Imageamento Tridimensional , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Músculo Esquelético/transplante , Tendões/transplante , Coxa da Perna , Adulto JovemRESUMO
Early diagnosis of severe infectious diseases is essential for timely implementation of lifesaving therapies. In a search for novel biomarkers in sepsis diagnosis we focused on polymorphonuclear neutrophils (PMNs). Notably, PMNs have their protein cargo readily stored in granules and following systemic stimulation, an immediate increase of neutrophil-borne proteins can be observed into the circulation of sepsis patients. We applied a combination of mass spectrometry (MS) based approaches, LC-MS/MS and selected reaction monitoring (SRM), to characterise and quantify the neutrophil proteome in healthy or disease conditions. With this approach we identified a neutrophil-derived protein abundance pattern in blood plasma consisting of 20 proteins that can be used as a protein signature for severe infectious diseases. Our results also show that SRM is highly sensitive, specific, and reproducible and, thus, a promising technology to study a complex, dynamic and multifactorial disease such as sepsis.
Assuntos
Proteínas Sanguíneas/metabolismo , Cromatografia Líquida , Neutrófilos/metabolismo , Proteômica/métodos , Sepse/sangue , Espectrometria de Massas em Tandem , Biomarcadores/metabolismo , Estudos de Casos e Controles , Progressão da Doença , Humanos , Neutrófilos/imunologia , Neutrófilos/microbiologia , Valor Preditivo dos Testes , Sepse/diagnóstico , Sepse/imunologia , Sepse/microbiologia , Índice de Gravidade de DoençaRESUMO
Overuse Achilles tendinopathy is a common and challenging problem in sports medicine. Little is known about the etiology of this disorder, and the development of a good animal model for overuse tendinopathy is essential for advancing insight into the disease mechanisms. Our aim was to test a previously proposed rat model for Achilles tendon overuse. Ten adult male Sprague-Dawley rats ran on a treadmill with 10° incline, 1 h/day, 5 days/wk (17-20 m/min) for 12 wk and were compared with 12 control rats. Histological, mechanical, and gene-expression changes were measured on the Achilles tendons after the intervention, and local tendon glucose-uptake was measured before and after the intervention with positron emission tomography. No differences were detected between runners and controls in tissue histology or in glucose uptake, indicating that tendon pathology was not induced. Greater tendon tissue modulus (P < 0.005) and failure stress/body weight (P < 0.02) in runners compared with controls further supported that tendons successfully adapted to uphill running. Several genes of interest were regulated after 12 wk of running. Expression of collagen III and insulin-like growth factor I was increased, while collagen I was unchanged, and decreases were seen in noncollagen matrix components (fibromodulin and biglycan), matrix degrading enzymes, transforming growth factor-ß1, and connective tissue growth factor. In conclusion, the tested model could not be validated as a model for Achilles tendinopathy, as the rats were able to adapt to 12 wk of uphill running without any signs of tendinopathy. Improved mechanical properties were observed, as well as changes in gene-expression that were distinctly different from what is seen in tendinopathy and in response to short-term tendon loading.
Assuntos
Tendão do Calcâneo/metabolismo , Fenômenos Biomecânicos/fisiologia , Regulação da Expressão Gênica/fisiologia , Condicionamento Físico Animal/métodos , Condicionamento Físico Animal/fisiologia , Corrida/fisiologia , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The glycocalyx or endocapillary layer on the luminal surface of microvessels has a major role in the exclusion of macromolecules from the underlying endothelial cells. Current structural evidence in the capillaries of frog mesentery indicates a regularity in the structure of the glycocalyx, with a center-to-center fiber spacing of 20 nm and a fiber width of 12 nm, which might explain the observed macromolecular filtering properties. In this study, we used electron micrographs of tissues prepared using perfusion fixation and tannic acid treatment. The digitized images were analyzed using autocorrelation to find common spacings and to establish whether similar structures, hence mechanisms, are present in the microvessel glycocalyces of a variety of mammalian tissues. Continuous glycocalyx layers in mammalian microvessels of choroid, renal tubules, glomerulus, and psoas muscle all showed similar lateral spacings at â¼19.5 nm (possibly in a quasitetragonal lattice) and longer spacings above 100 nm. Individual glycocalyx tufts above fenestrations in the first three of these tissues and also in stomach fundus and jejunum showed evidence for similar short-range structural regularity, but with more disorder. The fiber diameter was estimated as 18.8 (± 0.2) nm, but we believe this is an overestimate because of the staining method used. The implications of these findings are discussed.
Assuntos
Células Endoteliais/citologia , Glicocálix/metabolismo , Microvasos/citologia , Animais , Anuros , Capilares/citologia , Feminino , Masculino , Especificidade de Órgãos , Coelhos , RatosRESUMO
Knowledge about the effects of exercise on myofibrillar protein breakdown in human subjects is limited. Our purpose was to measure the changes in the degradation of myofibrillar proteins in response to different ways of eliciting muscle contractions using the local interstitial 3-methyl-histidine (3-MH) concentration as a marker for myofibrillar protein breakdown. Untrained males (n=8, 22-27 years, range) performed 210 maximal isokinetic eccentric contractions with each leg on an isokinetic dynamometer. One leg performed voluntary (VOL) and the other leg performed electrically induced contractions (ES). Microdialysis probes were placed in m. vastus lateralis in both the legs immediately after, and 1 and 3 days post-exercise. Interstitial 3-MH was higher in ES vs VOL immediately after exercise (P<0.05). One and 3 days post-exercise no difference between the two exercise types was observed. Only after ES did the histochemical stainings show significant disruption of cytoskeletal proteins. Furthermore, intracellular disruption and destroyed Z-lines were markedly more pronounced in ES vs VOL. In conclusion, the local level of interstitial 3-MH in the skeletal muscle was significantly enhanced after ES compared with VOL immediately after exercise, while the level of 3-MH did not change in the post-exercise period after VOL. These results indicate that the local myofibrillar breakdown is accelerated after ES associated with severe myofiber damage.
Assuntos
Estimulação Elétrica , Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Adulto , Biomarcadores , Dinamarca , Teste de Esforço , Humanos , Hidrólise , Masculino , Metilistidinas/análise , Adulto JovemRESUMO
CCR5 and CXCR3 are important molecules in regulating the migration of activated lymphocytes. Thus, the majority of tissue-infiltrating T cells found in the context of autoimmune conditions and viral infections express CCR5 and CXCR3, and the principal chemokine ligands are expressed within inflamed tissues. Accordingly, intervention studies have pointed to nonredundant roles of these receptors in models of allograft rejection, viral infection, and autoimmunity. In spite of this, considerable controversy exists, with many studies failing to support a role for CCR5 or CXCR3 in disease pathogenesis. One possible explanation is that different chemokine receptors may take over in the absence of any individual receptor, thus rendering individual receptors redundant. We have attempted to address this issue by analyzing CCR5(-/-), CXCR3(-/-), and CCR5/CXCR3(-/-) mice with regard to virus-induced liver inflammation, generation and recruitment of effector cells, virus control, and immunopathology. Our results indicate that CCR5 and CXCR3 are largely dispensable for tissue infiltration and virus control. In contrast, the T-cell response is accelerated in CCR5(-/-) and CCR5/CXCR3(-/-) mice and the absence of CCR5 is associated with the induction of CD8(+) T-cell-mediated immunopathology consisting of marked hepatic microvesicular steatosis.
Assuntos
Infecções por Arenaviridae/imunologia , Movimento Celular/imunologia , Fígado Gorduroso/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptores CCR5/imunologia , Receptores de Quimiocinas/imunologia , Animais , Infecções por Arenaviridae/complicações , Infecções por Arenaviridae/genética , Infecções por Arenaviridae/patologia , Autoimunidade/genética , Linfócitos T CD8-Positivos , Movimento Celular/genética , Quimiocinas/imunologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fígado Gorduroso/virologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Camundongos , Camundongos Knockout , Receptores CCR5/deficiência , Receptores CXCR3 , Receptores de Quimiocinas/deficiênciaRESUMO
Disruption to proteins within the myofibre after a single bout of unaccustomed eccentric exercise is hypothesized to induce delayed onset of muscle soreness and to be associated with an activation of satellite cells. This has been shown in animal models using electrical stimulation but not in humans using voluntary exercise. Untrained males (n=8, range 22-27 years) performed 210 maximal eccentric contractions with each leg on an isokinetic dynamometer, voluntarily (VOL) with one leg and electrically induced (ES) with the other leg. Assessments from the skeletal muscle were obtained prior to exercise and at 5, 24, 96 and 192 h postexercise. Muscle tenderness rose in VOL and ES after 24 h, and did not differ between groups. Maximal isometric contraction strength, rate of force development and impulse declined in the VOL leg from 4 h after exercise, but not in ES (except at 24 h). In contrast, a significant disruption of cytoskeletal proteins (desmin) and a rise of myogenic growth factors (myogenin) occurred only in ES. Intracellular disruption and destroyed Z-lines were markedly more pronounced in ES (40%) compared with VOL (10%). Likewise, the increase in satellite cell markers [neural cell adhesion molecule (N-CAM) and paired-box transcription factor (Pax-7)] was more pronounced in ES versus VOL. Finally, staining of the intramuscular connective tissue (tenascin C) was increased equally in ES and VOL after exercise. The present study demonstrates that in human muscle, the delayed onset of muscle soreness was not significantly different between the two treatments despite marked differences in intramuscular histological markers, in particular myofibre proteins and satellite cell markers. An increase in tenascin C expression in the midbelly of the skeletal muscle in both legs provides further evidence of a potential role for the extracellular matrix in the phenomenon of delayed onset of muscle soreness.
Assuntos
Contração Muscular/fisiologia , Músculo Esquelético/fisiopatologia , Miofibrilas/patologia , Miofibrilas/fisiologia , Tenascina/metabolismo , Adulto , Biomarcadores/metabolismo , Biópsia , Estimulação Elétrica/efeitos adversos , Exercício Físico/fisiologia , Matriz Extracelular/fisiologia , Humanos , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miofibrilas/ultraestrutura , Dor/fisiopatologia , Células Satélites de Músculo Esquelético/patologia , Células Satélites de Músculo Esquelético/fisiologia , Vimentina/metabolismoRESUMO
The present study examined the hypothesis that collagen fibril diameter and crimp angle in ruptured human Achilles tendons differed from that of intact ones. Tissue samples were obtained from the central core (distal core) and the posterior periphery (distal superficial) at the rupture site, and the proximally intact (proximal superficial) part of the tendon in 10 subjects (38+/-8 years) with a complete tendon rupture. For comparisons corresponding tissue samples were procured from age (38+/-7 years) and gender matched intact Achilles tendons during routine forensic autopsy. The cross-sectional area density and diameter distribution of fibrils were analyzed using stereological techniques of digitized electron microscopy biopsy cross-sections, while crimp angle was measured by the changing banding pattern of collagen fibers when rotated between crossed polars. Nine of 10 persons with tendon ruptures reported that the injury did not occur during exceedingly large forces, and none experienced any symptoms in the days or months prior to the injury. Fibril diameter distribution showed no region-specific differences in either the ruptured or intact tendons for either group. However, in the distal core there were fewer fibrils in the ruptured compared to the intact tendons in 60-150 nm range, P<0.01. Similarly, in the distal superficial portion there were fewer fibrils in the ruptured compared to the intact tendons in the 90-120 nm range, 2P<0.05, while there were no differences in the proximal superficial tendons. Crimp angle did not display any region-specific differences, or any difference between the rupture and intact tendons. In conclusion, these data suggest that although crimp morphology is unchanged there appears to be a site-specific loss of larger fibrils in the core and periphery of the Achilles tendon rupture site. Moreover, the lack of symptoms prior to the rupture suggests that clinical tendinopathy is not an etiological factor in complete tendon ruptures.
Assuntos
Tendão do Calcâneo/química , Tendão do Calcâneo/patologia , Colágenos Associados a Fibrilas/análise , Colágenos Associados a Fibrilas/química , Ruptura/patologia , Traumatismos dos Tendões/patologia , Tendão do Calcâneo/citologia , Adulto , Feminino , Humanos , Masculino , Microscopia Eletrônica , Microscopia de Polarização , Pessoa de Meia-Idade , Ruptura/metabolismo , Traumatismos dos Tendões/metabolismo , Resistência à TraçãoRESUMO
OBJECTIVE: The lungs have dual blood supply: The bronchial and the pulmonary circulation. The importance of bronchial circulation is disputed. The purpose of this study was to establish an experimental model to examine the importance of the bronchial artery. DESIGN: Comparative porcine experimental study. The surgical technique was evaluated in group A (n = 9). Group B (n = 8) underwent left bronchial arterial devascularization. In group C (n = 9) the left principal bronchus was devascularized, transsected, and reanastomosed. In groups B and C bronchial mucosal blood flow was studied with laser-Doppler velocimetry. Devascularization was controlled by angiography at section, and specimens were examined with conventional histology and scanning electron microscopy. The right bronchus served as control. RESULTS: In group B devascularization caused considerable, yet insignificant reduction in bronchial mucosal blood flow index (p = 0.1282) postoperatively, and after 1 week (p = 0.0678), insignificant histologic (p > 0.2) changes, and no scanning electron microscopy differences. In group C devascularization with bronchial transsection caused significant reduction in mucosal blood flow index (p = 0.0277) postoperatively and after 1 week (p = 0.0277), significant histologic changes (p = 0.0277), and insignificant (p = 0.069) changes in scanning electron microscopy. CONCLUSION: Bronchial arterial devascularization with transsection caused significant physiologic and morphologic changes, and a model with bronchial devascularization should include transsection.
Assuntos
Brônquios/irrigação sanguínea , Artérias Brônquicas/fisiologia , Modelos Animais , Fluxo Sanguíneo Regional/fisiologia , Angiografia , Animais , Brônquios/ultraestrutura , Feminino , Pulmão/cirurgia , Pulmão/ultraestrutura , Circulação Pulmonar/fisiologia , SuínosRESUMO
BACKGROUND: The bronchial artery may be vital to the bronchi and lung parenchyma, but results of lung transplantation have raised doubts. This study was performed to examine the effect of bronchial arterial devascularization on bronchial morphology after bronchial transsection and reanastomosis. METHODS: In 6 pigs (study group), the left main bronchus was transsected, reanastomosed, and devascularized. Six control pigs had the same operation without devascularization. After 1 week, bronchial arterial angiography was performed, and specimens were examined with conventional histology and scanning electron microscopy. RESULTS: Histology showed significant changes (inflammation, edema, and fibrosis) in bronchi and lung parenchyma of the study group compared with the unoperated side (p = 0.028) and with the control group (p = 0.050). Scanning electron microscopy showed significant ciliary denudation in the study group's left bronchus compared with the unoperated side (p = 0.043) and with the control group (p = 0.0071). CONCLUSIONS: The loss of cilia of the bronchial epithelium and the occurrence of inflammation, edema, and fibrosis in bronchi and lung parenchyma 1 week postoperatively were significantly related to the absence of the bronchial arterial circulation.
Assuntos
Brônquios/patologia , Artérias Brônquicas/fisiologia , Pulmão/patologia , Anastomose Cirúrgica , Animais , Brônquios/cirurgia , Cílios , Feminino , Microscopia Eletrônica de Varredura , Fluxo Sanguíneo Regional , SuínosRESUMO
A previously unknown ligament, the superficial anulus fibrosus ligament (SAFL), situated on the ventral part of the L5 intervertebral disc (ID) was observed and described from autopsy material. Twenty-eight cadaveric specimens from 12 black and 16 white persons aged 17-30 years were studied during routine forensic autopsies. The anterior longitudinal ligament was separated from the ID and the ventral part of the SAFL was visualized. The SAFL samples were removed, measured and studied with both conventional histology and examination by transmission electron microscopy. The SAFL was a completely separate ligament at the level of the L5-S1 ID situated between the anterior longitudinal ligament and the anulus fibrosus of the ID. The fibers of the ligament were vertically oriented. A difference in distance between the L5-S1 vertebral bodies ventrally was noted in the two groups studied (18.7 +/- 1.2 mm in the black vs. 15.2 +/- 1.0 mm in the white, p < 0.001), indicating a difference in the ventral thickness of the intervertebral disc. Also, there was a difference in the length (black: 17.7 +/- 1.6 mm vs. white: 14.1 +/- 1.1), thickness (black: 3.3 +/- 0.3 mm vs. white: 2.1 +/- 1.9), and the cross-sectional area (black: 58.2 +/- 6.7 mm(2) vs. white: 26.5 +/- 2.7 mm(2), p < 0.001) of the SAFL. Conventional light microscopy revealed no obvious differences. However, transmission electron microscopy revealed notably larger collagen fibril diameter in black than white subjects. In conclusion, the interbody distances were greater in the black group, indicating a greater intervertebral disc thickness, compared to that of the white. Furthermore, the SAFL was significantly longer and thicker in the black than in the white group. Albeit unsubstantiated, these race-specific macroscopic findings may have implications for understanding the etiology of various low back stress problems.
Assuntos
Ligamentos/anatomia & histologia , Coluna Vertebral/anatomia & histologia , Adolescente , Adulto , População Negra , Cadáver , Humanos , Disco Intervertebral/anatomia & histologia , Ligamentos/ultraestrutura , Vértebras Lombares/anatomia & histologia , Microscopia Eletrônica , Coluna Vertebral/ultraestrutura , População BrancaRESUMO
Airway epithelium explants from cystic fibrosis (CF) patients and non-CF subjects formed monolayered spheres, with the apical ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. With the use of two microelectrodes, transepithelial potential difference and changes in potential difference in response to passage of current pulses were recorded, and epithelial resistance and the equivalent short-circuit current were calculated. Non-CF control potential difference and short-circuit current values were significantly lower than the CF values, and amiloride inhibited both values. Fluid transport rates were calculated from repeated measurements of spheroid diameters. The results showed that 1) non-CF and CF spheroids absorbed fluid at identical rates (4.4 microl x cm(-2) x h(-1)), 2) amiloride inhibited fluid absorption to a lower residual level in non-CF than in CF spheroids, 3) Cl(-)-channel inhibitors increased fluid absorption in amiloride-treated non-CF spheroids to a level equal to that of amiloride-treated CF spheroids, 4) hydrochlorothiazide reduced the amiloride-insensitive fluid absorption in both non-CF and CF spheroids, and 5) osmotic water permeabilities were equal in non-CF and CF spheroids ( approximately 27 x 10(-7) cm x s(-1) x atm(-1)).
Assuntos
Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Cavidade Nasal/citologia , Cavidade Nasal/metabolismo , Água/metabolismo , Amilorida/farmacologia , Técnicas de Cultura de Células/métodos , Membrana Celular/metabolismo , Células Cultivadas , Diuréticos/farmacologia , Eletrofisiologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Hidroclorotiazida/farmacologia , Masculino , Osmose/efeitos dos fármacos , Inibidores de Simportadores de Cloreto de Sódio/farmacologia , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
A previous investigation indicated that the chief cells of the endolymphatic sac produce an endogenous inhibitor of sodium re-absorption in the kidneys, which has tentatively been named "saccin". In this study, the ultrastructure of the endolymphatic sac and in particular the chief cells is described, demonstrating that this organ fulfils the morphological criteria of a potential endocrine gland. Accordingly, the chief cells are shown to exhibit all the organelles and characteristics of cells that simultaneously synthesize, secrete, absorb and digest proteins.
Assuntos
Glândulas Endócrinas/fisiologia , Saco Endolinfático/fisiologia , Natriuréticos/metabolismo , Animais , Saco Endolinfático/citologia , Saco Endolinfático/ultraestrutura , Masculino , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Ratos , Ratos WistarRESUMO
PURPOSE: Production of reliable sections of fovea centralis of primate retina. METHODS: Rhesus monkey eyes were processed using a new technique known to minimize artefacts and cell-shrinkage in other tissues. An in vivo vascular perfusion-fixation technique with an isotonic fixative followed by a process of slow infiltration with small increments in concentrations were used. RESULTS: Tangential 1 microm thick serial-sections showed a very good morphology. An average cone-to-cone-center distance of 3.8 microm in the central part of the fovea centralis was found. A cone-to-cone-center distance of 2.2-2.6 microm is suggested in the literature. CONCLUSION: In spite of the limited study we believe that the new technique has resulted in less tissue shrinkage. Our results are discussed in relation to maximal possible resolution of the eye. Our calculations (for green light) support the idea that the structure of the fovea centralis and the refractive media of the eye are developed close to perfection.
Assuntos
Fóvea Central/citologia , Macaca mulatta/anatomia & histologia , Células Fotorreceptoras Retinianas Cones/citologia , Acuidade Visual , Animais , Cadáver , Fóvea Central/fisiologia , Humanos , Matemática , Fotomicrografia , Células Fotorreceptoras Retinianas Cones/fisiologiaRESUMO
In the present study, we describe a novel three-dimensional airway epithelial explant preparation and demonstrate its use for ion transport studies by electrophysiological technique. Suspension cultures of sheets of epithelial cells released by protease treatment from cystic fibrosis (CF) and non-CF nasal polyps developed free-floating, monolayered epithelial spheres, with the apical, ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. Microelectrode impalement of both non-CF and CF spheroids revealed lumen-positive transepithelial electrical potential differences (PDs) that were inhibited by amiloride, indicating that the spheroids were inflated due to amiloride-sensitive Na+ absorption followed by water. Transformation to a Cl- secretory state was achieved by addition of ATP to the bath, leading to the development of a diphenylamine-2-carboxylate-sensitive PD. A cAMP-induced increase in PD was seen in non-CF spheroids only. In response to hydrocortisone treatment, Na+ transport reflected by amiloride-sensitive PD increased and more so in CF than in non-CF spheres. We concluded that this preparation is a useful model for the airway surface epithelium and is suitable for studies of transport mechanisms and regulation.
Assuntos
Pólipos Nasais/metabolismo , Absorção/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Amilorida/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cloretos/metabolismo , AMP Cíclico/fisiologia , Fibrose Cística/complicações , Eletrofisiologia , Epitélio/metabolismo , Epitélio/fisiopatologia , Feminino , Humanos , Hidrocortisona/farmacologia , Íons , Masculino , Pólipos Nasais/complicações , Pólipos Nasais/patologia , Pólipos Nasais/fisiopatologia , Sódio/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , ortoaminobenzoatos/farmacologiaRESUMO
Using histological and ultrastructural techniques the aims of this study were to investigate whether the mineralization pattern and surface microanatomy of the caries-susceptible fissure enamel were different from those on the caries-inactive lingual surface. The material consisted of 31 unerupted third mandibular molars. The specimens were initially grouped into four categories: (1) without, (2) with initial, (3) with almost completed and (4) with completed root formation. One ground section with fissure-like morphology was selected from each tooth. Using water as a medium the observed birefringence was negative along the lingual and fissure transverses in specimens with almost completed and with completed root formation, while the observed birefringence was positive at different distances in the enamel in sections representing less maturation stages. Qualitative imbibition studies revealed hypomineralized enamel in the lower part of the fissures in specimens representing almost and completed root formation. Imbibed in quinoline, parts of the hypomineralized enamel behaved like a molecular sieve due to the presence of micropores, indicating that the structural arrangement is different from that in the enamel adjacent to this areas. After division of the sections into a lingual and a buccal part, SEM features were described from lower and upper parts of the buccal fissure wall and on lingual enamel in the area corresponding to the bottom part of the fissure. The surface microanatomy varied greatly. Negative developmental irregularities such as fissures and holes were associated with the immature enamel, while matured enamel - particularly fissures - housed many positive developmental irregularities such as enamel caps and protrusions. The crystal size in the mature specimens appeared smaller and more uniform than the crystals from the immature specimens. Apart from the occurrence of hypomineralized enamel in fissures and numerous positive developmental irregularities on the fissure surface, no major differences between fissure and lingual enamel were noticed, neither with respect to mineralization pattern during final stages of tooth development nor to the degree of surface porosity prior to tooth emergence.
Assuntos
Esmalte Dentário/ultraestrutura , Dente Serotino/ultraestrutura , Dente não Erupcionado/ultraestrutura , Cristalografia , Suscetibilidade à Cárie Dentária , Fissuras Dentárias/patologia , Técnicas de Preparação Histocitológica , Humanos , Mandíbula , Microscopia Eletrônica de Varredura , Odontogênese/fisiologia , Porosidade , Quinolinas , Calcificação de Dente , Raiz Dentária/fisiologia , ÁguaRESUMO
The immunosuppressant cyclosporine protects against the development of experimental transplant arteriosclerosis. To investigate the mechanism underlying this effect, aorta-allografted rabbits were randomly assigned to cyclosporine (n = 6) in the human therapeutic range or to its vehicle (n = 5). Perfusion fixation was performed 2 weeks after the transplantation, followed by light, scanning and transmission electron microscopy examination. Intimal proliferation was absent in native aortas, present in all grafts from vehicle-treated animals, and either absent or sparse in grafts from cyclosporine-treated rabbits. The endothelium of native aortas from both vehicle- and cyclosporine-treated rabbits was normal. Aortic allografts from cyclosporine-treated rabbits exhibited a normal endothelium with only a few adhering mononuclear cells, whereas aortic allografts from vehicle-treated rabbits exhibited an endothelial surface ranging from near-normal with only few adhering platelets and mononuclear cells, to an almost destroyed endothelium, lined with lymphocyte-like cells, monocytes/macrophages, platelets, erythrocytes and fibrin; in the subendothelial layer, mononuclear cells and smooth muscle cells were abundant. These results suggest that damage to the endothelial cells as well as invasion of lymphocytes, monocytes/macrophages and smooth muscle cells into the subendothelial space are important events during initiation of transplant arteriosclerosis, and that cyclosporine largely attenuates these early pathological changes.
Assuntos
Aorta Torácica/transplante , Arteriosclerose/etiologia , Arteriosclerose/prevenção & controle , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/ultraestrutura , Aorta Torácica/efeitos dos fármacos , Arteriosclerose/patologia , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Colesterol/sangue , Ciclosporina/administração & dosagem , Endotélio/efeitos dos fármacos , Endotélio/patologia , Endotélio/ultraestrutura , Rejeição de Enxerto/prevenção & controle , Imunossupressores/administração & dosagem , Masculino , Microscopia , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Veículos Farmacêuticos/uso terapêutico , Coelhos , Transplante Homólogo/imunologiaRESUMO
The permeability of capillaries is usually explained with reference to "small pores," permeable to water and small solutes, and "large pores," permeable to proteins. Fenestrated capillaries are far more permeable to water and small solutes than nonfenestrated capillaries; moreover, the permeability is highest in the capillaries with the most fenestrae. This suggests that the small pores reside in the fenestral diaphragms; however, no such pores have been illustrated using electron microscopy. In an attempt to clarify this controversy, fenestrated capillaries in the small intestine, stomach, and kidney of the rat were reinvestigated by electron microscopy. A new method of vascular perfusion-fixation was employed, which included the use of glutaraldehyde dissolved in an oxygen-carrying blood substitute (fluorocarbon) and delivered by a peristaltic pump controlled by pressure feedback. The investigation disclosed a prominent endocapillary surface coat, about 50-100 nm thick, and the existence of bush-like filamentous sieve plugs in the fenestrae. Each filamentous plug was composed of 20-40 filaments, each filament measuring 300-400 nm in length and 5-10 nm in thickness. The filamentous plugs have tentatively been named fascinae fenestrae, because they resemble fascines and may also function as such.
Assuntos
Citoesqueleto de Actina/fisiologia , Permeabilidade Capilar/fisiologia , Animais , Substitutos Sanguíneos , Capilares/patologia , Filtração , Mucosa Gástrica/irrigação sanguínea , Glutaral , Intestinos/irrigação sanguínea , Intestinos/ultraestrutura , Glomérulos Renais/irrigação sanguínea , Túbulos Renais/irrigação sanguínea , Microscopia Eletrônica , Microvilosidades/ultraestrutura , Porosidade , Ratos , Ratos WistarRESUMO
Cytoplasmic granules have been demonstrated in epithelial cells from the endolymphatic sac, an extraosseus part of the inner ear located in the posterior cranial fossa. Intravenously infused extracts from endolymphatic sacs in anesthetized rats elicited a potent natriuresis and diuresis without effects on blood pressure, glomerular filtration rate, or lithium clearance. Only a minor kaliuresis was observed. Extracts of dural tissue adjacent to the endolymphatic sacs had no effect. It is concluded that the endolymphatic sac contains as endogenous inhibitor of sodium reabsorption and could be the sensory organ/mediator of "cerebral" natriuresis. Furthermore, this substance, tentatively named saccin, may influence the homeostasis of the inner ear fluids and accordingly play a significant role in the pathogenesis of Mèniére's disease.