RESUMO
The angiopoietins and members of the vascular endothelial growth factor (VEGF) family are the only growth factors thought to be largely specific for vascular endothelial cells. Targeted gene inactivation studies in mice have shown that VEGF is necessary for the early stages of vascular development and that angiopoietin-1 is required for the later stages of vascular remodeling. Here it is shown that transgenic overexpression of angiopoietin-1 in the skin of mice produces larger, more numerous, and more highly branched vessels. These results raise the possibility that angiopoietins can be used, alone or in combination with VEGF, to promote therapeutic angiogenesis.
Assuntos
Glicoproteínas de Membrana/fisiologia , Neovascularização Fisiológica , Pele/irrigação sanguínea , Angiopoietina-1 , Animais , Capilares/anatomia & histologia , Capilares/ultraestrutura , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/ultraestrutura , Expressão Gênica , Queratinócitos/metabolismo , Linfocinas/genética , Linfocinas/fisiologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Pele/metabolismo , Transgenes , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Vênulas/anatomia & histologia , Vênulas/ultraestruturaRESUMO
Hamsters breathed, nose-only, for 13 weeks, 5 days/week, 6 hr/day, either man-made vitreous fiber (MMVF)10a, MMVF33, or long amosite asbestos at approximately 300 World Health Organization (WHO) fibers/cc or long amosite at 25 WHO fibers/cc. [World Health Organization fibers are longer than 5 microm and thicker than 3 microm, with aspect ratio >3.] After sacrifice, fiber burden was estimated (left lungs) by ashing and scanning electron microscopy (ashing/SEM) or (right middle lobes) by confocal laser scanning microscopy (CLSM) in situ. In situ CLSM also provided three-dimensional views of fibers retained, undisturbed, in lung tissue. Fibers of each type were lodged in alveoli and small airways, especially at airway bifurcations, and were seen fully or partly engulfed by alveolar macrophages. Amosite fibers penetrated into and through alveolar septa. Length densities of fibers in parenchyma (total length of fiber per unit volume of lung) were estimated stereologically from fiber transsections counted on two-dimensional optical sections and were 30.5, 25.3, 20.0, and 81.6 mm/mm3 for MMVF10a, MMVF33, and low- and high-dose amosite, respectively. Lengths of individual fibers were measured in three dimensions by tracking individual fibers through series of optical sections. Length distributions of amosite fibers aerosolized, but before inhalation versus after retention in the lung were similar, whether determined by ashing/SEM or in situ CLSM. In contrast, the fraction of short MMVF10a and MMVF33 fibers increased and the geometric mean fiber lengths of both MMVFs decreased by approximately 60% during retention. Most likely due to fiber deposition pattern and differences in sampling, fiber burdens [MMVF10a, MMVF33, and amosite (high dose; 269 WHO fibers/cc)] determined by ashing/SEM were 1.4, 1. 5, and 3.5 times greater, respectively, than those calculated from in situ CLSM data. In situ CLSM is able to provide detailed information about the anatomic sites of fiber retention and also fiber lengths and burdens in good agreement with ashing/SEM results.
Assuntos
Poluentes Atmosféricos/efeitos adversos , Amianto Amosita/efeitos adversos , Vidro , Pneumopatias/etiologia , Administração por Inalação , Aerossóis , Animais , Cricetinae , Masculino , Mesocricetus , Tamanho da PartículaRESUMO
We have quantified the fibrous collagen (predominantly type I) and elastin in four locations of perceived mechanical importance: one quasi-planar feature, the alveolar septum or wall (W), and three linear features, the junction (J) of three septa, the free edges (E) of septa, and the line along which two septa join at a distinct angle or bend (B). The frequencies of these four features on light micrographs and the areas of transections through collagen and elastin seen on electron micrographs were combined to give the volumes of collagen and elastin within each feature. We find that E and B have similar compositions and contain most (4/5) of the parenchymal elastin in their relatively heavy cables. The E and B are interconnected and similar in location and composition, and they may constitute a functional entity in which elastin provides tension over a range of lung volumes, opposing septal tensions. In J and W, elastin is typically sparse and fine. Calculations, however, suggest it contributes the dominant portion of septal tension at lower lung volumes. Elastin may be essential to stabilizing septal configuration. Collagen, on the other hand, is distributed relatively evenly throughout E, B, J, and W, consistent with the role of protecting all components against rupture.
Assuntos
Colágeno/análise , Elastina/análise , Alvéolos Pulmonares/análise , Animais , Tecido Conjuntivo/análise , Tecido Conjuntivo/ultraestrutura , Cães , Elasticidade , Medidas de Volume Pulmonar , Matemática , Alvéolos Pulmonares/ultraestrutura , Tensão SuperficialRESUMO
On the basis of microscopic appearance of excised lungs, it has been thought that alveolar septa may fold and unfold during deflation and inflation. We suspected that this appearance might depend heavily on the inflation history of the lung preparation. We therefore studied, by light and electron microscopy, dog, rabbit, and rat lungs fixed over a range of inflation pressures and after a variety of inflation histories. Septal folding, as suggested by the configurations of the air spaces, by the placement of the fine and coarse connective tissue elements, and by the pattern of infolding of alveolar epithelium, was readily seen with some inflation protocols but was absent with others. Pressure at fixation was not as important as events before fixation; deflation to 3 cmH2O did not induce folding, and inflation to 16 cmH2O did not undo the folds. This range corresponds with concepts of critical opening and closing pressures. We suggest that folds form de novo during experimental preparation; one need not postulate that septal folding was present in vivo.
Assuntos
Pulmão/anatomia & histologia , Alvéolos Pulmonares/anatomia & histologia , Mecânica Respiratória/fisiologia , Animais , Cães , Técnicas Histológicas , Técnicas In Vitro , Pulmão/fisiologia , Medidas de Volume Pulmonar , Microscopia Eletrônica , Alvéolos Pulmonares/fisiologia , CoelhosRESUMO
To determine the dihedral angle, alpha, at the characteristic three-way septal junctions of lung parenchyma, we examined photomicrographs of sections. The three angles, A, formed where three septal traces meet on section, were measured and found to range between approximately 50 and 170 degrees. Theoretical considerations predicted that the dispersion of alpha is much narrower than that of A. The mean of A and alpha is identically 120 degrees. The standard deviation of alpha was inferred from the cumulative distribution function of A. In lungs inflated to 30 cmH2O (VL30), the standard deviation of alpha was very small (approximately 2 degrees) and increased to approximately 6 degrees in lungs inflated to 0.4 VL30. These findings imply that at VL30 tensions exerted by septa are locally homogeneous (2% variation) and at lower lung volumes become less so (6% variation). At high distending pressures, tissue forces are thought to dominate interfacial forces, and therefore the local uniformity of tensions suggests a stress-responsive mechanism for forming or remodeling the connective tissues. The source of the local nonuniformity at lower volumes is unclear but could relate to differences in mechanical properties of alveolar duct and alveoli. Finally, local uniformity does not imply global uniformity.
Assuntos
Alvéolos Pulmonares/anatomia & histologia , Animais , Cães , MatemáticaRESUMO
We present a theoretical treatment of backscattered light from the interior of a lung illuminated by a thin beam of light normally incident on the pleural surface. An approximate formula is developed describing how the backscattered intensity varies with distance from the point of light entry. This is shown to depend markedly on the optical mean free path and on the effective extinction coefficient. We attempt to relate the optical mean free path to the mean alveolar size. This relationship is found to depend primarily on septal reflection and refraction. Reflection is treated quantitatively. Refraction is much more difficult and may have to be approached empirically. We present here the rudiments of a technique with implications for the possibility of dynamic stereology.
Assuntos
Luz , Pulmão , Espalhamento de Radiação , Pulmão/anatomia & histologia , Matemática , Óptica e FotônicaRESUMO
The pattern of light backscattered by lung tissue should depend strongly on the size of air spaces and equivalently on the internal surface area of the lung. To verify and apply this, we shone a laser beam into excised lungs through the pleural surface and measured the backscattered light surrounding the beam with a focused photodetector. The intensity, I, fell off as a function of distance, r, from the point of entry of light. The configurations of I(r) curves corresponded closely to theory over a 3-decade range of I. I(r) changed systematically with lung volume. The optical mean free path, lambda, was calculated from I(r) curves in a series of canine lobes fixed immediately after optical scanning and was compared with stereological measurement of mean linear intercept, Lm, an index of alveolar size. At high lung volumes the relation of lambda to Lm was consistent with reflection by alveolar septa. At lower lung volumes there appeared to be, additionally, a substantial refractive component. This technique is independent of current stereological methods and has the advantages of being noninvasive, continuous, and potentially applicable to dynamic events in unfixed lungs.
Assuntos
Luz , Pulmão/anatomia & histologia , Espalhamento de Radiação , Animais , Cães , Lasers , Óptica e Fotônica/instrumentaçãoRESUMO
We sought a technique to preserve lung tissue for micrography with air-space dimensions unchanged from the fresh state. In our hands, conventional techniques were problematical. Aware of the possibility that distortion might be caused by inadequate mechanical fixation of elastin, we dehydrated the still-inflated lung by intravascular perfusion with graded ethanols. Canine and rabbit lungs so prepared had straighter alveolar septa, greater air-space dimensions, and an improved correlation with light-scattering measurements. Bovine ligamentum nuchae (mostly elastin) was only partially fixed by glutaraldehyde or osmium tetroxide but was effectively stiffened by dehydration. We conclude that perfusion dehydration aids in the faithful preservation of parenchymal configuration, probably by mechanical fixation of elastin.
Assuntos
Dessecação , Técnicas Histológicas , Pulmão/anatomia & histologia , Perfusão , Animais , Bovinos , Cães , Elastina , Fixadores , Técnicas Histológicas/instrumentação , Ligamentos , Pescoço , CoelhosRESUMO
Previous studies of isolated piglet lungs suggested that local distending forces around bronchi might be relatively weak before postnatal growth and maturation. The present study used tantalum bronchograms to compare pressure-diameter relationships of bronchi in situ and after excision from the parenchyma in immature (3- to 7-day-old) and mature (3-mo-old) piglets. The mature group reproduced behavior that is well established in mature lungs from other species; i.e., bronchial diameters maintained a constant relationship to the parenchyma as the lungs were deflated from maximum to minimum volume. In sharp contrast, diameters failed to change until the immature lungs were deflated to <5 cmH(2)O transpulmonary pressure. Total percent change in bronchial diameter was then only 24% in the immature lungs compared with 47% in the mature lungs (P < 0.002). Total elastances of mature generation 3-8 bronchi did not change when they were excised from the parenchyma. However, in the same generations of immature bronchi, total elastances were lower after than before (1.06 vs. 1.60 cmH(2)O/%, P < 0.05) excision from the parenchyma. Elastances of the excised immature and mature bronchi were then the same (1.06 vs. 1.03 cmH(2)O/%, not significant). Because elastic moduli of the lung parenchyma are also similar in the two age groups, it was concluded that local features of airway-parenchyma coupling limited the generation of local parenchymal recoil around bronchi in the immature lungs.
Assuntos
Brônquios , Fatores Etários , Resistência das Vias Respiratórias/fisiologia , Animais , Animais Recém-Nascidos , Brônquios/anatomia & histologia , Brônquios/crescimento & desenvolvimento , Brônquios/fisiologia , Elasticidade , Feminino , Complacência Pulmonar/fisiologia , Medidas de Volume Pulmonar , SuínosRESUMO
To clarify the mechanics of alveolar parenchyma, we undertook a stereological and topological study in perfusion-fixed canine lungs of the borders of alveolar septa. We defined the principal borders as those along which one septum 1) joins two others (J), 2) joins one other at a distinct angle (B), or 3) joins no other structure (E). E and B borders are invariably reinforced with heavy connective tissue cables; J borders are not. Relative net lengths, determined from the number of traces per section area, were J, 45%; E, 19%; and B, 25%. These were remarkably constant over 10 canine lobes (5 animals, 4 volumes). Parenchyma, then, departs from the simple models that comprise only Js and Es. Bs are important; their net length exceeds that of Es. With lobe deflation, E shortened somewhat more than required to maintain geometric similarity, suggesting that the alveolar duct contracted disproportionately. A three-dimensional reconstruction was made from serial sections, and individual border segments were followed through the reconstruction. Typical lengths of individual J, B, and E borders were nearly equal. To characterize how the network of borders were interconnected, we counted the nodes at which they meet by class, e.g., EBE for the meeting of one B, two Es. The most common are JJJJ, 26%; EEEJ, 10%; EBJ, 24%; EBE, 8%; BBJJ, 12%. If parenchyma were constructed only from free-standing entrance rings and septal junctions, only JJJJ and EEEJ would be anticipated. The presence of EBJ, EBE, and BBJJ underscores parenchymal complexity. Only 7% of septa examined were bordered entirely by Js. Connective tissue cables were not confined to the alveolar duct's lumen but often extended to the primary septa at the periphery of the ductal unit. They rarely linked adjacent alveolar ducts; only 1 in 200 cable segments crossed from one duct to another. These observations support the concept that the parenchyma is an elastic network, characterized in part by a serial mechanical linkage from connective tissue cable to septal membrane to cable again.
Assuntos
Pulmão/anatomia & histologia , Alvéolos Pulmonares/anatomia & histologia , Animais , Membrana Celular , Cães , Matemática , PerfusãoRESUMO
Alveolar parenchyma comprises two interacting tensile systems: the cable system (a network of linear condensations of connective tissue) and the membrane system (a network of quasiplanar alveolar septa). Inferences can be drawn about the mechanics of this structure from it configuration. We reported earlier (E.H. Oldmixon, J.P. Butler, and F.G. Hoppin, Jr. J. Appl. Physiol. 64: 299-307, 1988) that the angles between alveolar septa at the common three-way junctions (J) are nearly uniform, indicating that septal tensions are also nearly uniform. We now report on the interseptal angles at the next most common class of septal junction (B), a structure where two septa meet along a segment of the cable system. We find, first, that the distributions of interseptal angles at B junctions have means > 120 degrees, are narrow, and have few, if any, angles < 120 degrees. The findings of uniform 120 degrees angles at J junctions and a cutoff below 120 degrees at B junctions are also characteristic of soap films supported on a frame, which follows the physical principle of surface area minimization. We suggest that this principle may be operative in parenchymal development and remodeling.
Assuntos
Pulmão/anatomia & histologia , Animais , Fenômenos Biomecânicos , Tecido Conjuntivo/anatomia & histologia , Tecido Conjuntivo/fisiologia , Cães , Pulmão/crescimento & desenvolvimento , Pulmão/fisiologia , Medidas de Volume Pulmonar , Modelos Biológicos , Alvéolos Pulmonares/fisiologia , Propriedades de SuperfícieRESUMO
We have investigated the basis and implications of pneumoconstriction by measuring disposition and quantities of alpha-smooth muscle actin in rat and guinea pig lungs and modeling its effects on lung recoil and compliance. A robust marker of contractility, alpha-smooth muscle actin appears in smooth muscle or myofibroblast-like cells in pleura, airways, blood vessels, and alveolar ductal tissues. In each site, we measured its transected area by immunofluorescent staining and frequency-modulated scanning confocal microscopy. We incorporated these data in a model of the parenchyma consisting of an extensive elastic network with embedded contractile structures. We conclude that contraction at any one of these sites alone can decrease parenchymal compliance by 20-30% during tidal breathing. This is due mostly to the stiffness of activated contractile elements undergoing passive cycling; constant muscle tension would have little effect. The magnitude of the effect corresponds with known responses of the lung to hypocapnia, consistent with a homeostatic function in which gas exchange is defended by redistributing ventilation away from overventilated units.
Assuntos
Actinas/metabolismo , Pulmão/fisiologia , Modelos Biológicos , Animais , Feminino , Cobaias , Técnicas In Vitro , Pulmão/citologia , Pulmão/metabolismo , Complacência Pulmonar/fisiologia , Músculo Liso/citologia , Músculo Liso/metabolismo , Músculo Liso/ultraestrutura , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/ultraestrutura , Concentração Osmolar , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
A version of Mallory's phloxine-methylene blue-azure II technique suitable for large epoxy sections is described. Phloxine B (C.I. 45410) and a yellow-green interference filter (546-548 nm transmission) combine to give high contrast monochrome images. By comparing light micrographs of lung parenchyma entirely unstained or stained only with phloxine B against electron micrographs of the same material, it is seen that phloxine B emphasizes essentially only elastin and collagen fiber bundles. The technique has produced images useful for investigating lung parenchyma architecture and micromechanics.
Assuntos
Colágeno/análise , Elastina/análise , Pulmão/análise , Coloração e Rotulagem/métodos , Animais , Corantes Azur , Cães , Azul de Eosina I , Resinas Epóxi , Pulmão/citologia , Azul de Metileno , MicrotomiaRESUMO
Confocal scanning laser microscopy makes it possible to obtain series of optical sections in precise registration. Certain studies of lung parenchyma, however, require both the fine resolution obtainable with high-numerical-aperture (NA) objectives and the extensive fields of view that usually would be achieved only with low-NA objectives. This article presents a technique that resolves this conflict by using a sequence of operations: (i) to correct intensity variations on individual sections due to non-uniform illumination/detection characteristics of the microscope; (ii) to correct intensity variations between successive sections in a series due to, for example, depth-related absorption or step changes in detector sensitivity; (iii) to adjust adjacent, overlapping stacks of sections to a common intensity level; and (iv) to fuse a group of such overlapping stacks into a single series of larger sections. This resulting stack may contain, for example, a complete cross-section of an alveolar ductal unit about 500 microns or more in diameter at about 1-micron pixel resolution.
Assuntos
Pulmão/anatomia & histologia , Microscopia/métodos , Algoritmos , Animais , Cães , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodosRESUMO
Pressure-volume characteristics of the lung have been thought to be due primarily to the properties of the network of alveolar septa. However, Hajji et al. (J. Appl. Physiol.: Respirat . Environ. Exercise Physiol. 47: 175-181, 1979) attributed a substantial role to the visceral pleura. Seeking a structural explanation for this result, we compared the relative amounts of collagen fibrils and elastin fibers in the visceral pleura and alveolar parenchyma using stereological measurements in five canine lobes. We found about one-fifth as much collagen and one-tenth as much elastin in the pleura as in the alveolar parenchyma. This structural result confirms the functional conclusions of Hajji et al. We argue that such a substantial structure is not needed for protection against overinflation but may have to do with stabilization of lobe shape or handling of frictional forces.
Assuntos
Colágeno/metabolismo , Elastina/metabolismo , Pulmão/metabolismo , Pleura/metabolismo , Animais , Cães , Complacência Pulmonar , Microscopia Eletrônica , Alvéolos Pulmonares/metabolismo , RespiraçãoRESUMO
The distribution of the lengths of airspace chords in pulmonary parenchyma characterizes many architectural features of the alveoli and alveolar ducts. Laborious to obtain manually, the distributions and density functions may be acquired semi-automatically by video microscopy, digitization and image processing. The accuracy of the estimation is influenced by the microscopical methods and also by the techniques used (i) to convert the digitized greyscale picture to a two-valued image, (ii) to collect the chord lengths and (iii) to compensate for finite field widths. The last problem arises because some chords are completely visible within a field while others are only partially seen, since one of the two air-tissue boundaries lies outside the field of view. This error systematically biases the observed distribution. This paper contains solutions to hardware, software and analytic problems encountered while developing the capability to measure airspace chord length density functions semi-automatically. Formulas for estimating the true chord length density function from samples of observed chord lengths are presented. Also given are formulas for the estimation of the first and second moments of the true chord length distribution from the means of observed chord lengths. These techniques of image preparation and analysis should be suitable for characterizing particle, grain or cell size distributions, especially where many profiles fall partially outside the field of view.
Assuntos
Alvéolos Pulmonares/anatomia & histologia , Animais , Cães , Processamento de Imagem Assistida por Computador , Computação Matemática , Microscopia/métodos , Gravação em VídeoRESUMO
At present, cytotoxicity measurements using the fluorescent cytoprint assay are based on achieving complete cell death in cultures of drug-sensitive tumors. Thus, the usefulness of the assay would be extended if partial effects of chemotherapeutic drugs could be quantified. In this study, we addressed the issue by developing and validating a thresholding algorithm for automatic image processing that can be used to quantify the areas occupied by viable (i.e., fluorescent) micro-organs in the culture.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Processamento de Imagem Assistida por Computador/instrumentação , Validação de Programas de Computador , Software , Humanos , Microscopia de FluorescênciaRESUMO
The relative amounts of radioactively labelled disaccharide-peptide monomers and peptide-cross-linked dimers and trimers found in the peptidoglycan of Streptococcus faecalis ATCC 9790 were compared to the relative amounts to be expected from two different polymerization mechanisms (random condensation and monomer addition). Data from continuously-labeled, exponentially-growing cells are consistent with a monomer addition cross-linking process, not with a random condensation cross-linking mechanism. This conclusion was supported by data obtained from analyses of cells labeled during valine starvation (and wall thickening), recovery from valine starvation, and pulse and pulse-chase labeling of walls from exponentially-growing cultures.