Longitudinal extension of the pulmonary trunk during breathing in young rabbits: a possible factor in elastin fragmentation.

Corrosion casts of the pulmonary trunk and major branches of the pulmonary arteries of 39 rabbits were made from a silicone polymer. In half of the rabbits the casts were made with the lungs expanded and half with the lungs collapsed. The length of various segments of the casts were measured from magnified photographs. It was found that in rabbits less than 23 days old the pulmonary trunk is significantly longer when the lungs are expanded than when they are collapsed. This suggests that a repeated longitudinal extension of the vessel occurs during breathing. This effect disappears after 30 days of age, possibly because of elastin fragmentation. Between the ages of 23 and 30 days the growth in length and diameter of the pulmonary trunk undergoes a rapid acceleration. This may also be the result of elastin fragmentation. Whereas it may be that repeated longitudinal stress in the pulmonary trunk during breathing causes elastin in its media to fragment, one cannot exclude the possibility that other factors such as growth are responsible.     

The effect of some terrestrial oligochaeta on nitrogenase activity in the soil

Nitrogenase activity, assayed by the acetylene reduction technique, was estimated on control soils and soils which contained several species of earthworms. The worms and casts were separately assayed for nitrogenase activity. The results indicated that nitrogenase activity was associated with gut content or body surface of worms and also their casts. Nitrogen fixation was also enhanced in soils enriched with enchytracids in the presence of leaf litter.     

Plastic casts of embryonic respiratory and cardiovascular system: a technique.

This report describes a technique for producing plastic casts of the cardiovascular and respiratory systems of the chick embryo at Stage 36 or older. For casts of the cardiovascular system, polymerization compound is injected in the right ventricle filling the heart, venous, and arterial system. A two stage injection produced more detailed casts of the pulmonary vasculature. For respiratory system casts, the compound is injected in the trachea filling the bronchi and air sacs. Casts are prepared by tissue corrosion in 10% potassium hydroxide. This technique is an alternative to serial section reconstruction in the study of developing cardiovascular and respiratory system.     

Radon dosimetry applications ofin vitro tracheobronchial-deposition measurements

Deposition efficiencies of monodisperse ammonium fluorescein aerosols have been measured in simulated human lungs made of replica laryngeal casts combined with trachebronchial systems. Other tests, with radiolabelled submicron-sized particles, combined the larynges with replica tracheobronchial casts. The laryngeal casts had internal flow rate-specific geometries. Data indicate thatin vitro bifurcations have ?hot spots? or highly localized deposits, particularly at carinal ridges, suggesting that epithelial cells at airway branching sitesin vitro receive increased exposures to inhaled particulate matter. For dosimetry purposes, therefore, the lung should be likened to a series of Y-shaped airway junctures. The data have risk assessment applications for ambient radon progeny and radioactive airborne particles found in uranium mining and milling operations.     

Lung tissue engineering and preservation of alveolar microstructure using a novel casting method

Abstract

We used a rat model to decellularize and seed alveolar cells on a three-dimensional lung scaffold to preserve alveolar microarchitecture. We verified the preservation of terminal respiratory structure by casting and by scanning electron microscopy (SEM) of the casts after decellularization. Whole lungs were obtained from 12 healthy Sprague-Dawley rats, cannulated through the trachea under sterile conditions, and decellularized using a detergent-based method. Casting of both natural and decellularized lungs was performed to verify preservation of the inner microstructure of scaffolds for further cell seeding. Alveolar cell seeding was performed using green fluorescent protein (GFP) lung cells and non-GFP lung cells, and a peristaltic pump. We assessed cell seeding using histological and immunohistochemical staining, and enzymatic evaluation. All cellular components were removed completely from the scaffolds, and histological staining and SEM of casts were used to verify the preservation of tissue structure. Tensile tests verified conservation of biomechanical properties. The hydroxyproline content of decellularized lungs was similar to native lung. Histological and immunohistochemical evaluations showed effective cell seeding on decellularized matrices. Enzymatic measurement of trypsin and alpha 1 antitrypsin suggested the potential functional properties of the regenerated lungs. Casts produced by our method have satisfactory geometrical properties for further cell seeding of lung scaffolds. Preservation of micro-architecture and terminal alveoli that was confirmed by SEM of lung casts increases the probability of an effective cell seeding process.     

Volume of the conducting airways calculated from morphometric parameters

A method of calculating the volume of a tree distal to a cut at the origin of a branch, using branching, diameter and length ratios, has been developed. The method was applied to bronchial tree casts from human, dog, sheep, hamster, and rat lungs. It was found that the exponenta in the equation weight=k×diameter ^a is approximately equal to 3.0 in sheep lung casts, as found by Hooper (1977), but it is greater than 3.0 in casts from the other four species.     

Comparison of theoretical and experimental particle diffusion data within human airway casts

The dose delivered to airway cells is a critical factor whether one is addressing the therapeutic (i.e., positive) effects of inhaled pharmacologic agents or the toxic (i.e., negative) effects of pollutants. In this study, theoretical models describing particle deposition have been compared with experimental data from the literature. In the simulations, airways can be either roughor smooth-walled to be consistent with human lungs which can be either lined by cartilaginous rings (i.e., rough) or muscle (i.e., smooth). Particle motion for rough-walled airways within generations I=1–6 is calculated using the formula proposed by Martonen et al. (1). For smooth-walled airways within generations I=7–10, particle motion is calculated using the formula proposed by Martonen et al. (2). Theoretical predictions of particle deposition efficiencies are not only in agreement with the overall best fit empirical correlation presented by Cohen and Asgharian (3) over a wide range of dimensionless diffusion parameters, but also match individual experimental measurements (only available in I=1–6) with regard to effects of the parameters of particle size, flow rate, and airway dimensions. The mean difference in the ratio of experimental-to-theoretical particle diffusion values is 0.9 for a flow rate of 18 L/min and 1.1 for a flow rate of 34 L/min (i.e., the difference is only about 10%) within the upper airways of the casts (airway generations I=1–6), the mean difference for the whole casts was much greater. This may be attributed to the uncertainty of flow conditions in the peripheral airways as a result of the trimmmed nature of the casts. Overall, the findings suggest that the model can be a valuable component of aerosol therapy and risk assessment protocols, especially to address effects of enhanced deposition of pharmacologic drugs and radionuclides at sites within the human tracheobronchial tree.     

A new method to make vascular and bronchial casts of voluminous organs

Vascular and bronchial endocasts represent a useful instrument to study the ramification pattern of these structures. Casts have been made from different materials, such as waxes in ancient times and, more recently, silicon-like compounds or resins (see e.g. Mercox) to study the finest details. These techniques are valuable for small specimens, whereas they are inadequate for very large organs, where technical difficulties require the development of specific instrumentation. In this study we present a new simple injection technique, based on expanded polyurethane, which allows preparing vascular and bronchial trees for macroscopic and microscopic studies. The new injection technique is very easy to carry out, since the propulsion is provided by compressed air, and it does not require special instrumentation. To this aim, endocasts of the entire tracheal-bronchial tree and casts of vascular kidney from different animals were prepared. The specimens have a very low weight, show the finest ramifications, and are very stable and resistant to mechanical stress. To examine microscopically the details of the casts, specimens from the kidney cast were also analyzed by scanning electron microscopy, revealing good preservation of microcirculatory structures, functional sphincters and endothelial cell impressions. Therefore, the technique may be useful for macroscopic studies of large specimens, retaining sufficiently fine details.     

Lung morphology in rodents (Mammalia, Rodentia) and its implications for systematics

A new nomenclature of the lung lobes and of the bronchial tree is presented, with which the lungs in 40 species of 11 rodent families are described. Whole, fixed lungs and silicone casts of the bronchial tree are tested for 23 characters, based on the distribution of lung lobes, the number and geometry of first order bronchi, the pulmonary blood supply, and lung symmetry. Ten lung morphotypes are recognized, seven of them representing one or more families: Castor type (Castoridae), Cryptomys type (Bathyergidae), Ctenodactylus type (Ctenodactylidae), Eliomys type (Gliridae), Myocastor type (Myocastoridae), Octodon type (Octodontidae and Echimyidae) and Rattus type (Sciuridae, Muridae pt. and Dipodidae). The Hydromys type is found only in Hydromys chrysogaster (Muridae), while Galea type A and B both appear in Galea musteloides (Caviidae). The data are phylogenetically analyzed by the program PAUP 4.0 using as outgroup Lagomorpha or Insectivora. On the species level, there are no well-resolved cladograms. On the family level, the cladograms do not contradict traditional rodent systematics with one exception: the Caviidae do not fall within Caviomorpha or even within the Hystricomorpha, but form a sister group to Dipodidae (Myomorpha). This appears to be a result of convergence. The lungs of Gliridae are more similar to those of Muridae than to those of Sciuridae. Included in the ingroup, Oryctolagus (Lagomorpha) forms a clade with Caviidae + Dipodidae. Thus, the "Glires hypothesis" is neither supported nor refuted.     

Chronic bronchial dilatations in different colonies of laboratory rats

Bronchiectasis occurred naturally in 12-month-old spontaneously diabetic eSS male rats. The lungs of 3 and 6-month-old eSS rats were compared in eumetabolic eSS rats from three inbred lines consisting of inbred spontaneously diabetic eSS derived from IIM strain; these were compared with eumetabolic, outbred Wistar rats, paired by sex and age. Acrylic casts of bronchial tree were obtained after injection of a plastic substance. The casts were pruned to focus on the first four bronchial branchings. Diameter and volume of the conductive bronchial tree were determined using a binocular magnifier. Histological sections were obtained. All lines showed multiple bronchiectasis, mostly fusiform, bronchial dilatation and inflammatory response with lymphocytic infiltrates. These symptoms were much more severe in 180-day-old eSS males. Bacteria were isolated from the lungs in 70% of cases (n = 32), except in eSS rats. Pseudomonas spp. (38%) and Gram-positive cocci as coagulase-negative Staphylococcus spp. (20%) were detected. Neither pathogenic bacteria nor saprophyte fungi were found. Although all lines were affected, diabetes in eSS appears to be an aggravating factor.     

A technique for quantitating airway size from bronchial casts

To develop a technique for quantitating the size of airways at various positions in the bronchial tree, we analyzed casts of formalin-fixed excised lungs of five mature male ferrets. The left lower lobe of each cast was dissected, the diameter and position of each terminal bronchiole were entered into a computer programmed to reconstruct the airway system, and the cross-sectional areas of 120 conducting airways were measured. The fraction of the lobe served by each measured airway was estimated by dividing the sum of the squared diameters of the terminal bronchioles subtended by that airway by the summed squared diameters of all terminal bronchioles in the lobe. In each cast the relationship between an airway's cross-sectional area (Y) and the fraction of the lobe it was estimated to subtend (X) was described (0.91 less than R2 less than 0.95) by the expression ln(Y) = A + B ln(X) + C [ln(X)]2. Linear regression of ln(Y) on ln(X) for 30-50 airways estimated to serve fractions of the lobe around each of three arbitrarily selected levels (airways serving 0.7, 2.2, and 9.5% of the lobe) was adequate to characterize the area of airways at each level in each of the five animals with 95% confidence intervals narrower than 8% of the estimated area. Variability of airway size at each level among the five casts was modest, suggesting that this technique identified analagous airways in the various animals. Interindividual variability did not increase when the data were reanalyzed with terminal units defined on the basis of airway diameters rather than on the morphological identification of terminal bronchioles.     

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.     

A replication technique for scanning electron microscopy: Applications for anthropologists

Scanning electron microscopy has become an increasingly useful tool for anthropologists, particularly because of the development of improved methods of replicating specimens. One of the best replication techniques involves silicone-based dental impression materials to make negative impressions, in conjunction with epoxy resins, which are used to make positives or casts. The technique outlined here is particularly useful for anthropologists. Using this technique allows the examination of bone surfaces, teeth, and fossils for taphonomic, microwear, and experimental studies. Reproduction of detail is faithful at magnifications of × 1,500 to × 2,000, routinely giving resolutions of .1 to .25 μm.     

Development of the vascular bed in the rabbit ear: scanning electron microscopy of vascular corrosion casts

The development of the vascular bed in the rabbit ear was investigated using vascular corrosion casts from animals of various ages. Examination of the casts revealed that the arrangement of the major auricular arteries and veins was determined before birth and was maintained during postnatal growth of the ear. Furthermore, the number of arteries branching off the central ear artery and the lateral arteries did not increase with increasing ear length. Scanning electron microscopic examination of lateral segments of adult ear casts revealed many anastomoses between marginal arteries and veins. These arteriovenous anastomoses (AVAs) occurred singly, in pairs, or in clusters of three to six. Their size and shape were variable, even in the same cast. The central segment of many AVA casts showed surface impressions of endothelial cell nuclei which were different from the impressions on adjacent arteries and veins. Arteriovenous anastomoses were also detected in ear casts from animals as young as 8 days. The density of AVAs in lateral ear segments ranged from 95-165 cm-2 in 8- to 11-day-old rabbits to 80-115 cm-2 in adults. However, estimates of the total number of AVAs in the lateral ear margin indicated that AVAs continued to be formed at a steady rate during growth of the ear. During the early neonatal period the cutaneous capillary plexuses developed prominent tufts projecting toward the skin surface, which were apparently associated with developing hair follicles. These capillary tufts were not seen in casts from fetal or adult rabbits.     

Image analysis of oronasal fistulas in cleft palate patients acquired with an intraoral camera

The aim of this study was to examine the clinical technique of using an intraoral camera to monitor the size of residual oronasal fistulas in cleft lip-cleft palate patients, to assess its repeatability on study casts and patients, and to compare its use with other methods. Seventeen plaster study casts of cleft palate patients with oronasal fistulas obtained from a 5-year series of 160 patients were used. For the clinical study, 13 patients presenting in a clinic prospectively over a 1-year period were imaged twice by the camera. The area of each fistula on each study cast was measured in the laboratory first using a previously described graph paper and caliper technique and second with the intraoral camera. Images were imported into a computer and subjected to image enhancement and area measurement. The camera was calibrated by imaging a standard periodontal probe within the fistula area. The measurements were repeated using a double-blind technique on randomly renumbered casts to assess the repeatability of measurement of the methods. The clinical images were randomly and blindly numbered and subjected to image enhancement and processing in the same way as for the study casts. Area measurements were computed. Statistical analysis of repeatability of measurement using a paired sample t test showed no significant difference between measurements, indicating a lack of systematic error. An intraclass correlation coefficient of 0.97 for the graph paper and 0.84 for the camera method showed acceptable random error between the repeated records for each of the two methods. The graph paper method remained slightly more repeatable. The mean fistula area of the study casts between each method was not statistically different when compared with a paired samples t test (p = 0.08). The methods were compared using the limits of agreement technique, which showed clinically acceptable repeatability. The clinical study of repeated measures showed no systematic differences when subjected to a t test (p = 0.109) and little random error with an intraclass correlation coefficient of 0.98. The fistula size seen in the clinical study ranged from 18.54 to 271.55 mm. Direct measurements subsequently taken on 13 patients in the clinic without study models showed a wide variation in the size of residual fistulas presenting in a multidisciplinary clinic. It was concluded that an intraoral camera method could be used in place of the previous graph paper method and could be developed for clinical and scientific purposes. This technique may offer advantages over the graph paper method, as it facilitates easy visualization of oronasal fistulas and objective fistulas size determination and permits easy storage of data in clinical records.     

An immunocytochemical method for quantification of lung tissue fibronectin

A technique to quantify tissue fibronectin was developed, using peroxidase-antiperoxidase immunocytochemistry and automated scanning light microscopy. This technique was developed using isolated perfused rat lungs, some of which were subjected to acute oxidant lung injury. Both injured and control lungs were perfused with solutions containing heterologous fibronectin. The technique clearly demonstrated differences in the amount of tissue fibronectin in injured and noninjured lung as well as differences between lungs exposed to fibronectin and those not exposed. The described technique offers a reliable method for quantifying tissue fibronectin and is sensitive enough to detect differences in tissue fibronectin under experimental conditions of acute lung injury.     

Vascular release of nonheme iron in perfused rabbit lungs

In this study, we hypothesized that the lung actively releases excess iron into the circulation to regulate iron homeostasis. We measured nonheme iron (NHFe) in the perfusate of control isolated perfused rabbit lungs and lungs with ischemia-reperfusion (I/R) ventilated with normoxic (21% O(2)) or hypoxic (95% N(2)) gas mixtures. Some were perfused with bicarbonate-free (HEPES) buffer or treated with the anion exchange inhibitor DIDS. The control lungs released approximately 0.25 microg/ml of NHFe or 20% of the total lung NHFe into the vascular space that was not complexed with ferritin, transferrin, or lactoferrin or bleomycin reactive. The I/R lungs released a similar amount of NHFe during ischemia and some bleomycin-detectable iron during reperfusion. NHFe release was attenuated by approximately 50% in both control and ischemic lungs by hypoxia and by >90% in control lungs and approximately 60% in ischemic lungs by DIDS and HEPES. Reperfusion injury was not affected by DIDS or HEPES but was attenuated by hypoxia. These results indicate that biologically nonreactive nonheme iron is released rapidly by the lung into the vascular space via mechanisms that are linked to bicarbonate exchange. During prolonged ischemia, redox-active iron is also released into the vascular compartment by other mechanisms and may contribute to lung injury.     

Investigation of influencing variables on the computer-aided simulation of contacts in dynamic occlusion based on optically digitized plaster casts]

In dentistry, mechanical articulators with which mandibular movements can be reproduced in dentals casts play a major role. Commonly used semiadjustable articulators, however, have major limitations: On the one hand, the movement of the mandible is not reproduced exactly, on the other, they do not provide time-related information on jaw movement. Both problems can be solved by replacing the mechanical articulator by a digital simulation ("virtual articulator") based on digitized plaster casts and electronically recorded masticatory movements. We present a system for the 3D measurement of plaster casts in a skull-related, anatomical coordinate system using the fringe projection technique, and electronically recorded condylar movements. Using numerical algorithms, the contacts between upper and low jaw, and the angle of rotation of the temporomandibular joint can be computed for each movement in dynamic occlusion. Taking the data recorded from a patient as an example, the influence of the accuracy of the digitization of plaster casts on the computation of the rotation of the temporomandibular joint is discussed in relation to the anatomy of the masticatory apparatus.     

Studies on the functional morphology of rat ocular vessels with scanning electron microscopy

Tests are still lacking about the suitability of scanning electron microscopy (SEM) of vascular resin casts to show different functional states of peripheral blood vessels. With the aid of a vitalmicroscopic device, we tried to elaborate a vascular casting method using the model of the albino rat iris vasculature. Functional variations of the vasculature were induced by local application of epinephrine to one eye using the untreated fellow eye as a control. It was found that if our modification of Araldite plastic is injected via a systemic access and without preceding rinsing with fixatives or salt solutions there is a good correlation between the vessel diameters seen in SEM of resin casts and the vessel diameters found in the vitalmicroscopic observations. Thus, this method appears also suitable for studying the effect of vasoactive substances.     

Acute cerebral artery constriction in the spontaneously hypertensive rat following blood and plasma administration into the subarachnoid space

The purpose of the present study was to demonstrate, using a vascular casting technique, acute vasoconstrictive changes in the cerebral vasculature 1 h following whole-blood or plasma infusion into the subarachnoid space of conscious spontaneously hypertensive rats. Vascular casts from animals infused (over 20 min) with 0.45 ml of heparinized autologous arterial blood or plasma exhibited incomplete filling, while casts from saline-infused controls exhibited virtually no filling defects. Significant elevations in intracranial pressure were noted in blood, but not in plasma- or saline-infused rats. Two characteristic forms of constriction occurred, depending upon the vessel lumen diameter. Vessels with lumen diameters >100 µm were flattened longitudinally with deep endothelial nuclear imprints, while smaller vessels had focal circular constrictions resembling beads. Arterial cast filling terminated in vessels with lumen diameters from 70 to 20 µm with focal signs of constriction at or near the point of cast termination. The results indicate that the presence of both blood and plasma in the subarachnoid space produces acute small-artery constriction. This phenomenon is due to a noncellular blood component and does not correlate with increases in intracranial pressure.