Reproducible uniform equibiaxial stretch of precision-cut lung slices.

Simulating ventilator-induced lung injury (VILI) in the laboratory requires stretching of lung alveolar tissue. Whereas precision-cut lung slices (PCLSs) are widely used for studying paracrine signaling pathways in the lungs, their use in stretch studies is very limited because of the technical challenge of fixing them to a stretchable substrate, stretching them uniformly, or holding them in a stretch device without causing rupture. We describe a novel method for attaching PCLSs to silicone membranes by stitching them together in a star-shaped pattern. Using a device that was previously designed in our laboratory for stretching primary alveolar epithelial cell monolayers, we demonstrate that in the central region of the PCLSs stretch is uniform, equibiaxial, and, after a short preconditioning period, also reproducible. The stitched and stretched PCLSs showed equal or better viability outcomes after 60 min of cyclic stretch at different magnitudes of physiological stretch compared with primary pulmonary alveolar epithelial cell monolayers. Preparing and stitching the PCLSs before stretch is relatively easy to perform, yields repeatable outcomes, and can be used with tissue from any species. Together with the ensuring uniform and equibiaxial stretch, the proposed methods provide an optimal model for VILI studies with PCLSs.

Design of an adaptive randomized clinical trial of intravenous citrulline for sickle cell pain crisis in the emergency department.

Background: Vaso-occlusive pain crisis (VOC) is the most frequent cause for Emergency Department (ED) visits and hospital admissions for patients with sickle cell disease (SCD). Nitric oxide plays a critical role in the pathogenesis of vaso-occlusion. The amino acid, citrulline, is the main endothelial nitric oxide booster that offers the potential to ameliorate vaso-occlusion and decrease the risk of hospitalization. Objective: In this two-part study, the goal of the first part is to determine the pharmacokinetic profile of intravenous (IV) l-citrulline and optimal dose for the second part of the study, which is to determine the efficacy and tolerability of the intervention in patients with SCD. Design: A phase I/IIA open-label dose-finding study with subsequent double-blind, placebo-controlled, randomized Study of l-citrulline in children and adolescents with SCD presenting to the ED in VOC. Methods: Part 1: Subjects experiencing VOC are enrolled in an open-label, ascending dose of IV l-citrulline to identify the optimum dose with endpoints of pharmacokinetic parameters, pain scores, reduction of opioid use, quality of life, proportion admitted to the hospital for treatment of pain, readmission rates, and assessment of adverse events. Part 2 of the trial is a double-blind, placebo-controlled adaptive "pick-the-winner" design to evaluate the efficacy and tolerability of IV l-citrulline in patients with SCD while receiving standard of care therapy for VOC. Summary: This ED based sickle cell adaptive trial will determine the optimal dose for IV citrulline and whether the intervention improves outcome as a potential novel therapy for VOC in SCD.

Frequency and peak stretch magnitude affect alveolar epithelial permeability.

The present study measured stretch-induced changes in transepithelial permeability to uncharged tracers (1.5-5.5 A) using cultured monolayers of alveolar epithelial type-I like cells. Cultured alveolar epithelial cells were subjected to uniform cyclic (0, 0.25 and 1.0 Hz) biaxial stretch from 0% to 12, 25 or 37% change in surface area (DeltaSA) for 1 h. Significant changes in permeability of cell monolayers were observed when stretched from 0% to 37% DeltaSA at all frequencies, and from 0% to 25% DeltaSA only at high frequency (1 Hz), but not at all when stretched from 0% to 12% DeltaSA compared with unstretched controls. At stretch oscillation amplitudes of 25 and 37% DeltaSA, imposed at 1 Hz, tracer permeability increased compared with that at 0.25 Hz. Cells subjected to a single stretch cycle at 37% DeltaSA (0.25 Hz), to simulate a deep sigh, were not distinguishable from unstretched controls. Reducing stretch oscillation amplitude while maintaining a peak stretch of 37% DeltaSA (0.25 Hz) via the application of a simulated post-end-expiratory pressure did not protect barrier properties. In conclusion, peak stretch magnitude and stretch frequency were the primary determining factors for epithelial barrier dysfunction, as opposed to oscillation amplitude.

Diaphragm mechanics in dogs with unilateral emphysema.

We studied dogs with unilateral papain-induced emphysema to answer two questions: (1) Do emphysema lung-apposed hemidiaphragm (DiE) and normal lung-apposed hemidiaphragm (DiN) have equal capacities for lowering lung surface pressure? and (2) Are side-to-side differences in intrathoracic pressure the result of unequal force outputs by DiE and DiN or are they caused by differences in their mechanical efficiency as pressure generators? After the airways of the emphysematous and normal lungs were intubated with a dual lumen endotracheal tube, both phrenic nerves were maximally stimulated at rates between 1 and 50 Hz and the changes in airway occlusion pressure (delta PaoE,N) and diaphragm length (sonomicrometry) were recorded. In all animals, delta PaoN exceeded delta PaoE. Differences in pressure ranged from 1.2 +/- 0.6 cm H2O during a twitch to 6.0 +/- 2.9 cm H2O during a 50-Hz tetanus. Midcostal bundles of DiE shortened less than corresponding bundles of DiN, but both reached the same active length relative to their optimal lengths, which were measured in vitro. There was no significant difference in fiber type distribution, fiber cross-sectional area, or maximal isometric tetanic tensions among midcostal regions of DiE and DiN. We conclude that unilateral hyperinflation impairs the mechanical efficiency of the apposing hemidiaphragm as a pressure generator.

How do normals move during prolonged wheelchair-sitting?

The clinical practice for minimizing the risk of pressure sores (PS) is to relief pressures under bony prominences of immobilized patients by changing their postures frequently. The US Department of Health advises to relief sitting pressures at least every 1 hour, and every 15 minutes for individuals who are body-abled. Surprisingly, there is paucity of information in the literature concerning motion of healthy subjects during prolonged sitting, which can be compared with these recommendations. Considering that healthy individuals are able to sit for hours without suffering injuries, such information seems particularly important. Accordingly, our objective was to measure frequency of postural changes and extent of motion during postural changes among healthy subjects who sit in a wheelchair (N=10), in order to provide information that is missing in the literature of PS biomechanics. Subjects were asked to sit comfortably for 90 minutes, during which their trunk's frontal and sagittal motions and sitting pressures were measured. We found that normals change their posture every 9 +/- 6 minutes in the sagittal plane, and independently, every 6 +/- 2 minutes in the frontal plane. Shoulders, thoracic-spine and lumbar-spine frontal plane motions were 8 +/- 4 degrees , 14 +/- 7 degrees and 15 +/- 7 degrees , respectively, and sagittal trunk-thigh movement was 10.3 +/- 7 degrees . The frequency of postural changes in normals, measured herein, was higher than frequencies reported in the literature for patients who suffered PS. This small study population therefore supports the hypothesis that prolonged immobilization contributes to PS onset.

Local influence of cell viability on stretch-induced permeability of alveolar epithelial cell monolayers.

Ventilator induced lung injury (VILI), often attributed to over-distension of the alveolar epithelial cell layer, can trigger loss of barrier function. Alveolar epithelial cell monolayers can be used as an idealized in vitro model of the pulmonary epithelium, with cell death and tight junction disruption and permeability employed to estimate stretch-induced changes in barrier function. We adapted a method published for vascular endothelial permeability, compare its sensitivity with our previously published method, and determine the relationship between breeches in barrier properties after stretch and regions of cell death After 4-5 days in culture, primary rat alveolar epithelial cells seeded on plasma treated polydimethylsiloxane membrane coated with biotin-labeled fibronectin, or fibronectin alone were stretched in the presence of FITC-tagged streptavidin (biotin-labeled membrane) or BODIPY-ouabain. We found that the FITC-labeling method was a more sensitive indicator of permeability disruption, with significantly larger positively stained areas visible in the presence of stretch and with ATP production inhibitor Antimycin-A. Triple-stained images with Hoescht (nuclei), Ethidium Homodimer (EthD, damaged cell nuclei) and FITC (permeable regions) were used to determine that within permeable regions intact cells were positioned closer to damaged cells than in non-permeable regions. We concluded that local cell death may be an important contributor to barrier integrity.

Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.

Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was

Regional variation in canine diaphragm thickness.

To quantify the relationship between both regional and overall diaphragm morphometry and body weight in the dog, diaphragm thickness was measured in five regions of the costal diaphragm and three regions of the crural diaphragm in 40 healthy dogs (8-40 kg). Surface area of the diaphragm, diaphragm weight, and body weight were also determined. Diaphragm surface area and weight varied linearly with body weight, but there was no significant correlation between overall diaphragm thickness and body weight. Diaphragm thickness varied significantly between regions, and three regions had systematic left-to-right differences as well. Because diaphragm geometry influences the diaphragm's function as a pressure generator, regional differences in thickness may alter the relationship between the force developed by the activation of a particular region of the diaphragm and its action on the respiratory system.

Effects of unilateral hyperinflation on the interpulmonary distribution of pleural pressure.

Motivated by single lung transplantation, we studied the mechanics of the chest wall during single lung inflations in recumbent dogs and baboons and determined how pleural pressure (Ppl) is coupled between the hemithoraces. In one set of experiments, the distribution of Ppl was inferred from known volumes and elastic properties of each lung. In a second set of experiments, costal pleural liquid pressure (Pplcos) was measured with rib capsules. Both methods revealed that the increase in Ppl over the ipsilateral or inflated lung (delta Ppli) is greater than that over the contralateral or noninflated lung (delta Pplc). Mean d(delta Pplc)/d(delta Ppli) and its 95% confidence interval was 0.7 +/- 0.1 in dogs and 0.5 +/- 0.1 in baboons. In a third set of experiments in three dogs and three baboons, we prevented sternal displacement and exposed the abdominal diaphragm to atmospheric pressure during unilateral lung inflation. These interventions had no significant effect on Ppl coupling between the hemithoraces. We conclude that lungs of unequal size and mechanical properties need not be exposed to the same surface pressure, because thoracic midline structures and the lungs themselves resist displacement and deformation.

Regional ventilation in statically and dynamically hyperinflated dogs.

Using the parenchymal marker technique in normal anesthetized dogs, we compared the dynamics of regional lung expansion between two ventilation strategies designed to increase mean thoracic volume. Dynamic hyperinflation (DH was produced by ventilating the lungs at a rate of 50 breaths/min and with a duty cycle of 0.5. Static hyperinflation (SH) was produced through the application of extrinsic positive end-expiratory pressure while the lungs were ventilated at a rate of 15 breaths/min and with a duty cycle of 0.15. Regional tidal volume (VT,r), regional functional residual volume, and the time delay between regional expansion and the flow signal at the common airway were computed for up to 100 regions/lobe in 5 animals. Ventilation strategy had no effect on the overall variance of VT,r within lobes. Although the VT,r measured during SH correlated with VT,r measured during DH, the average correlation coefficient was only 0.69. Ventilation rate-related differences in VT,r and regional functional residual capacity varied with the regional time delay in ways qualitatively consistent with parallel inhomogeneity of unit time constants. However, a large component of frequency-dependent behavior remains unexplained by established mechanisms. We conclude that DH and SH should not be considered equivalent lung unit recruitment strategies.

Shape of the chest wall in the prone and supine anesthetized dog.

The shape of the passive chest wall of six anesthetized dogs was determined at total lung capacity (TLC) and functional residual capacity (FRC) in the prone and supine body positions by use of volumetric-computed tomographic images. The transverse cross-sectional areas of the rib cage, mediastinum, and diaphragm were calculated every 1.6 mm along the length of the thorax. The changes in the volume and the axial distribution of transverse area of the three chest wall components with lung volume and body position were evaluated. The decrease of the transverse area within the rib cage between TLC and FRC, as a fraction of the area at TLC, was uniform from the apex of the thorax to the base. The volume of the mediastinum increased slightly between TLC and FRC (14% of its TLC volume supine and 20% prone), squeezing the lung between it and the rib cage. In the transverse plane, the heart was positioned in the midthorax and moved little between TLC and FRC. The shape, position, and displacement of the diaphragm were described by contour plots. In both postures, the diaphragm was flatter at FRC than at TLC, because of larger displacements in the dorsal than in the ventral region of the diaphragm. Rotation from the prone to supine body position produced a lever motion of the diaphragm, displacing the dorsal portion of the diaphragm cephalad and the ventral portion caudad. In five of the six dogs, bilateral isovolume pneumothorax was induced in the supine body position while intrathoracic gas volume was held constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar epithelial surface area-volume relationship in isolated rat lungs.

In vitro studies of the alveolar epithelial response to deformation require knowledge of the in situ mechanical environment of these cells. Because of the presence of tissue folding and crumpling, previous measurements of the alveolar surface area available for gas exchange are not equivalent to the epithelial surface area. To identify epithelial deformations in uniformly inflated lungs representative of the in vivo condition, we studied isolated Sprague-Dawley rat lungs (n = 31) fixed by perfusion with glutaraldehyde on deflation after cycling three times at high lung volume (10-25 cmH2O). The epithelial basement membrane in 45 electron micrographs (x12,000)/rat was traced, digitally scanned, and analyzed. Epithelial basement membrane surface area (EBMSA) was computed from a morphometric relationship. EBMSA was found to increase 5, 16, 12, and 40% relative to EBMSA at 24% total lung capacity at lung volumes of 42, 60, 82, and 100% total lung capacity, respectively. The increases in EBMSA suggest that epithelial cells undergo significant deformations with large inflations and that alveolar basement membrane deformation may contribute to lung recoil at high lung pressures.

Deformation of the dog lung in the chest wall.

Data on the shape of the chest wall at total lung capacity (TLC) and functional residual capacity (FRC) were used as boundary conditions in an analysis of the deformation of the dog lung. The lung was modeled as an elastic body, and the deformation of the lung from TLC to FRC caused by the change in chest wall shape and gravity were calculated. Parenchymal distortions, distributions of regional volume at FRC as a fraction of the volume at TLC, and distributions of surface pressure at FRC are reported. In the prone dog there are minor variations in fractional volume along the cephalocaudal axis. In transverse planes opposing deformations are caused by the change of shape of the transverse section and the gravitational force on the lung, and the resultant fractional volume and pleural pressure distributions are nearly uniform. In the supine dog, there is a small cephalocaudal gradient in fractional volume, with lower fractional volume caudally. In transverse sections the heart and abdomen extend farther dorsally at FRC, squeezing the lung beneath them. The gradients in fractional volume and pleural pressure caused by shape changes are in the same direction as the gradients caused by the direct gravitational force on the lung, and these two factors contribute about equally to the large resultant vertical gradients in fractional volume and pleural pressure. In the prone position the heart and upper abdomen rest on the rib cage. In the supine posture much of their weight is carried by the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of body position and lung volume on in situ operating length of canine diaphragm.

The performance of the diaphragm is influenced by its in situ length relative to its optimal force-generating length (Lo). Lead markers were sutured to the abdominal surface of the diaphragm along bundles of the left ventral, middle, and dorsal regions of the costal diaphragm and the left crural diaphragm of six beagle dogs. After 2-3 wk postoperative recovery, the dogs were anesthetized, paralyzed, and scanned prone and supine in the Dynamic Spatial Reconstructor (DSR) at a total lung capacity (TLC), functional residual capacity (FRC), and residual volume (RV). The location of each marker was digitized from the reconstructed DSR images, and in situ lengths were determined. After an overdose of anesthetic had been administered to the dogs, each marked diaphragm bundle was removed, mounted in a 37 degrees C in vitro chamber, and adjusted to Lo (maximum tetanic force). The operating length of the diaphragm, or in situ length expressed as percent Lo, varied from region to region at the lung volumes studied; variability was least at RV and increased with increasing lung volume. At FRC, all regions of the diaphragm was shorter in the prone posture compared with the supine, but there was no clear gravity-dependent vertical gradient of in situ length in either posture. Because in vitro length-tension characteristics were similar for all diaphragm regions, regional in vivo length differences indicate that the diaphragm's potential to generate maximal force is nonuniform.

Zone of apposition in the passive diaphragm of the dog.

We determined the regional area of the diaphragmatic zone of apposition (ZAP) as well as the regional craniocaudal extent of the ZAP (ZAPht) of the passive diaphragm in six paralyzed anesthetized beagle dogs (8-12 kg) at residual lung volume (RV), functional residual capacity (FRC), FRC + 0.25 and FRC + 0.5 inspiratory capacity, and total lung capacity (TLC) in prone and supine postures. To identify the caudal boundary of the ZAP, 17 lead markers (1 mm) were sutured to the abdominal side of the costal and crural diaphragms around the diaphragm insertion on the chest wall. Two weeks later, the dogs' caudal thoraces were scanned by the use of the dynamic spatial reconstructor (DSR), a prototype fast volumetric X-ray computer tomographic scanner, developed at the Mayo Clinic. The three-dimensional spatial coordinates of the markers were identified (+/- 1.4 mm), and the cranial boundary of the ZAP was determined from 30-40 1.4-mm-thick sagittal and coronal slices in each DSR image. We interpolated the DSR data to find the position of the cranial and caudal boundaries of the ZAP every 5 degrees around the thorax and computed the distribution of regional variation of area of the ZAP and ZAPht as well as the total area of ZAP. The ZAPht and area of ZAP increased as lung volume decreased and were largest near the lateral extremes of the rib cage. We measured the surface area of the rib cage cephaled to the ZAP (AL) in both postures in another six beagle dogs (12-16 kg) of similar stature, scanned previously in the DSR. We estimated the entire rib cage surface area (Arc = AZAP + AL). The AZAP as a percentage of Arc increased more than threefold as lung volume decreased from TLC to RV, from approximately 9 to 29% of Arc.

Geometry and kinematics of dog ribs.

Five anesthetized supine beagle dogs were scanned using a fast, multislice computed tomographic X-ray technique to determine the orientation of the ribs at total lung capacity (TLC) and functional residual capacity (FRC). A plane was fit to each rib using a coordinate system in which the z-axis was aligned approximately cephalocaudally and the x-z-plane coincided with the sagittal midplane. The orientation of each plane was described by "pump-handle" and "bucket-handle" angles. The ribs rotated downward and inward during a passive deflation of the lungs from TLC to FRC. Rib displacement was not uniform: bucket-handle motion was predominant in the upper ribs, and pump- and bucket-handle motions were equal in the lower ribs. The change in the pump-handle angles between TLC and FRC was approximately 6 degrees for ribs 3-8, and the change in the bucket-handle angles decreased with rib number from 16 degrees for rib 3 to 6 degrees for rib 8. Rib shape was described by fitting an ellipse to the data for each rib; the ribs became larger and more circular with increasing rib number.

Finite-element analysis of stress in the canine diaphragm.

Stress in the diaphragm, transdiaphragmatic pressure, and diaphragm shape are interrelated by a balance of forces. Using precise in vivo measurements of diaphragm shape and transdiaphragmatic pressure distribution in combination with finite-element analysis (ANSYS), we determined the direction and magnitude of stress in the passive diaphragm at relaxation volume. Lead spheres sutured along muscle bundles identified muscle bundle location and orientation in vivo. The x, y, and z coordinates of the lead spheres and entire surface of the diaphragm, excluding the zone of apposition, were determined to within 1.4 mm. Thin shell elements were used to construct a finite-element model of the diaphragm with a 2.1- to 4.2-mm internodal spacing. The diaphragm was assumed to have a uniform thickness of 2.5 mm, and magnitude and direction of the principal stresses were computed. The results show that 1) diaphragm stress is nonuniform and anisotropic (i.e., varies both with location on diaphragm surface and direction examined), 2) largest stress (sigma 1) is aligned with muscle bundles and is two to four times larger than sigma 2 (perpendicular to sigma 1 in diaphragm plane), and 3) stress along the muscle bundles is larger in vivo under conditions of biaxial stress than at same length in vitro under uniaxial stress. Although diaphragm stress and tension have often been assumed to be uniform, our finding that stress is oriented primarily along the muscle fibers should be considered in future models of the diaphragm.(ABSTRACT TRUNCATED AT 250 WORDS)

Static lung-lung interactions in unilateral emphysema.

Motivated by the introduction of single-lung transplantation into clinical practice, we compared the static mechanical properties of the respiratory system in six supine dogs before (at baseline) with those after the induction of unilateral emphysema. Relaxation volume (Vrel), total lung capacity (TLC), and static compliance of the emphysematous lung increased to 214 +/- 68, 186 +/- 39, and 253 +/- 95% (SD) of baseline, respectively. Vrel of the nonemphysematous lung fell to 81 +/- 28% of baseline, with no significant change in TLC of the nonemphysematous lung or its pressure-volume relationship, indicating that unilateral hyperinflation does not cause dropout of contralateral lung units. After unilateral emphysema, the chest wall shifted to a higher unstressed or neutral volume (when pleural pressure equals atmospheric pressure) in three of six animals, minimizing the anticipated decrease in lung recoil pressure at the higher respiratory system Vrel. The pattern of relative lung emptying in the intact dog and in the excised lungs was similar during stepwise deflations from TLC, suggesting that mean pleural pressure of the hemithoraces is equal. We conclude that in the dog the static volume distribution between emphysematous and nonemphysematous lungs is determined only by differences in lung recoil and compliance.

Effect of body position on regional diaphragm function in dogs.

The in situ lengths of muscle bundles of the crural and three regions of the costal diaphragm between origin and insertion were determined with a video roentgenographic technique in dogs. At total lung capacity (TLC) in both the prone and supine positions, the length of the diaphragm is not significantly different from the unstressed excised length, suggesting that the diaphragm is not under tension at TLC and that there is a hydrostatic gradient of pleural pressure on the diaphragmatic surface. Except for the ventral region of the costal diaphragm, which does not change length at lung volumes greater than 70% TLC, all other regions are stretched during passive deflations from TLC. Therefore below TLC the diaphragm is under passive tension and supports a transdiaphragmatic pressure (Pdi). The length of the diaphragm relative to its unstressed length is not uniform at functional residual capacity (FRC) and does not follow a strict vertical gradient that reverses when the animal is changed from the supine to the prone position. By inference, the length of muscle bundles is determined by factors other than the vertical gradient of Pdi. During mechanical ventilation, regional shortening is identical to the passive deflation length-volume relationship near FRC. Prone and supine FRC is the same, but the diaphragm is slightly shorter in the prone position. In both positions, during spontaneous ventilation there are no consistent differences in regional fractional shortening, despite regional differences in initial length relative to unstressed length.

MRI study of regional variations of pharyngeal wall compliance in cats.

Upper airway compliance indicates the potential of the airway to collapse and is relevant to the pathogenesis of obstructive sleep apnea. We hypothesized that compliance would vary over the rostral-to-caudal extent of the pharyngeal airway. In a paralyzed isolated upper airway preparation in cats, we controlled static upper airway pressure during magnetic resonance imaging (MRI, 0.391-mm resolution). We measured cross-sectional area and anteroposterior and lateral dimensions from three-dimensional reconstructed MRIs in axial slices orthogonal to the airway centerline. High-retropalatal (HRP), midretropalatal (MRP), and hypopharyngeal (HYP) regions were defined. Regional compliance was significantly increased from rostral to caudal regions as follows: HRP < MRP < HYP (P < 0.0001), and compliance differences among regions were directly related to collapsibility. Thus our findings in the isolated upper airway of the cat support the hypothesis that regional differences in pharyngeal compliance exist and suggest that baseline regional variations in compliance and collapsibility may be an important factor in the pathogenesis and treatment of obstructive sleep apnea.