Effect of positive airway pressure on capillary transit time in rabbit lung

We used fluorescence videomicroscopy to measure the passage of fluorescent dye through the subpleural microcirculation of the lung. With the rabbit in the left lateral decubitus position, the subpleural microcirculation was viewed either through a transparent parietal pleural window located in the superior part of the chest or directly with the chest open. There was no physical contact with the chest or lung. The rabbit was anesthetized, paralyzed, and mechanically ventilated with 100% O2. The dye was injected into the right ventricle during a 2-min apneic period to eliminate lung movement due to ventilation. The video signal of the passage of the dye was analyzed frame by frame by use of digital image processing to compensate for cardiogenic oscillations of the lung surface. Gray scale levels of an arteriole and adjacent venule were measured every 1/30 s. Capillary transit time was determined from the difference between the concentration-weighted mean time values of the arteriolar and venular dye dilution curves. We studied the effect of airway pressure (0-20 cmH2O) on transit time. Cardiac output was measured at different airway pressures by the thermal dilution technique. Capillary transit time averaged 0.60 s at functional residual capacity. Right ventricular-to-arteriolar transit time was four times as large as the capillary transit time. An increase in airway pressure from 0-5 to 20 cmH2O resulted in a fourfold increase in both capillary and arterial transit times and a threefold decrease in cardiac output.     

Detection of microspheres in vivo using multispectral optoacoustic tomography

We introduce a new approach to detect individual microparticles that contain NIR fluorescent dye by multispectral optoacoustic tomography in the context of the hemoglobin-rich environment within murine liver. We encapsulated a near infrared (NIR) fluorescent dye within polystyrene microspheres, then injected them into the ileocolic vein, which drains to the liver. NIR absorption was determined using multispectral optoacoustic tomography. To quantitate the minimum diameter of microspheres, we used both colorimetric and spatial information to segment the regions in which the microspheres appear. Regional diameter was estimated by doubling the maximum regional distance. We found that the minimum microsphere size threshold for detection by multispectral optoacoustic tomography images is 78.9 µm.     

Comparison of direct and indirect measurements of pulmonary capillary transit times

Using in vivo microscopy, we made direct measurements of pulmonary capillary transit time by determining the time required for fluorescent dye to pass from an arteriole to a venule on the dependent surface of the dog lung. Concurrently, in the same animals, pulmonary capillary transit time was measured indirectly in the entire lung using the diffusing capacity method (capillary blood volume divided by cardiac output). Transit times by each method were the same in a group of five dogs [direct: 1.75 +/- 0.27 (SE) s; indirect: 1.85 +/- 0.33 s; P = 0.7]. The similarity of these transit times is important, because the widely used indirect determinations based on diffusing capacity are now shown to coincide with direct measurements and also because it demonstrates that measurements of capillary transit times on the surface of the dependent lung bear a useful relationship to measurements on the capillaries in the rest of the lung.     

Penetration of fluorescent microspheres into the NEEM (North Eemian) Greenland ice core to assess the probability of microbial contamination

The validation of microbiological results from non-aseptically drilled deep ice cores is challenging because exogenous microbial cells can be transported into the core interior and compromise the existing microbial populations. The NEEM (North Eemian) ice core in Greenland provided a first-time opportunity to use fluorescent microspheres as tracers for assessing potential microbial contamination of glacial ice. We developed specific procedures to coat the surfaces of selected NEEM core samples representing bubbly (93–100 m), brittle (633–644 m) and clathrated (1,730 and 2,050 m) ice with melamine-based carboxylated fluorescent microspheres and tracked periodically their penetration into the core interior for 2.5 years using flow cytometry. Sufficient ice surface coating was achieved by immersing retrieved cores in plastic bags containing suspensions of pre-counted 1- and 10-μm microspheres or by down-hole microsphere deployment in plastic sleeves attached to the drill barrel and liberated during drilling. We examined the relationship between microspheres penetration and ice core depth, structure and time after coating. One consistent observation for all cores (except the brittle ice) was that removing a few millimeters of the outer layer drastically reduced microsphere counts, independent of timing, indicating that penetration was mostly limited to the surface layers. Any deeper penetration was found to be microsphere size dependent. The brittle ice showed significant microsphere penetration possibly due to microfractures. Overall, the use of fluorescent microspheres as tracers and microbial surrogates proved to be a sensitive approach for testing potential contamination during deep core projects.     

Differentiation of HL-60 promyelocytic leukemia cells: simultaneous determination of phagocytic activity and cell cycle distribution by flow cytometry

Phagocytosis of fluorescent microspheres by HL-60 promyelocytic leukemia cells following induction of differentiation with dimethyl sulfoxide (DMSO) was monitored using flow cytometry. Initiation of phagocytic capability following initiation of differentiation with 1.5% DMSO coincided with the attainment of respiratory burst activity as measured by NBT (nitro blue tetrazolium) reduction; the degree of phagocytic activity was dependent upon parameters such as microsphere size, microsphere number, and exposure time. Ingestion of fluorescent microspheres did not interfere with the measurement of DNA content using propidium iodide; thus, simultaneous determination of phagocytic activity and the cell cycle phase was possible. Accumulation of cells in the G1/G0 phase of the cell cycle following DMSO treatment was correlated with the acquisition of the capacity to phagocytize. Analysis of two-parameter correlated data also indicated that phagocytosis is coupled with residence in the G1/G0 phase of the cell cycle, further suggesting that the ability to phagocytize fluorescent microspheres is associated with end-stage differentiation.     

Physiological neutrophil sequestration in the lung: visual evidence for localization in capillaries

Although the lung is known to be a major site of neutrophil margination, the anatomic location of these sequestered cells within the lung is controversial. To determine the site of margination and the kinetics of neutrophil transit through the pulmonary microvasculature, we infused fluorescein isothiocyanate-labeled canine neutrophils into the pulmonary arteries of 10 anesthetized normal dogs and made fluorescence videomicroscopic observations of the subpleural pulmonary microcirculation through a window inserted into the chest wall. The site of fluorescent neutrophil sequestration was exclusively in the pulmonary capillaries with a total of 951 labeled cells impeded in the capillary bed for a minimum of 2 s. No cells were delayed in the arterioles or venules. Transit times of individual neutrophils varied over a wide range from less than 2 s to greater than 20 min with an exponential distribution skewed toward rapid transit times. These observations indicate that neutrophil margination occurs in the pulmonary capillaries with neutrophils impeded for variable periods of time on each pass through the lung. The resulting wide distribution of transit times may determine the dynamic equilibrium between circulating and marginated neutrophils.     

Fringe-scan flow cytometry

We describe the development of a scanning flow cytometer capable of measuring the distribution of fluorescent dye along objects with a spatial resolution of 0.7 micron. The heart of this instrument, called a fringe-scan flow cytometer, is an interference field (i.e., a series of intense planes of illumination) produced by the intersection of two laser beams. Fluorescence profiles (i.e., records showing the intensity of fluorescence measured at 20 ns intervals) are recorded during the passage of objects through the fringe field. The shape of the fringe field is determined by recording light scatter profiles as 0.25 micron diameter microspheres traverse the field. The distribution of the fluorescent dye along each object passing through the fringe field is estimated from the recorded fluorescence profile using Fourier deconvolution. We show that the distribution of fluorescent dye along microsphere doublets and along propidium iodide stained human chromosomes can be determined accurately using fringe-scan flow cytometry. The accuracy of fringe-scan shape analysis was determined by comparing fluorescence profiles estimated from fringe-scan profiles for microspheres and chromosomes with fluorescence profiles for the same objects measured using slit-scan flow cytometry.     

Intravital microscopic observations of 15-microm microspheres lodging in the pulmonary microcirculation.

Vascular infusions of 15-microm-diameter microspheres are used to study pulmonary blood flow distribution. The sites of microsphere lodging and their effects on microvascular perfusion are debated but unknown. Using intravital microscopy of the subpleural surface of rat lungs, we directly observed deposition of fluorescent microspheres. In a pump-perfused lung model, approximately 0.5 million microspheres were infused over 30 s into the pulmonary artery of seven rats. Microsphere lodging was analyzed for the location in the microvasculature and the effect on local flow after lodging. On average, we observed 3.2 microspheres per 160 alveolar facets. The microspheres always entered the arterioles as singlets and lodged at the inlets to capillaries, either in alveolar corner vessels or small arterioles. In all cases, blood flow continued either around the microspheres or into the capillaries via adjacent pathways. We conclude that 15-microm-diameter microspheres, in doses in excess of those used in typical studies, have no significant impact on pulmonary capillary blood flow distribution.     

Validation of fluorescent-labeled microspheres for measurement of relative blood flow in severely injured lungs.

The aim of the study was to validate a nonradioactive method for relative blood flow measurements in severely injured lungs that avoids labor-intensive tissue processing. The use of fluorescent-labeled microspheres was compared with the standard radiolabeled-microsphere method. In seven sheep, lung injury was established by using oleic acid. Five pairs of radio- and fluorescent-labeled microspheres were injected before and after established lung injury. Across all animals, 175 pieces were selected randomly. The radioactivity of each piece was determined by using a scintillation counter. The fluorescent dye was extracted from each piece with a solvent without digestion or filtering. The fluorescence was determined with an automated fluorescent spectrophotometer. Perfusion was calculated for each piece from both the radioactivity and fluorescence and volume normalized. Correlations between flow determined by the two methods were in the range from 0.987 +/- 0.007 (SD) to 0.991 +/- 0.002 (SD) after 9 days of soaking. Thus the fluorescent microsphere technique is a valuable tool for investigating regional perfusion in severely injured lungs and can replace radioactivity.     

Novel, fluorescent, magnetic, polysaccharide-based microsphere for orientation, tracing, and anticoagulation: preparation and characterization

A fluorescent, magnetic composite poly(styrene-maleic anhydride) microsphere, suitable for conjugation with polysaccharide, was synthesized using magnetite/europium phthalate particles as seeds by copolymerization of styrene and maleic anhydride. The magnetite/europium phthalate particles were wrapped up by poly(ethylene glycol), which improved the affinity between the seed particles and the monomers. The composite microspheres obtained, with a diameter of 0.15-0.7 microm, contain 586-1013 microg of magnetite/g of microsphere and 0.5-16 mmol surface anhydride groups/g of microsphere. Heparin was conjugated with the reactive surface anhydride groups on the surface of the microspheres by covalent binding to obtain a fluorescent, magnetic, polysaccharide-based microsphere. The microspheres not only retain their bioactivities but also provide magnetic susceptibility and fluorescence. They can be used as a carrier with magnetic orientation and fluorescence tracer for potent drug targeting. The orientation, tracer, and anticoagulation of the fluorescence, magnetic, polysaccharide-based microspheres were studied. The anticoagulant activity of the microspheres and heparin binding capacity reached 54,212.8 U and 607.1 mg/g of dry microspheres. The activity recovery was 50.2%. The anticoagulant activity of the microspheres increases with the increase of the conjugated heparin on the surface of the microspheres and the decrease of the microsphere size. Furthermore, The fluorescent, magnetic, polysaccharide-based microspheres can be easily transported to a given position in a magnetic field and traced via their fluorescence.     

Transit time dispersion in the pulmonary arterial tree

Knowledge of the contributions of arterialand venous transit time dispersion to the pulmonary vascular transittime distribution is important for understanding lung function and forinterpreting various kinds of data containing information aboutpulmonary function. Thus, to determine the dispersion of blood transittimes occurring within the pulmonary arterial and venous trees, imagesof a bolus of contrast medium passing through the vasculature ofpump-perfused dog lung lobes were acquired by using an X-ray microfocalangiography system. Time-absorbance curves from the lobar artery andvein and from selected locations within the intrapulmonary arterial tree were measured from the images. Overall dispersion within the lunglobe was determined from the difference in the first and second moments(mean transit time and variance, respectively) of the inlet arterialand outlet venous time-absorbance curves. Moments at selected locationswithin the arterial tree were also calculated and compared with thoseof the lobar artery curve. Transit times for the arterial pathwaysupstream from the smallest measured arteries (200-µm diameter) wereless than ~20% of the total lung lobe mean transit time. Transittime variance among these arterial pathways (interpathway dispersion)was less than ~5% of the total variance imparted on the bolus as itpassed through the lung lobe. On average, the dispersion that occurredalong a given pathway (intrapathway dispersion) was negligible. Similar results were obtained for the venous tree. Taken together, the resultssuggest that most of the variation in transit time in theintrapulmonary vasculature occurs within the pulmonary capillary bedrather than in conducting arteries or veins.

     

Fluorescent microsphere technique to measure cerebral blood flow in the rat

The present protocol describes a method for parallel measurement of cerebral blood flow (CBF) using fluorescent microspheres and structural assessment of the same material. The method is based on the standard microsphere technique, embolizing capillaries proportional to the blood flow, but requires dissolution of the tissue to retrieve the microspheres. To link the blood flow to the tissue morphology we modified the technique to fluorescent microspheres, which are quantified in cryo- or vibratome sections, allowing structural analysis by, for example, immunohistochemistry or standard histology. The protocol takes 8 h 50 min, without pauses, to complete, but additional flow measurements or specific protocols can increase the time needed.     

Effects of lung inflation and blood flow on capillary transit time in isolated rabbit lungs.

In a previous study, direct measurements of pulmonary capillary transit time by fluorescence video microscopy in anesthetized rabbits showed that chest inflation increased capillary transit time and decreased cardiac output. In isolated perfused rabbit lungs we measured the effect of lung volume, left atrial pressure (Pla), and blood flow on capillary transit time. At constant blood flow and constant transpulmonary pressure, a bolus of fluorescent dye was injected into the pulmonary artery and the passage of the dye through the subpleural microcirculation was recorded via the video microscope on videotape. During playback of the video signals, the light emitted from an arteriole and adjacent venule was measured using a video photoanalyzer. Capillary transit time was the difference between the mean time values of the arteriolar and venular dye dilution curves. We measured capillary transit time in three groups of lungs. In group 1, with airway pressure (Paw) at 5 cmH2O, transit time was measured at blood flow of approximately 80, approximately 40, and approximately 20 ml.min-1.kg-1. At each blood flow level, Pla was varied from 0 (Pla less than Paw, zone 2) to 11 cmH2O (Pla greater than Paw, zone 3). In group 2, at constant Paw of 15 cmH2O, Pla was varied from 0 (zone 2) to 22 cmH2O (zone 3) at the same three blood flow levels. In group 3, at each of the three blood flow levels, Paw was varied from 5 to 15 cmH2O while Pla was maintained at 0 cmH2O (zone 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

外用红色诺卡氏菌细胞壁骨架效力测试实验方法的研究

摸索改进外用红色诺卡氏菌（Nocardia rubra）细胞壁骨架的两种效力测试方法,即流式细胞术-荧光微球法、流式细胞术-免疫双标法（荧光微球+荧光抗体）,并与国家食品药品监督管理局原审批方法进行比较,经过统计学评估,评价两种方法的可行性。通过抽取小鼠免疫后获得的腹腔液注入到流式细胞仪,在发射光的荧光通路中,检测巨噬细胞的荧光强度,测定未吞噬荧光微球的巨噬细胞和吞噬荧光微球的巨噬细胞的比例,全部数据经统计学分析,按公式计算吞噬率及吞噬指数完成流式细胞术检测过程。分别采用荧光微球以及荧光微球+荧光抗体两种方式上机检测巨噬细胞吞噬结果。测得的结果与原检验方法——人工显微镜观察法的结果进行比较,确定评估方法的有效性和准确性。结果表明,显微镜人工计数法与流式细胞术-免疫双标法（荧光微球+荧光抗体）的3次实验结果与正常对照组比较,吞噬率明显升高（P<0.01）,吞噬指数明显增大（P<0.01）,有显著性差异,判定结果为阳性;3次流式细胞术-荧光微球法实验,结果不稳定。流式细胞术-免疫双标法（荧光微球+荧光抗体）由于加入了成熟小鼠巨噬细胞表面特异性标志物抗体F480荧光抗体,可有效标记小鼠巨噬细胞,加强了流式细胞仪收集巨噬细胞的准确性,两者结合应用更能准确反映供试品对小鼠巨噬细胞吞噬功能的作用。利用此方法检测外用红色诺卡氏菌细胞壁骨架增强免疫力的结果和药品原检验方法的统计学结果一致。该方法获取的巨噬细胞数量远多于原方法,加入的荧光微球及荧光抗体,能更准确反应供试品对小鼠巨噬细胞的吞噬作用。该方法灵敏性高、稳定性好,并且易操作,可作为原实验方法的改良参考。     

Production of microspheres with surface amino groups from blends of Poly(Lactide-co-glycolide) and Poly(epsilon-CBZ-L-lysine) and use for encapsulation.

Microspheres were formed from blends of the biodegradable polymer poly(DL-lactic-co-glycolic acid) (PLGA) together with poly(epsilon-CBZ-L-lysine) (PCBZL) by a double-emulsification/solvent evaporation technique. The size of the microspheres formed by this method was dependent both on the total concentration of the polymers and on the ratio of PLGA to PCBZL. The use of the microspheres for encapsulation was demonstrated by the inclusion of a solution of Texas Red fluorescent dye. Lysine epsilon-amino groups on the surface of the microspheres were deprotected by acid hydrolysis or lithium/liquid ammonia reduction. Acid hydrolysis damaged the surface of the microspheres as assessed by scanning electron microscopy, whereas deprotection by lithium/ammonia produced less damage and allowed the retention of encapsulated dye solution. The surface lysine groups made available on the surface of the microspheres could be used to covalently link a variety of biologically active molecules to alter their in vivo properties and allow targeting to specific cell types.     

Synthesis and Characterization of Dual-Functionalized Core-Shell Fluorescent Microspheres for Bioconjugation and Cellular Delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins.     

High-resolution imaging reveals a limit in spatial resolution of blood flow measurements by microspheres

Density of 15-microm microspheres after left atrial application is the standard measure of regional perfusion. In the heart, substantial differences in microsphere density are seen at spatial resolutions <5 ml, implying perfusion heterogeneity. Microsphere deposition imaging permits a superior evaluation of the distribution pattern. Therefore, fluorescent microspheres (FMS) were applied, FMS deposition in the canine heart was imaged by epifluorescence microscopy in vitro, and the patterns were observed compared with MR images of iron oxide microspheres (IMS) obtained in vivo and in vitro. FMS deposition in myocardial slices revealed the following: 1) a nonrandom distribution, with sequentially applied FMS of different color stacked within the same vessel, 2) general FMS clustering, and 3) rather large areas devoid of FMS (n = 3). This pattern was also seen in reconstructed three-dimensional images (<1 nl resolution) of FMS distribution (n = 4). Surprisingly, the deposition pattern of sequentially applied FMS remained virtually identical over 3 days. Augmenting flow by intracoronary adenosine (>2 microM) enhanced local microsphere density, but did not alter the deposition pattern (n = 3). The nonrandom, temporally stable pattern was quantitatively confirmed by a three-dimensional intermicrosphere distance analysis of sequentially applied FMS. T2-weighted short-axis MR images (2-microl resolution) of IMS revealed similar patterns in vivo and in vitro (n = 6), as seen with FMS. The observed temporally stable microsphere patterns are not consistent with the notion that microsphere deposition is solely governed by blood flow. We propose that at high spatial resolution (<2 microl) structural aspects of the vascular network dominate microsphere distribution, resulting in the organized patterns observed.     

Shear stress gradient over endothelial cells in a curved microchannel system.

Our purpose was to test a scale model of the microcirculation by measuring the shear forces to which endothelial cells were exposed, and comparing this to computer simulations. In vitro experiments were performed to measure the 2-dimensional projected velocity profile along endothelial cell lined microchannels (D-shaped, 10-30 microns radius, n = 15), or in microchannels without endothelial cells (n = 18). Microchannels were perfused with fluorescently labeled microspheres (0.5 micron dia., < 1%) suspended in cell culture media. The velocity of individual microspheres was obtained off-line (videorecording), using an interactive software program; velocity was determined as the distance traveled in one video field (1/60 s). Mass balance was verified in the microchannels by comparing the microsphere velocities to the perfusion pump rate. In confluent endothelial cell lined microchannels, a velocity profile was obtained as microspheres passed an endothelial cell nucleus (identified by fluorescent dye), and again, for a paired region 100 microns away without nuclei (cytoplasm region). The velocity profile was significantly shifted and sharpened by the endothelial cell nucleus, as anticipated. Over the nucleus, data are consistent with a normal sized nucleus extending into the lumen, further confirming that this scale model can be used to determine the wall shear stress to which endothelial cells are exposed. Using the experimental bulk phase fluid parameters as boundary conditions, we used computational fluid dynamics (CFD) to predict the expected wall shear stress gradient along an endothelial cell lined D-shaped tube. The wall shear stress gradient over the nucleus was 2-fold greater in the radial versus axial directions, and was sensitive to lateral versus midline positioned nuclei.     

Pulmonary microcirculatory kinetics of neutrophils deficient in leukocyte adhesion-promoting glycoproteins

The mechanism that causes neutrophils to sequester in the pulmonary circulation is unknown. Because the CD11/CD18 glycoprotein family on the surface membrane of neutrophils participates in many adhesive interactions with the endothelium, we investigated the role of these proteins in the intravascular sequestration of pulmonary neutrophils. Neutrophils were isolated from normal dogs and from the only living dog known to have leukocyte adhesion deficiency disease, an inherited deficiency of the CD11/CD18 adhesion family. The neutrophils were labeled with fluorescein dye, injected into normal recipient dogs, and their passage through the pulmonary microcirculation was recorded by in vivo videofluorescence microscopy through a transparent thoracic window. Transit times for normal and deficient neutrophils were similar over a wide range of hemo-dynamic conditions. Activation by zymosan-activated plasma, which increases the surface membrane expression of CD11/CD18, prolonged the transit of normal neutrophils but did not alter the transit time of the deficient neutrophils. These results indicate that neutrophil CD11/CD18 adhesion-promoting glycoproteins are not involved in the normal pulmonary sequestration of neutrophils but have a significant role in the arrest of activated neutrophils in the pulmonary capillaries.     

Hydraulic conductivity of canine parietal pleura in vivo

The hydraulic conductivity (Lp) of the parietal pleura was measured in vivo in spontaneously breathing anesthetized dogs in either the supine (n = 8) or the prone (n = 7) position and in an excised portion of the chest wall in which the pleura and its adjacent tissue were intact (n = 3). A capsule was glued to the exposed parietal pleura after the intercostal muscles were removed. The capsule was filled with either autologous plasma or isotonic saline. Transpleural fluid flow (V) was measured at several transpleural hydrostatic pressures (delta P) from the rate of meniscus movement within a graduated pipette connected to the capsule. Delta P was defined as the measured difference between capsule and pleural liquid pressures. The Lp of the parietal pleura was calculated from the slope of the line relating V to delta P by use of linear regression analysis. Lp in vivo averaged 1.36 X 10(-3) +/- 0.45 X 10(-3) (SD) ml.h-1.cmH2O-1.cm-2, regardless of whether the capsule was filled with plasma or saline and irrespective of body position. This value was not significantly different from that measured in the excised chest wall preparation (1.43 X 10(-3) +/- 1.1 X 10(-3) ml.h-1.cmH2O-1.cm-2). The parietal pleura offers little resistance to transpleural protein movement, because there was no observed difference between plasma and saline. We conclude that because the Lp for intact parietal pleura and extrapleural interstitium is approximately 100 times smaller than that previously measured in isolated stripped pleural preparations, removal of parietal pleural results in a damaged preparation.