Static and dynamic imaging of alveoli using optical coherence tomography needle probes

Imaging of alveoli in situ has for the most part been infeasible due to the high resolution required to discern individual alveoli and limited access to alveoli beneath the lung surface. In this study, we present a novel technique to image alveoli using optical coherence tomography (OCT). We propose the use of OCT needle probes, where the distal imaging probe has been miniaturized and encased within a hypodermic needle (as small as 30-gauge, outer diameter 310 μm), allowing insertion deep within the lung tissue with minimal tissue distortion. Such probes enable imaging at a resolution of ～12 μm within a three-dimensional cylindrical field of view with diameter ～1.5 mm centered on the needle tip. The imaging technique is demonstrated on excised lungs from three different species: adult rats, fetal sheep, and adult pigs. OCT needle probes were used to image alveoli, small bronchioles, and blood vessels, and results were matched to histological sections. We also present the first dynamic OCT images acquired with an OCT needle probe, allowing tracking of individual alveoli during simulated cyclical lung inflation and deflation.     

Lipid-assisted microinjection: introducing material into the cytosol and membranes of small cells.

The microinjection of synthetic molecules, proteins, and nucleic acids into the cytosol of living cells is a powerful technique in cell biology. However, the insertion of a glass micropipette into the cell is a potentially damaging event, which presents significant problems, especially for small mammalian cells (spherical diameter = 2-15 micron), especially if they are only loosely adherent. The current technique is therefore limited to cells that are both sufficiently large or robust and firmly attached to a substrate. We describe here a modification of the standard technique that overcomes some of the problems associated with conventional microinjection but that does not involve the insertion of a micropipette deep into the cell cytoplasm. Instead, this method depends on lipid fusion at the micropipette tip to form a continuous but temporary conductance pathway between the interiors of the micropipette and cell. This technique thus also provides a novel method of transferring lipids and lipid-associated molecules to the plasma membrane of cells.     

Size distribution of recruited alveolar volumes in airway reopening.

In 11 isolated dog lung lobes, we studied the size distribution of recruited alveolar volumes that become available for gas exchange during inflation from the collapsed state. Three catheters were wedged into 2-mm-diameter airways at total lung capacity. Small-amplitude pseudorandom pressure oscillations between 1 and 47 Hz were led into the catheters, and the input impedances of the regions subtended by the catheters were continuously recorded using a wave tube technique during inflation from -5 cm H(2)O transpulmonary pressure to total lung capacity. The impedance data were fit with a model to obtain regional tissue elastance (Eti) as a function of inflation. First, Eti was high and decreased in discrete jumps as more groups of alveoli were recruited. By assuming that the number of opened alveoli is inversely proportional to Eti, we calculated from the jumps in Eti the distribution of the discrete increments in the number of opened alveoli. This distribution was in good agreement with model simulations in which airways open in cascade or avalanches. Implications for mechanical ventilation may be found in these results.     

Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor

Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A I M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, ³⁵SO₄ was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3 M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3–7.2, to intracellular concentrations of at least 40–200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4–8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.     

A simple, rapid, aseptic method for filling micropipettes by centrifugation

A centrifugation method for sterilizing, storing, and filling micropipettes is described. Each micropipette is held in a centrifuge tube by a rubber stopper which clamps the butt end of the micropipette. The pipettes are sterilized and aseptically stored. A pipette is filled by injecting solution into the butt end of the micropipette. The micropipette is returned to a suspended position in the centrifuge tube and the liquid is rapidly forced into the micropipette tip by centrifugation. The technique is simpler and more rapid than presently used centrifugation methods.     

Arguments against and alternatives for an extracellular surfactant layer in the alveoli of mammalian lung

It is generally believed that lung alveoli contain an extracellular aqueous layer of surfactant material, which is allegedly required to prevent alveolar collapse at small lung volume; the surfactant's major constituent is a fully saturated phospholipid, referred to as dipalmitoyl lecithin or DPL. I herein demonstrate that the surfactant hypothesis of alveolar stability is fundamentally wrong. Although DPL is synthesized inside type II epithelial cells and stored in the typical inclusion bodies therein and lowers surface tension to zero in the surface balance, there is no evidence to the effect that type II cells secrete the DPL surfactant into the aqueous intra-alveolar layer which is shown by electron microscopy in support of the surfactant theory. To the contrary, all the evidence indicates that, when seen, such an extracellular layer is an artifact. This is probably upon the damage glutaraldehyde inflicts onto alveolar structures during fixation of air-inflated lung tissue. Furthermore, several cogent arguments invalidate the belief that an extracellular layer of DPL and serum proteins is present in the alveoli of normal lung. In light of these arguments, a surface tension role of DPL in alveolar stability is excluded. Three hypotheses for an alternative role of DPL in respiration mechanics are proposed. They are: (a) alveolar clearance by viscolytic and surfactant action (bubble or foam formation) on the aqueous systems which are present in lung alveoli during edema and in prenatal life and which would otherwise be impervious to air; (b) homeostasis of blood palmitate in normal lung; (c) modulation of the elasticity of terminal lung tissue by the intact inclusion bodies and parts thereof inside type II cells in normal lung.     

Function of alveoli as a result of evolutionary development of respiratory system in mammals

Reaction of hemoglobin oxygenation is known to occur for 40 femtoseconds (40 × 10^?15 s). However, the process of oxygen diffusion to hemoglobin under physiologic conditions decelerated this reaction approximately billion times. In mammalian lungs, blood is moving at a high rate and in a relatively high amount. The human lung mass is as low as 600 g, but the complete cardiac output approaches 6 l/min. In rat, from 20 to 40 ml of blood is passed for one minute through the lung whose mass is about 1.5 g. Such blood flow rate is possible, as in lungs of high animals there exists a dense network of relatively large microvessels with diameter from 20 to 40 μm and more. In spite of a large volume and a high blood flow rate hampering oxygen diffusion, the complete blood oxygenation occurs in lung alveoli. This is due to peculiar mechanisms that facilitate markedly the oxygen diffusion and that developed in alveoli of mammals in the course of many million years of evolution of their respiratory system. Thus, alveolus is not a bubble with air, but a complex tool of fight with inertness of diffusion. It is interesting that in lungs of the low vertebrates, neither such system of blood vessels nor alveoli exist, and their blood flow rate is much lower than in mammals.     

Theoretical modeling of the interaction between alveoli during inflation and deflation in normal and diseased lungs

Alveolar recruitment is a central strategy in the ventilation of patients with acute lung injury and other lung diseases associated with alveolar collapse and atelectasis. However, biomechanical insights into the opening and collapse of individual alveoli are still limited. A better understanding of alveolar recruitment and the interaction between alveoli in intact and injured lungs is of crucial relevance for the evaluation of the potential efficacy of ventilation strategies. We simulated human alveolar biomechanics in normal and injured lungs. We used a basic simulation model for the biomechanical behavior of virtual single alveoli to compute parameterized pressure–volume curves. Based on these curves, we analyzed the interaction and stability in a system composed of two alveoli. We introduced different values for surface tension and tissue properties to simulate different forms of lung injury. The data obtained predict that alveoli with identical properties can coexist with both different volumes and with equal volumes depending on the pressure. Alveoli in injured lungs with increased surface tension will collapse at normal breathing pressures. However, recruitment maneuvers and positive endexpiratory pressure can stabilize those alveoli, but coexisting unaffected alveoli might be overdistended. In injured alveoli with reduced compliance collapse is less likely, alveoli are expected to remain open, but with a smaller volume. Expanding them to normal size would overdistend coexisting unaffected alveoli. The present simulation model yields novel insights into the interaction between alveoli and may thus increase our understanding of the prospects of recruitment maneuvers in different forms of lung injury.     

Morphologic aspects of alveolar microcirculation.

Direct observations of the flow direction and connections between arteries and veins in the mammal lung are difficult. When we divide the lung into smaller units like acini or segments we can observe a central supply of the unit with arterial blood that has venous drainage at different points of the periphery. Consideration of the situation prevailing at birth strongly suggests a preferential flow direction through paths located in primary septa at the bottom of alveoli. Capillaries of the secondary septa placed between alveoli open to the same duct represent collaterals of the mainstream flow filled only if pressure conditions permit. Another significant feature is the presence of pleated alveolar septa. While capillaries inside the interalveolar wall mostly appear flat or collapsed, the capillaries of the pleated alveolar corners are always wide open. Often they show openings into a small venule placed inside the pleated area, which strongly suggests that the pleated areas contain the venous side of the capillaries.     

Passive deformation analysis of human leukocytes

The following analysis presents an experimental and theoretical study of the passive viscoelastic behavior of human leukocytes. Individual neutrophils in EDTA were observed both during their partial aspiration into a small micropipette and after expulsion from a large micropipette where the cell had been totally aspirated and deformed into a sausage shape. To analyze the data, a passive model of leukocyte rheology has been developed consisting of a cortical shell containing a Maxwell fluid which describes the average properties of the cell cytoplasm. The cortical shell represents a crosslinked actin layer near the surface of the cell and is assumed to be under pre-stressed tension. This model can reproduce the results of experiments using micropipette for both short-time small deformation and slow recovery data after large deformation. In addition, a finite element scheme has been established for the same model which shows close agreement with the analytical solution.     

Lung protein leaks in ventilated lambs: effects of gestational age

To study the protein permeability properties of the ventilated premature lung, we delivered groups of eight lambs at 122 and 135 days gestational age and ventilated the lambs equivalently. The lambs at 122 days gestational age had been treated with natural sheep surfactant at birth, and both groups of lambs had similar pH and blood gas values to 3 h of age. Three groups of lambs at 146 days gestational age also were studied for comparison; four lambs were ventilated to normalized PCO2 values, four lambs were ventilated equivalently to the premature lambs with supplemental CO2 used to normalize PCO2 values, and four lambs were treated with natural surfactant and ventilated similarly to the preterm lambs. The percent recovery into an alveolar wash and lung tissue of 131I-albumin given by intravascular injection and of 125I-albumin given into the airways was measured in each animal after killing at 3 h of age. Full-term lambs had a small bidirectional leak of albumin to and from the alveoli and lung tissue. The recovery of intravascular 131I-albumin in the alveolar wash was 5.8- and 4.1-fold higher in lambs at 122 and 135 days gestational age, respectively, than in full-term lambs. The loss of 125I-albumin from the airways and alveoli also increased as gestational age decreased. The bidirectional flux of albumin to and from the alveoli increased as gestational age decreased in the prematurely delivered and ventilated lambs.     

Characterizing Cell Adhesion by Using Micropipette Aspiration

We have developed a technique to directly quantify cell-substrate adhesion force using micropipette aspiration. The micropipette is positioned perpendicular to the surface of an adherent cell and a constant-rate aspiration pressure is applied. Since the micropipette diameter and the aspiration pressure are our control parameters, we have direct knowledge of the aspiration force, whereas the cell behavior is monitored either in brightfield or interference reflection microscopy. This setup thus allows us to explore a range of geometric parameters, such as projected cell area, adhesion area, or pipette size, as well as dynamical parameters such as the loading rate. We find that cell detachment is a well-defined event occurring at a critical aspiration pressure, and that the detachment force scales with the cell adhesion area (for a given micropipette diameter and loading rate), which defines a critical stress. Taking into account the cell adhesion area, intrinsic parameters of the adhesion bonds, and the loading rate, a minimal model provides an expression for the critical stress that helps rationalize our experimental results.     

An Apparatus for Measuring Volumes of Small Objects

An apparatus for measuring volumes of small objects such as tissue blocks is described. The apparatus measures volumes by fluid displacement and consists of a micropipette adapted to fit the mouth of an Erleiuneyer flask, a Luer adaptor fused to the side of the flask, and a glass syringe. When assembled with fluid enclosed, the fluid rises to a low level in the micropipette. Withdrawal of fluid into the syringe lowers the fluid level below the mouth of the flask. The micropipette is raised, the object to be measured is placed in the flask, and the micropipette is joined to the flask again. Fluid returned to the flask from the syringe rises to a higher level in the micropipette. The difference between the two fluid levels equals the volume of the object measured.

This apparatus gives reproducible measurements and can be calibrated for absolute volume determination. It is inexpensive to construct and easy to use.     

A model of postnatal formation of alveoli in rat lung.

In rats, and many other species, most lung alveoli are formed after birth. Septation of the large air saccules existing at birth has been considered as the main mechanism for alveoli formation. However, other undefined means of alveolarization have also been postulated to account for the large increase in gas-exchange surface area that takes place in the lung as the rat grows larger. Moreover, recent results show that the majority of alveoli in rat lung are formed by means other than septation of saccules existing at birth, but these mechanisms have not been identified up to the present. In this study, a mathematical model of alveolarization in rat lung is presented. The model is based on three postulates: (a) new saccules continue to be formed up to adulthood according to certain rules; (b) all these saccules subsequently septate generating a certain number of alveoli; (c) once formed, the saccules (and alveoli) do not change in volume, but newly-formed saccules are larger than the preceding ones according to a given law. The model accurately predicts the experimentally-known values at different ages of total alveolar volume, alveolar number, volume of the average alveolus, gas-exchange surface area, and alveolar volume distribution for normal rats and for rats in which septation is inhibited by treatment with dexamethasone or hypoxia during the early postnatal weeks of life.     

Morphometric deformations of small airways and alveoli under quasi-static inflation process

Localized morphometric deformations of small airways and alveoli during respiration have many biomedical and physiological implications. We developed fast synchrotron radiation CT system to visualize the small airways and alveoli of an intact mouse lung without fixation and dehydration, and analyzed their localized morphometric deformations between functional residual capacity (FRC) and total lung capacity (TLC). In the diameter behavior, the averaged and range values were significantly larger for smaller airways (68.8%, range: 0.36-0.89) than larger airways (45.2%, range: 0.40-0.57). These results indicated that the airway did not deformed in same manner and that these morphological differences characterized the heterogeneous lung function.     

Growth of the small airways and alveoli from childhood to the adult lung measured by aerosol-derived airway morphometry.

Understanding the human development of pulmonary air spaces is important for calculating the dose from exposure to inhaled materials as a function of age. We have measured, in vivo, the air space caliber of the small airways and alveoli at their natural full distension [total lung capacity (TLC)] by aerosol-derived airway morphometry in 53 children of age 6-22 yr and 59 adults of age 23-80 yr. Aerosol-derived airway morphometry utilizes the gravitational settling time of inhaled inert particles to infer the vertical distance necessary to produce the observed loss of particles to the airway surfaces at sequential depths into the lung. Previously, we identified anatomical features of the lung: the caliber of the transitional bronchioles [transitional effective air space dimension (EADtrans)]; the mean linear dimension of the alveoli (EADmin); and a measure of conducting airway volume [volumetric lung depth (VLDtrans)]. In the present study, we found that EADmin increased with age, from 184 microm at age 6 to 231 microm at age 22, generally accounting for the increase in TLC observed over this age range. EADtrans did not increase with TLC, averaging 572 microm, but increased with subject age and height when the entire age range of 6-80 yr is included {EADtrans (microm)=0.012[height (cm)]x[age (yr)]+508; P=0.007}. VLDtrans scaled linearly with lung volume, but VLDtrans relative to TLC did not change with age, averaging 7.04+/-1.55% of TLC. The data indicate that from childhood (age of 6 yr) to adulthood a constant number of respiratory units is maintained while both the smallest bronchioles and alveoli expand in size to produce the increased lung volume with increased age and height.     

An ultrastructural study of the blood/air barrier in the guinea-pig

Horseradish peroxidase (HRP) was intravenously injected into guinea-pigs to ultrastructurally examine the permeability of the blood/air barrier. Adults were given 300 mg/kg of the tracer in a small volume of saline, anesthetized and sacrificed at intervals by either intratracheal filling or right ventricular perfusion with 3% glutaraldehyde. The reaction product had passed through endothelial clefts and accumulated in the interstitium as early as 1.5 min after injection. This same degree of penetration occurred with either fixation method used. Tight junctions between pneumocytes prevented passage of the reaction product into alveoli. Pinocytotic vesicles were numerous in both endothelial and epithelial cells, but did not significantly contribute to tracer transport. Ten minutes post-injection was selected as optimal for this model since the highest concentration of tracer was found in the tissues at this time.     

Discovery of potent and selective matrix metalloprotease 12 inhibitors for the potential treatment of chronic obstructive pulmonary disease (COPD)

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with irreversible progressive airflow limitation. Matrix metalloproteinase-12 (MMP-12) has been characterized to be one of the major proteolytic enzymes to induce airway remodeling, destruction of elastin and the aberrant remodeling of damaged alveoli in COPD and asthma. The goal of this project is to develop and identify an orally potent and selective small molecule inhibitor of MMP-12 for treatment of COPD and asthma. Syntheses and structure-activity relationship (SAR) studies of a series of dibenzofuran (DBF) sulfonamides as MMP-12 inhibitors are described. Potent inhibitors of MMP-12 with excellent selectivity against other MMPs were identified. Compound 26 (MMP118), which exhibits excellent oral efficacy in the MMP-12 induced ear-swelling inflammation and lung inflammation mouse models, had been successfully advanced into Development Track status.     

Non-invasive microstructure and morphology investigation of the mouse lung: qualitative description and quantitative measurement

Background

Early detection of lung cancer is known to improve the chances of successful treatment. However, lungs are soft tissues with complex three-dimensional configuration. Conventional X-ray imaging is based purely on absorption resulting in very low contrast when imaging soft tissues without contrast agents. It is difficult to obtain adequate information of lung lesions from conventional X-ray imaging.

Methods

In this study, a recently emerged imaging technique, in-line X-ray phase contrast imaging (IL-XPCI) was used. This powerful technique enabled high-resolution investigations of soft tissues without contrast agents. We applied IL-XPCI to observe the lungs in an intact mouse for the purpose of defining quantitatively the micro-structures in lung.

Findings

The three-dimensional model of the lung was successfully established, which provided an excellent view of lung airways. We highlighted the use of IL-XPCI in the visualization and assessment of alveoli which had rarely been studied in three dimensions (3D). The precise view of individual alveolus was achieved. The morphological parameters, such as diameter and alveolar surface area were measured. These parameters were of great importance in the diagnosis of diseases related to alveolus and alveolar scar.

Conclusion

Our results indicated that IL-XPCI had the ability to represent complex anatomical structures in lung. This offered a new perspective on the diagnosis of respiratory disease and may guide future work in the study of respiratory mechanism on the alveoli level.     

Viscoelastic properties of transformed cells: role in tumor cell progression and metastasis formation

The micropipette aspiration technique was used to investigate the deformation properties of a panel of nontransformed and transformed rat fibroblasts derived from the same normal cell line. In this method, a step negative pressure is applied to the cell via a micropipette and the aspiration distance into the pipette as a function of time is determined using video techniques. A standard solid viscoelastic model was then used to analyze the viscoelastic properties of the cell. From these results, it is concluded that a direct correlation exists between an increase in deformability and progression of the transformed phenotype from a nontumorigenic cell line into a tumorigenic, metastatic cell line.