Optical clearing: impact of optical and dielectric properties of clearing solutions on pulmonary tissue mechanics.

Optical clearing allows tissue visualization under preservation of organ integrity. Optical clearing of organs with a physiological change in three-dimensional geometry (such as the lung) would additionally allow visualization of macroscopic and microscopic tissue geometry. A prerequisite, however, is the preservation of the native tissue mechanics of the optically cleared lung tissue. We investigated the impact of optical and dielectric properties of clearing solutions on biomechanics and clearing potency in porcine tissue strips of healthy lungs. After fixation, bleaching, and rehydration, four methods of optical clearing were investigated using eight different protocols. The mechanical and optical properties of the cleared lung tissue strips were investigated by uniaxial tensile testing and by analyzing optical transparency and translucency for red, green, and blue light before, during, and after the biochemical optical clearing process. Fresh tissue strips were used as controls. Best balance between efficient clearing and preserved mechanics was found for clearing with a 1:1 mixture of dimethyl sulfoxide (DMSO) and aniline. Our findings show that 1) the degree of tissue transparency and translucency correlated with the refractive index of the clearing solution index (r = 0.976, P = 0.0004; and r = 0.91, P = 0.0046, respectively), 2) tissue mechanics were affected by dehydration and the type of clearing solution, and 3) tissue biomechanics and geometry correlated with the dielectric constant of the clearing solution (r = -0.98, P < 0.00001; and r = 0.69, P = 0.013, respectively). We show that the lower the dielectric constant of the clearing solutions, the larger the effect on tissue stiffness. This suggests that the dielectric constant is an important measure in determining the effect of a clearing solution on lung tissue biomechanics. Optimal tissue transparency requires complete tissue dehydration and a refractive index of 1.55 of the clearing solution.NEW & NOTEWORTHY Investigating optical clearing in porcine lung tissue strips, we found that refractive index and dielectric constant of the clearing solution affected tissue clearing and biomechanics. By documenting the impact of the composition of the clearing solution on clearing potency and preservation of tissue mechanics, our results help to compose optimal clearing solutions. In addition, the results allow conclusions on the molecular interaction of solvents with collagen fibers in tissue, thereby consolidating existing theories about the functionality of collagen.

The Equilibration of PCO2 in Pigs Is Independent of Lung Injury and Hemodynamics.

OBJECTIVES: In mechanical ventilation, normoventilation in terms of PCO2 can be achieved by titration of the respiratory rate and/or tidal volume. Although a linear relationship has been found between changes in respiratory rate and resulting changes in end-tidal cO2 (△PetCO2) as well as between changes in respiratory rate and equilibration time (teq) for mechanically ventilated patients without lung injury, it is unclear whether a similar relationship holds for acute lung injury or altered hemodynamics. DESIGN: We performed a prospective randomized controlled animal study of the change in PetCO2 with changes in respiratory rate in a lung-healthy, lung-injury, lung-healthy + altered hemodynamics, and lung-injury + altered hemodynamics pig model. SETTING: University research laboratory. SUBJECTS: Twenty mechanically ventilated pigs. INTERVENTIONS: Moderate lung injury was induced by injection of oleic acid in 10 randomly assigned pigs, and after the first round of measurements, cardiac output was increased by approximately 30% by constant administration of noradrenalin in both groups. MEASUREMENTS AND MAIN RESULTS: We systematically increased and decreased changes in respiratory rate according to a set protocol: +2, -4, +6, -8, +10, -12, +14 breaths/min and awaited equilibration of Petco2. We found a linear relationship between changes in respiratory rate and △PetCO2 as well as between changes in respiratory rate and teq. A two-sample t test resulted in no significant differences between the lung injury and healthy control group before or after hemodynamic intervention. Furthermore, exponential extrapolation allowed prediction of the new PetCO2 equilibrium and teq after 5.7 ± 5.6 min. CONCLUSIONS: The transition between PetCO2 equilibria after changes in respiratory rate might not be dependent on moderate lung injury or cardiac output but on the metabolic production or capacity of cO2 stores. Linear relationships previously found for lung-healthy patients and early prediction of PetCO2 equilibration could therefore also be used for the titration of respiratory rate on the PetCO2 for a wider range of pathologies by the physician or an automated ventilation system.

Endomicroscopic analysis of time- and pressure-dependent area of subpleural alveoli in mechanically ventilated rats.

We investigated the effects of recruitment maneuvers on subpleural alveolar area in healthy rats. 36 mechanically ventilated rats were allocated to either ZEEP-group or PEEP - 5cmH2O - group. The subpleural alveoli were observed using a transthoracal endoscopic imaging technique. Two consecutive low-flow maneuvers up to 30cmH2O peak pressure each were performed, interrupted by 5s plateau phases at four different pressure levels. Alveolar area change at maneuver peak pressures and during the plateau phases was calculated and respiratory system compliance before and after the maneuvers was analyzed. In both groups alveolar area at the second peak of the maneuver did not differ significantly compared to the first peak. During the plateau phases there was a slight increase in alveolar area. After the maneuvers, compliance increased by 30% in ZEEP group and 20% in PEEP group. We conclude that the volume insufflated by the low-flow recruitment maneuver is distributed to deeper but not to subpleural lung regions.

Time and volume dependence of dead space in healthy and surfactant-depleted rat lungs during spontaneous breathing and mechanical ventilation.

Volumetric capnography is a standard method to determine pulmonary dead space. Hereby, measured carbon dioxide (CO2) in exhaled gas volume is analyzed using the single-breath diagram for CO2. Unfortunately, most existing CO2 sensors do not work with the low tidal volumes found in small animals. Therefore, in this study, we developed a new mainstream capnograph designed for the utilization in small animals like rats. The sensor was used for determination of dead space volume in healthy and surfactant-depleted rats (n = 62) during spontaneous breathing (SB) and mechanical ventilation (MV) at three different tidal volumes: 5, 8, and 11 ml/kg. Absolute dead space and wasted ventilation (dead space volume in relation to tidal volume) were determined over a period of 1 h. Dead space increase and reversibility of the increase was investigated during MV with different tidal volumes and during SB. During SB, the dead space volume was 0.21 ± 0.14 ml and increased significantly at MV to 0.39 ± 0.03 ml at a tidal volume of 5 ml/kg and to 0.6 ± 0.08 ml at a tidal volume of 8 and 11 ml/kg. Dead space and wasted ventilation during MV increased with tidal volume. This increase was mostly reversible by switching back to SB. Surfactant depletion had no further influence on the dead space increase during MV, but impaired the reversibility of the dead space increase.

Endoscopic imaging to assess alveolar mechanics during quasi-static and dynamic ventilatory conditions in rats with noninjured and injured lungs.

OBJECTIVES: Although global respiratory mechanics are usually used to determine the settings of mechanical ventilation, this approach does not adequately take into account alveolar mechanics. However, it should be expected that the ventilatory condition (quasi-static vs. dynamic) and lung condition (noninjured vs. injured) affect alveolar mechanics in a clinically relevant way. Accordingly, the aim of this study was to investigate alveolar mechanics during quasi-static and dynamic ventilatory maneuvers in noninjured and injured lungs. We hypothesized that alveolar mechanics vary with ventilatory and lung conditions. DESIGN: Prospective animal study. SETTING: Animal research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Alveolar mechanics (derived from alveolar size and airway pressure) were determined in noninjured (n = 9) and in lungs lavaged with saline (n = 8) at quasi-static (low flow at a peak pressure of 40 cm H2O) and dynamic ventilatory maneuvers (increase and decrease in positive end-expiratory pressure from 0 to 15 and back to 0 cm H2O in steps of 3 cm H2O). Alveoli were recorded endoscopically and alveolar mechanics were extracted using automated tracking of alveolar contours. MEASUREMENTS AND MAIN RESULTS: The increase in alveolar size during quasi-static maneuvers was significantly greater than during dynamic maneuvers in noninjured (mean difference 18%, p < 0.001) but not in injured lungs (mean difference 3%, p = 0.293). During dynamic maneuvers, slope of the intratidal alveolar pressure/area curve (reflecting distensibility) decreased with increasing positive end-expiratory pressure (p = 0.001) independent of lung condition (noninjured and injured lungs). In contrast, independent of positive end-expiratory pressure but dependent on lung condition, the maximal tidal change in alveolar size was greater by an average of 40% in injured compared with noninjured lungs (p = 0.028). CONCLUSIONS: Alveolar mechanics during mechanical ventilation differed between quasi-static and dynamic conditions and varied with lung condition. Our data thus confirm that analysis of respiratory system mechanics under dynamic conditions is preferable to analysis during static conditions.

Time-dependent recruitment effects in ventilated healthy and lung-injured rats: "recruitment-memory".

We investigated the sustained effects of recruitment manoeuvres in terms of "recruitment memory" in healthy and lung injured rats. 46 ventilated rats were allocated to either the control (sham) or the lavage group. Two consecutive low-flow manoeuvres were performed before sham/lavage and hourly during a 2-h-observation period. The slopes of the inspiratory limbs of the two resulting pressure-volume loops were translated into compliance-volume curves. The difference between the two compliance curves was smaller after lavage (root-mean-square deviation: 0.065 ml/cm H2O control group, 0.038 ml/cm H2O lavage group; p<0.05) and stayed small during the whole experiment. In the control group, the deviation was small after sham manoeuvre but increased throughout the experiment. Compliance gain after recruitment was higher in the control group (0.1 ml/cm H2O) compared to the lavage group (0.02 ml/cm H2O, p<0.05). We conclude that lung lavage led to alveolar collapse not susceptible to recruitment manoeuvres. On the contrary in healthy lungs recruitment manoeuvres led to persistent lung recruitment which we interpret as recruitment memory.

Locally measured shear moduli of pulmonary tissue and global lung mechanics in mechanically ventilated rats.

This study was aimed at measuring shear moduli in vivo in mechanically ventilated rats and comparing them to global lung mechanics. Wistar rats (n = 28) were anesthetized, tracheally intubated, and mechanically ventilated in supine position. The animals were randomly assigned to the healthy control or the lung injury group where lung injury was induced by bronchoalveolar lavage. The respiratory system elastance E(rs) was analyzed based on the single compartment resistance/elastance lung model using multiple linear regression analysis. The shear modulus (G) of alveolar parenchyma was studied using a newly developed endoscopic system with adjustable pressure at the tip that was designed to induce local mechanostimulation. The data analysis was then carried out with an inverse finite element method. G was determined at continuous positive airway pressure (CPAP) levels of 15, 17, 20, and 30 mbar. The resulting shear moduli of lungs in healthy animals increased from 3.3 ± 1.4 kPa at 15 mbar CPAP to 5.8 ± 2.4 kPa at 30 mbar CPAP (P = 0.012), whereas G was ~2.5 kPa at all CPAP levels for the lung-injured animals. Regression analysis showed a negative correlation between G and relative E(rs) in the control group (r = -0.73, P = 0.008 at CPAP = 20 mbar) and no significant correlation in the lung injury group. These results suggest that the locally measured G were inversely associated with the elastance of the respiratory system. Rejecting the study hypothesis the researchers concluded that low global respiratory system elastance is related to high local resistance against tissue deformation.

Effects of intra-abdominal pressure on respiratory system mechanics in mechanically ventilated rats.

We investigated the effects of intra-abdominal pressure on respiratory system compliance at different PEEP levels. 20 ventilated rats underwent four pressure levels (7, 9, 11, 13 mm Hg) of helium pneumoperitoneum and were allocated to one of the four PEEP groups (0, 3, 6 and 9 cm H(2)O). From the expiratory pressure-volume curve the mathematical inflection point (MIP) was calculated. Volume-dependent compliance was analyzed using the SLICE-method. MIP-pressure correlated to the intra-abdominal pressure (r(2)=0.94, p<0.001). Peak inspiratory pressure increased with intra-abdominal pressure, and was lower after recruitment-maneuvers (p<0.001). The compliance gain following recruitment-maneuvers depended on PEEP, intra-abdominal pressure and intratidal volume (all p<0.001). Mean arterial pressure was independent of PEEP (p=0.068) and intra-abdominal pressure (p=0.293). Volume-dependent compliance courses varied according to PEEP and intra-abdominal pressure. The level of intra-abdominal pressure alters respiratory system mechanics in healthy lungs. Intratidal compliance can be used to guide the PEEP adjustment in intra-abdominal hypertension. If counterbalanced by PEEP, elevated intra-abdominal pressure has no negative effects on oxygenation or hemodynamics.

Morphometry of subpleural alveoli may be greatly biased by local pressure changes induced by the microscopic device.

Microscopy of subpleural alveoli has become an important technique to analyze alveolar morphology during mechanical ventilation. Mere contact of a microscope with the lung, however, may alter the local pressure at the pleural surface and thus the transmural pressure of the alveoli under view. The effect of local pleural pressure changes on alveolar morphology during microscopy has not been systematically evaluated hitherto. We developed a new microscopic device enabling control of the pressure directly at the field of view. In 6 isolated rat lungs we systematically varied the transmural pressure of subpleural alveoli by varying both the local pleural pressure and the alveolar pressure. Results show fixation pressure, alveolar pressure and local pleural pressure significantly influenced alveolar size and the number of alveoli per field of view. Our study demonstrates the important impact of local pleural pressure on the morphology of subpleural alveoli. We conclude that local pressures need to be determined during microscopy of subpleural alveoli to avoid misinterpretation of changes in alveolar geometry.

Intravital microscopy of subpleural alveoli via transthoracic endoscopy.

Transfer of too high mechanical energy from the ventilator to the lung's alveolar tissue is the main cause for ventilator-induced lung injury (VILI). To investigate the effects of cyclic energy transfer to the alveoli, we introduce a new method of transthoracic endoscopy that provides morphological as well as functional information about alveolar geometry and mechanics. We evaluate the new endoscopic method to continuously record images of focused subpleural alveoli. The method is evaluated by using finite element modeling techniques and by direct observation of subpleural alveoli both in isolated rat lungs as well as in intact animals (rats). The results confirm the overall low invasiveness of the endoscopic method insofar as the mechanical influences on the recorded alveoli are only marginal. It is, hence, a suited method for intravital microscopy in the rat model as well as in larger animals.