Comparison of airway responses in sheep of different age in precision-cut lung slices (PCLS).

BACKGROUND: Animal models should display important characteristics of the human disease. Sheep have been considered particularly useful to study allergic airway responses to common natural antigens causing human asthma. A rationale of this study was to establish a model of ovine precision-cut lung slices (PCLS) for the in vitro measurement of airway responses in newborn and adult animals. We hypothesized that differences in airway reactivity in sheep are present at different ages. METHODS: Lambs were delivered spontaneously at term (147d) and adult sheep lived till 18 months. Viability of PCLS was confirmed by the MTT-test. To study airway provocations cumulative concentration-response curves were performed with different allergic response mediators and biogenic amines. In addition, electric field stimulation, passive sensitization with house dust mite (HDM) and mast cells staining were evaluated. RESULTS: PCLS from sheep were viable for at least three days. PCLS of newborn and adult sheep responded equally strong to methacholine and endothelin-1. The responses to serotonin, leukotriene D4 and U46619 differed with age. No airway contraction was evoked by histamine, except after cimetidine pretreatment. In response to EFS, airways in PCLS from adult and newborn sheep strongly contracted and these contractions were atropine sensitive. Passive sensitization with HDM evoked a weak early allergic response in PCLS from adult and newborn sheep, which notably was prolonged in airways from adult sheep. Only few mast cells were found in the lungs of non-sensitized sheep at both ages. CONCLUSION: PCLS from sheep lungs represent a useful tool to study pharmacological airway responses for at least three days. Sheep seem well suited to study mechanisms of cholinergic airway contraction. The notable differences between newborn and adult sheep demonstrate the importance of age in such studies.

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.

A method to measure mechanical properties of pulmonary epithelial cell layers.

The lung has a huge inner alveolar surface composed of epithelial cell layers. The knowledge about mechanical properties of lung epithelia is helpful to understand the complex lung mechanics and biomechanical interactions. Methods have been developed to determine mechanical indices (e.g., tissue elasticity) which are both very complex and in need of costly equipment. Therefore, in this study, a mechanostimulator is presented to dynamically stimulate lung epithelial cell monolayers in order to determine their mechanical properties based on a simple mathematical model. First, the method was evaluated by comparison to classical tensile testing using silicone membranes as substitute for biological tissue. Second, human pulmonary epithelial cells (A549 cell line) were grown on flexible silicone membranes and stretched at a defined magnitude. Equal secant moduli were determined in the mechanostimulator and in a conventional tension testing machine (0.49 ± 0.05 MPa and 0.51 ± 0.03 MPa, respectively). The elasticity of the cell monolayer could be calculated by the volume-pressure relationship resulting from inflation of the membrane-cell construct. The secant modulus of the A549 cell layer was calculated as 0.04 ± 0.008 MPa. These findings suggest that the mechanostimulator may represent an adequate device to determine mechanical properties of cell layers.

Mechanostimulation, electrostimulation and force measurement in an in vitro model of the isolated rat diaphragm.

In an in vitro model of the entire rat diaphragm, diaphragmatic contraction forces at defined preload levels were investigated. A total of 24 excised rat diaphragms were electrically stimulated inside a two-chamber strain-applicator. The resulting contraction forces were determined on eight adjusted preload levels via measuring the elicited pressure in the chamber below the diaphragm. Subsequently, diaphragms were exposed for 6 h to one of four treatments: (1) control, (2) cyclic mechanical stretch, (3) intermittent electrical stimulation or (4) combination of cyclic mechanical stretch and electrical stimulation. Diaphragmatic contraction force increased from 116 ± 21 mN at the lowest preload level to 775 ± 85 mN at the maximal preload level. After 6 h maximal muscle contraction forces were smallest after non-electrostimulated treatment (control: 81 ± 15 mN, mechanical deflection: 94 ± 12 mN) and largest after electrostimulation treatment (mere electrostimulation: 165 ± 20 mN, combined mechano- and electro-stimulation: 164 ± 14 mN). We conclude that our model allows force measurements on isolated rat diaphragms. Furthermore, we conclude that by intermediate electrical stimulation diaphragmatic force generation was better preserved than by mechanical stimulation.

Pressure support improves oxygenation and lung protection compared to pressure-controlled ventilation and is further improved by random variation of pressure support.

OBJECTIVES: To explore whether 1) conventional pressure support ventilation improves lung function and attenuates the pulmonary inflammatory response compared to pressure-controlled ventilation and 2) random variation of pressure support levels (noisy pressure support ventilation) adds further beneficial effects to pressure support ventilation. DESIGN: Three-arm, randomized, experimental study. SETTING: University hospital research facility. SUBJECTS: Twenty-four juvenile pigs. INTERVENTIONS: Acute lung injury was induced by surfactant depletion. Animals were randomly assigned to 6 hrs of mechanical ventilation (n = 8 per group) with either 1) pressure-controlled ventilation, 2) pressure support ventilation, or 3) noisy pressure support ventilation. During noisy pressure support ventilation, the pressure support varied randomly, with values following a normal distribution. In all groups, the driving pressures were set to achieve a mean tidal volume of 6 mL/kg. At the end of experiments, animals were killed and lungs extracted for histologic and biochemical analysis. MEASUREMENTS AND MAIN RESULTS: Respiratory, gas-exchange, and hemodynamics variables were assessed hourly. The diffuse alveolar damage and the inflammatory response of lungs were quantified. Pressure support ventilation and noisy pressure support ventilation improved gas exchange and were associated with reduced histologic damage and interleukin-6 concentrations in lung tissue compared to pressure-controlled ventilation. Noisy pressure support ventilation further improved gas exchange and decreased the inspiratory effort while reducing alveolar edema and inflammatory infiltration compared to pressure support ventilation. CONCLUSIONS: In this model of acute lung injury, pressure support ventilation and noisy pressure support ventilation attenuated pulmonary inflammatory response and improved gas exchange as compared to pressure-controlled ventilation. Noisy pressure support ventilation further improved gas exchange, reduced the inspiratory effort, and attenuated alveolar edema and inflammatory infiltration as compared to conventional pressure support ventilation.

Variable tidal volumes improve lung protective ventilation strategies in experimental lung injury.

RATIONALE: Noisy ventilation with variable Vt may improve respiratory function in acute lung injury. OBJECTIVES: To determine the impact of noisy ventilation on respiratory function and its biological effects on lung parenchyma compared with conventional protective mechanical ventilation strategies. METHODS: In a porcine surfactant depletion model of lung injury, we randomly combined noisy ventilation with the ARDS Network protocol or the open lung approach (n = 9 per group). MEASUREMENTS AND MAIN RESULTS: Respiratory mechanics, gas exchange, and distribution of pulmonary blood flow were measured at intervals over a 6-hour period. Postmortem, lung tissue was analyzed to determine histological damage, mechanical stress, and inflammation. We found that, at comparable minute ventilation, noisy ventilation (1) improved arterial oxygenation and reduced mean inspiratory peak airway pressure and elastance of the respiratory system compared with the ARDS Network protocol and the open lung approach, (2) redistributed pulmonary blood flow to caudal zones compared with the ARDS Network protocol and to peripheral ones compared with the open lung approach, (3) reduced histological damage in comparison to both protective ventilation strategies, and (4) did not increase lung inflammation or mechanical stress. CONCLUSIONS: Noisy ventilation with variable Vt and fixed respiratory frequency improves respiratory function and reduces histological damage compared with standard protective ventilation strategies.

Detrimental role of CC chemokine receptor 4 in murine polymicrobial sepsis.

CC chemokine receptor 4 (CCR4) and its two ligands, CCL17 and CCL22, are critically involved in different immune processes. In models of lipopolysaccharide-induced shock, CCR4-deficient (CCR4(-/-)) mice showed improved survival rates associated with attenuated proinflammatory cytokine release. Using CCR4(-/-) mice with a C57BL/6 background, this study describes for the first time the role of CCR4 in a murine model of polymicrobial abdominal sepsis, the colon ascendens stent peritonitis (CASP). CASP-induced sepsis led to a massive downregulation of CCR4 in lymphoid and nonlymphoid tissues, whereas the expression of CCL17 and CCL22 was independent of the presence of CCR4. After CASP, CCR4(-/-) animals showed a strongly enhanced bacterial clearance in several organs but not in the peritoneal lavage fluid and the blood. In addition, significantly reduced levels of proinflammatory cytokines/chemokines were measured in organ supernatants as well as in the sera of CCR4(-/-) mice. CCR4 deficiency consequently resulted in an attenuated severity of systemic sepsis and a strongly improved survival rate after CASP or CASP with intervention. Thus, our data provide clear evidence that CCR4 plays a strictly detrimental role in the course of polymicrobial sepsis.

Pumpless extracorporeal lung assist for protective mechanical ventilation in experimental lung injury.

OBJECTIVE: To test the hypothesis that ventilation with 3 mL/kg tidal volume combined with extracorporeal CO2 removal by arteriovenous interventional lung assist reduces ventilator-associated organ injury in experimental acute lung injury when compared with ventilation with 6 mL/kg tidal volume without interventional lung assist. DESIGN: Prospective, randomized, controlled trial. SETTING: A university research laboratory. SUBJECTS: A total of 14 pigs weighing 46 +/- 4 kg (mean +/- sd). INTERVENTIONS: Acute lung injury was induced by repeated lung lavages until Pao2 was <100 mm Hg, with Fio2 of 1.0 and positive end-expiratory pressure of 5 cm H2O, for 1 hr without additional lavages. Animals were randomized to an interventional group with a tidal volume of 3 mL/kg with interventional lung assist (n = 7) or to a control group with a tidal volume of 6 mL/kg without interventional lung assist (n = 7) for 24 hrs. Organ function in vivo was determined by laboratory analyses, including calculations of pulmonary ventilation/perfusion distribution. Histologic assessment of organ injury was performed post mortem after 24 hrs. MEASUREMENTS AND MAIN RESULTS: In both groups, gas exchange improved in the course of the study (p < .05). However, in contrast to control animals, animals with lower tidal volumes and interventional lung assist had severe ventilation/perfusion mismatch, as indicated by increased perfusion to lung areas with a low ventilation/perfusion ratio (p < .05). Other variables of organ function in vivo and results of histologic examination post mortem did not reveal any statistical difference between groups. CONCLUSIONS: Combined ventilation with lower tidal volumes and extracorporeal CO2 removal as compared with traditional low tidal volumes without extracorporeal CO2 removal is not associated with differences in organ injury. Obviously, ventilation with tidal volumes of <6 mL/kg may cause pulmonary de-recruitment when positive end-expiratory pressure is not adequately increased.