Airway Smooth Muscle Sensitivity to Methacholine in Precision-Cut Lung Slices (PCLS) from Ovalbumin-induced Asthmatic Mice

Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness (AHR) and reversible airway obstruction. Methacholine (MCh) is widely used in broncho-provocation test to evaluate airway resistance. For experimental investigation, ovalbumin-induced sensitization is frequently used in rodents (Ova-asthma). However, albeit the inflammatory histology and AHR in vivo, it remains unclear whether the MCh sensitivity of airway smooth muscle isolated from Ova-asthma is persistently changed. In this study, the contractions of airways in precision-cut lung slices (PCLS) from control, Ova-asthma, and IL-13 overexpressed transgenic mice (IL-13TG) were compared by analyzing the airway lumen space (AW). The airway resistance in vivo was measured using plethysmograph. AHR and increased inflammatory cells in BAL fluid were confirmed in Ova-asthma and IL-13TG mice. In the PCLS from all three groups, MCh concentration-dependent narrowing of airway lumen (DeltaAW) was observed. In contrast to the AHR in vivo, the EC50 of MCh for DeltaAW from Ova-asthma and IL-13TG were not different from control, indicating unchanged sensitivity to MCh. Although the AW recovery upon MCh-washout showed sluggish tendency in Ova-asthma, the change was also statistically insignificant. Membrane depolarization-induced DeltaAW by 60 mM K+ (60K-contraction) was larger in IL-13TG than control, whereas 60K-contraction of Ova-asthma was unaffected. Furthermore, serotonin-induced DeltaAW of Ova-asthma was smaller than control and IL-13TG. Taken together, the AHR in Ova-asthma and IL-13TG are not reflected in the contractility of isolated airways from PCLS. The AHR of the model animals seems to require intrinsic agonists or inflammatory microenvironment that is washable during tissue preparation.

Requirement of Pretone by Thromboxane A2 for Hypoxic Pulmonary Vasoconstriction in Precision-cut Lung Slices of Rat

Hypoxic pulmonary vasoconstriction (HPV) is physiologically important response for preventing mismatching between ventilation and perfusion in lungs. The HPV of isolated pulmonary arteries (HPV-PA) usually require a partial pretone by thromboxane agonist (U46619). Because the HPV of ventilated/perfused lungs (HPV-lung) can be triggered without pretone conditioning, we suspected that a putative tissue factor might be responsible for the pretone of HPV. Here we investigated whether HPV can be also observed in precision-cut lung slices (PCLS) from rats. The HPV in PCLS also required partial contraction by U46619. In addition, K+ channel blockers (4AP and TEA) required U46619-pretone to induce significant contraction of PA in PCLS. In contrast, the airways in PCLS showed reversible contraction in response to the K+ channel blockers without pretone conditioning. Also, the airways showed no hypoxic constriction but a relaxation under the partial pretone by U46619. The airways in PCLS showed reliable, concentration-dependent contraction by metacholine (EC50, ~210 nM). In summary, the HPV in PCLS is more similar to isolated PA than V/P lungs. The metacholine-induced constriction of bronchioles suggested that the PLCS might be also useful for studying airway physiology in situ.

The establishment of the method of precise lung slicing and the evaluation of neutral red assay for measuring viability of lung slices / 中国药理学通报

Aim To establish the metho d of precise lung slicing and examine the validity of neutral red(NR) assay for measuring lung slices viability.Method Inflated with 1% low melting agaro se solution, lung lobes were cut into slices by shaking microtome. With the thic kness of 400,500,600,700 ?m,lung slices were cultured in medium pH 6.8,7 .0,7.2, 7.4, respectively. After 1 h pre-incubation, lung slices were cont inuously submerged in 24-well plate, incubated for 0,2,4,6 h,respectively. NR assay, MTT assay, LDH leakage and SOD activity were used to assess the slices viability under different slices thickness, medium pH and culturing time. Result When the slice thickness was 600 ?m and medium pH was 7.0, t he viability of slices maintained best and steady for 6 h. There were positive c orrelations between NR uptake and MTT reduction in slices under different thickn ess(r 1 = 0.91, P

The establishment of lung slice fibrosis induced by transforming growth factor-?_1 / 中国药理学通报

Aim In order to provide the experimental basis to investigate the pathologic mechanisms and drug treatment of pulmonary fibrosis,establish the lung slice fibrosis model induced by transforming growth factor-?_1 (TGF-?_1) . Methods Lung was isolated and inflated with 0.4 % agarose solution, then was cut into slices. The lung slice viability was assessed through lactate dehydrogenase (LDH) leakage and MTT assay after incubation of 1, 3, 5, 7, 9 days. The sub-optimal time and dose of TGF-?_1- induced lung slice fibrosis were investigated via measurement of hydroxyproline (HYP), and lung slice fibrosis was examined with HE and Masson staining. Results The lung slice was viable for up to 9 days. The sub-optimal time and dose of TGF-?_1-induced lung slice fibrosis were 7 days and 2.5 ?g?L~ -1 respectively. Meanwhile, hydrocortisone did not decrease the HYP levels in lung slices of TGF-?_1-induced fibrosis. Conclusion TGF-?_1 (2.5 ?g?L~ -1 ,?7d) induced lung slice fibrosis, and hydrocortisone did not exert advantageous effect on this process.