Korean J Physiol Pharmacol.  2015 Jan;19(1):65-71. 10.4196/kjpp.2015.19.1.65.

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

• Kim HJ
• Kim Y
• Park SJ
• Bae B
• Kang HR
• Cho SH
• Yoo HY
• Nam JH
• Kim WK
• Kim SJ

• KMID: 2071821
• DOI: http://doi.org/10.4196/kjpp.2015.19.1.65

Abstract

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.

Keyword

Airway; Asthma; Lung slice; Smooth muscle

MeSH Terms

Airway Obstruction
Airway Resistance
Animals
Asthma
Interleukin-13
Lung*
Membranes
Methacholine Chloride*
Mice*
Mice, Transgenic
Muscle, Smooth*
Rodentia
Interleukin-13
Methacholine Chloride

Figure

• 

  Fig. 1 Airway inflammation and hyperresponsiveness in murine asthma model. Increased total and differential cell count in bronchoalveolar lavage (BAL) fluid (A) and enhanced airway hyperresponsiveness (Penh) [15] (B) in Ova sensitized and challenged murine Ova-asthma and IL-13TG compared with untreated control. Lung histology shows increased peribronchial and perivascular infiltration of inflammatory cells in Ova-asthma and IL-13TG (C).

• 

  Fig. 2 Representative cases of PCLS experiment. Images of mouse lung slices taken from PCLS preparations; Control (A), Ova-asthma (B), and IL-13TG (C). Representative recordings of airway area (pixel number, AW) changes in response to 60K-induced depolarization and different concentrations of MCh in Control (D), Ova-asthma (E), IL-13 TG (F).

• 

  Fig. 3 AW change (ΔAW) during 60K-contraction and 100 µM MCh application. Summary of ΔAW in Control (n=15), Ova-asthma (n=18) and IL-13TG (n=4) are displayed as bar graphs (mean±SEM). Numbers of tested slices are also directly indicated above the figure. Ova-asthma had a tendency of higher sensitivity to 60 K (***p<0.001) in both ΔAW area (pixel number) changes (A) and ΔAW normalized to each basal (B, ΔAW/AWbasal). However, there were no differences in the maximum stimulation of muscarinic receptor among three groups.

• 

  Fig. 4 Pharmacological sensitivity of AW responses to muscarinic stimulations. Concentration-response curves to differential concentrations of MCh. For comparison between groups, ΔAW normalized to each basal (A, ΔAW/AWbasal) and to maximum ΔAW-induced by 100 µM MCh (B, ΔAW/AWMChMax). Number of tested slices for the each group is same with that of figure 3.

• 

  Fig. 5 Recovery AW after repetitive and incremental application of MCh. AWs were measured at 3 min after washout of various concentrations of MCh (see Fig. 2D-F). Then the measured AW was normalized to the initial AW in each slice (AW/AWini, A and B) or to the AW measured just before the application of MCh (C, AW/AWbasal). Comparison of control vs. Ova-asthma (A), and of control vs. IL-13TG (B). In C, summary of AW/AWbasal (%) for 1 and 10 µM of MCh in Ova-asthma and IL-13TG are shown as bar graphs. Albeit the apparent difference between control and Ova-asthma at 1 µM of MCh (A), statistical comparison shows p>0.05. Numbers of tested slices are directly indicated in figure.

• 

  Fig. 6 Serotonin (5-HT)-induced airway constriction in PCLS. Representative responses of AWs from each group (A) and their summary (B, Control; n=6, Ova-asthma; n=6). AW change was normalized to basal AW in each slice. Ova-asthma showed significantly attenuated constriction to 5-HT (***p<0.001).

Reference

1. Sumino K, Sugar EA, Irvin CG, Kaminsky DA, Shade D, Wei CY, Holbrook JT, Wise RA, Castro M. American Lung Association Asthma Clinical Research Centers. Methacholine challenge test: diagnostic characteristics in asthmatic patients receiving controller medications. J Allergy Clin Immunol. 2012; 130:69–75.e6. PMID: 22465214.

2. Dixon C. The bronchial challenge test: a new direction in asthmatic management. J Natl Med Assoc. 1983; 75:199–204. PMID: 6827612.

3. Wills-Karp M. Interleukin-13 in asthma pathogenesis. Immunol Rev. 2004; 202:175–190. PMID: 15546393.

4. Matsumoto H, Hirata Y, Otsuka K, Iwata T, Inazumi A, Niimi A, Ito I, Ogawa E, Muro S, Sakai H, Chin K, Oku Y, Mishima M. Interleukin-13 enhanced Ca²⁺ oscillations in airway smooth muscle cells. Cytokine. 2012; 57:19–24. PMID: 22078634.

5. Kasaian MT, Miller DK. IL-13 as a therapeutic target for respiratory disease. Biochem Pharmacol. 2008; 76:147–155. PMID: 18502398.

6. Shin YS, Takeda K, Gelfand EW. Understanding asthma using animal models. Allergy Asthma Immunol Res. 2009; 1:10–18. PMID: 20224665.

7. Mullane K, Williams M. Animal models of asthma: reprise or reboot? Biochem Pharmacol. 2014; 87:131–139. PMID: 23831953.

8. Held HD, Martin C, Uhlig S. Characterization of airway and vascular responses in murine lungs. Br J Pharmacol. 1999; 126:1191–1199. PMID: 10205008.

9. Blacquière MJ, Hylkema MN, Postma DS, Geerlings M, Timens W, Melgert BN. Airway inflammation and remodeling in two mouse models of asthma: comparison of males and females. Int Arch Allergy Immunol. 2010; 153:173–181. PMID: 20413985.

10. Park SJ, Yoo HY, Kim HJ, Kim JK, Zhang YH, Kim SJ. Requirement of pretone by thromboxane A(2) for hypoxic pulmonary vasoconstriction in precision-cut lung slices of rat. Korean J Physiol Pharmacol. 2012; 16:59–64. PMID: 22416221.

11. Martin C, Uhlig S, Ullrich V. Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices. Eur Respir J. 1996; 9:2479–2487. PMID: 8980957.

12. Henjakovic M, Martin C, Hoymann HG, Sewald K, Ressmeyer AR, Dassow C, Pohlmann G, Krug N, Uhlig S, Braun A. Ex vivo lung function measurements in precision-cut lung slices (PCLS) from chemical allergen-sensitized mice represent a suitable alternative to in vivo studies. Toxicol Sci. 2008; 106:444–453. PMID: 18775882.

13. Chew AD, Hirota JA, Ellis R, Wattie J, Inman MD, Janssen LJ. Effects of allergen on airway narrowing dynamics as assessed by lung-slice technique. Eur Respir J. 2008; 31:532–538. PMID: 18032442.

14. Kim YK, Oh SY, Jeon SG, Park HW, Lee SY, Chun EY, Bang B, Lee HS, Oh MH, Kim YS, Kim JH, Gho YS, Cho SH, Min KU, Kim YY, Zhu Z. Airway exposure levels of lipopolysaccharide determine type 1 versus type 2 experimental asthma. J Immunol. 2007; 178:5375–5382. PMID: 17404323.

15. Hamelmann E, Schwarze J, Takeda K, Oshiba A, Larsen GL, Irvin CG, Gelfand EW. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med. 1997; 156:766–775. PMID: 9309991.

16. Erle DJ, Sheppard D. The cell biology of asthma. J Cell Biol. 2014; 205:621–631. PMID: 24914235.

17. Gosling M, Poll C, Li S. TRP channels in airway smooth muscle as therapeutic targets. Naunyn Schmiedebergs Arch Pharmacol. 2005; 371:277–284. PMID: 15917982.

18. Gao YD, Zou JJ, Zheng JW, Shang M, Chen X, Geng S, Yang J. Promoting effects of IL-13 on Ca²⁺ release and store-operated Ca²⁺ entry in airway smooth muscle cells. Pulm Pharmacol Ther. 2010; 23:182–189. PMID: 20045483.

19. Gao YD, Zheng JW, Li P, Cheng M, Yang J. Store-operated Ca²⁺ entry is involved in transforming growth factor-β1 facilitated proliferation of rat airway smooth muscle cells. J Asthma. 2013; 50:439–448. PMID: 23452113.

20. Suganuma N, Ito S, Aso H, Kondo M, Sato M, Sokabe M, Hasegawa Y. STIM1 regulates platelet-derived growth factorinduced migration and Ca²⁺ influx in human airway smooth muscle cells. PLoS One. 2012; 7:e45056. PMID: 22984609.

21. Ressmeyer AR, Larsson AK, Vollmer E, Dahlèn SE, Uhlig S, Martin C. Characterisation of guinea pig precision-cut lung slices: comparison with human tissues. Eur Respir J. 2006; 28:603–611. PMID: 16737991.

22. Jung YJ, Suh EC, Lee KE. Oxygen/Glucose Deprivation and Reperfusion Cause Modifications of Postsynaptic Morphology and Activity in the CA3 Area of Organotypic Hippocampal Slice Cultures. Korean J Physiol Pharmacol. 2012; 16:423–429. PMID: 23269905.

23. Wohlsen A, Martin C, Vollmer E, Branscheid D, Magnussen H, Becker WM, Lepp U, Uhlig S. The early allergic response in small airways of human precision-cut lung slices. Eur Respir J. 2003; 21:1024–1032. PMID: 12797499.