Cigarette Smoke Exposure as a Model of Inflammation Associated with COPD

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation resulting from inflammation?driven pathologies in the lungs that are a consequence of smoking over many years. Given that the disease is increasing globally, understanding the mechanism by which cigarette smoke (CS) causes lung inflammation and exploiting that knowledge to develop effective treatments is urgently required. Animal models of CS exposure are commonly used to examine the inflammatory processes that may be involved in the development of COPD. The protocols described in this unit detail the development of preclinical models of CS?driven lung inflammation. These systems can be utilized to investigate the role of various biological pathways in CS?mediated inflammation and to assess the efficacy of new therapeutic strategies for treating COPD. Curr. Protoc. Pharmacol. 60:14.24.1?14.24.18. © 2013 by John Wiley & Sons, Inc.

Keywords: animal models; airways; COPD; emphysema; inflammation; lung

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Introduction
Basic Protocol 1: Setup of the Cigarette Smoke Exposure System
Basic Protocol 2: Cigarette Smoke Exposure Protocol
Basic Protocol 3: Determination of Lung Inflammation Following CS Exposure
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Setup of the Cigarette Smoke Exposure System

Materials

136‐liter exposure chambers (Teague Enterprises)
Dayton 2C820 extraction unit (Grainger Industrial Supply)
Electric fan (12 V DC, 6‐cm diameter, RS Components)
Perspex smoke extraction box (300 mm × 200 mm × 150 mm) (custom made in‐house)
Time‐set cigarette pinch valve (C Lee Machining)
Strong adhesive tape
AC/DC adapter (2 to 12 V; RS Components Ltd.)
Ashtray (100 mm × 10 mm × 40 mm)
Rubber bungs/stoppers, 30‐mm (VWR International)
Masterflex silicone tubing L/S 15 (Cole‐Parmer)
PVC hose 30‐mm diameter (4 m)

Basic Protocol 2: Cigarette Smoke Exposure Protocol

Materials

Male C57BL/6 mice (approx. 18 to 20 g), Sprague Dawley rats (approx. 200 to 225 g), or Dunkin‐Hartley guinea pigs (approx. 225 to 275 g)
Glycerol (Sigma‐Aldrich)
70% ethanol
Trigene solution

Appropriate animal housing cages [i.e., use of individually vented cages (IVCs) reduces the smell of smoke outside of the eages]
Filtered research cigarettes [University of Kentucky Research Cigarettes (http://www.ca.uky.edu/refcig/) #3R4F]
Large food container (for cigarettes)
Bench coat (VWR International)
Stainless steel animal smoking cages (400 mm × 320 mm × 150 mm; Adnor)
CS exposure system ( protocol 1 )
Flow Meter, 4 liters/min (Dwyer, cat. no. VFB‐65)
Flashlight
Pocket lighter
Stopwatch or laboratory timer
Metal forceps (small size)
Total suspended particulate sampling unit (Teague Enterprises)
- Air sampling pump
- Dry gas meter
- Filter holder
- Tubing
PallFlex 25‐mm total suspended particulate (TSP) membrane filters (VWR International, Emfab #TX40H120‐WW)
Cotton‐tipped applicators (VWR International)

Basic Protocol 3: Determination of Lung Inflammation Following CS Exposure

Materials

Mice exposed to CS ( protocol 2 )
Pentobarbitone
RPMI 1640 medium + GlutaMAX‐I (RPMI; Invitrogen)
Fetal bovine serum (FBS; Gibco, Invitrogen)
Liquid nitrogen (optional)
Collagenase (Roche Diagnostics)
DNase (Roche Diagnostics)
Penicillin/streptomycin stock (25,000 U/ml penicillin, 25 mg/ml streptomycin; Roche Diagnostics)
Reference blood samples
Wright‐Giemsa stain (Sigma)

1‐ml syringes, with and without 23‐gauge needles
Surgical tools (forceps, small scissors, fine scissors)
Cannula with luer connection
Microcentrifuge tubes (2 ml)
McIlwain tissue chopper (Campden Instruments)
Grant OLS 200 shaking water bath (Camlab)
70‐µm cell sieve
Sysmex cell counter (Sysmex, Milton Keynes)
Cytospin centrifuge (Shandon)
Hema‐tek 2000 automated slide stainer (Ames)

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Figures

Figure 5.64.1 Diagram of the cigarette smoke exposure system: front view.

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Figure 5.64.2 Diagram of the cigarette smoke exposure system: rear view.

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Figure 5.64.3 (A ) Rear view of the cigarette smoke exposure system. (B ) Rear view with detailed connections (inlets/outlets). Numbers 1, 2, and 3 in (B) correspond to the connections described in , steps 8 to 10.

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Figure 5.64.4 Front view of total suspended particulate (TSP) sampling equipment with details of components and connections.

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Figure 5.64.5 Characterization of the airway inflammation after cigarette smoke (CS) challenge. (A ) Male C57BL/6 mice were challenged for 3 days with CS (250, 500, or 750 ml/min, 1 hr, twice daily, 4 hr apart) or ambient air. Bronchoalveolar lavage fluid (BALF) samples were collected 24 hr after the final challenge. Graph illustrates the numbers of neutrophils in the BALF; the arrow indicates the CS challenge level (500 ml/min) chosen for further work. Data are presented as mean ± SE of n = 6 to 8 observations. (B ) Mice were challenged for 3 days with CS (500 ml/min, 1 hr, twice daily) or ambient air. BALF samples were collected at increasing time points after the final challenge. Graph illustrates the numbers of neutrophils in the BALF. Data are presented as mean ± SE of n = 6 to 8 observations. Figure adapted from Eltom et al. ().

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Figure 5.64.6 Characterization of the airway inflammation after cigarette smoke (CS) challenge. Mice were challenged for 3 to 28 days with CS (500 ml/min, 1 hr, twice daily, 4 hr apart) or ambient air. Bronchoalveolar lavage fluid (BALF) samples were collected 24 hr after the final challenge. The numbers of neutrophils, macrophages, and lymphocytes are shown in (A ), (B ), and (C ), respectively. Data are presented as mean ± SE of n = 6 to 8 observations. Figure adapted from Eltom et al. ().

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Figure 5.64.7 Characterization of the airway inflammation after cigarette smoke (CS) challenge. Mice were challenged for 14 days with CS (500 ml/min, 1 hr, twice daily, 4 hr apart) or ambient air. Bronchoalveolar lavage fluid (BALF) samples were collected at various time points after the final challenge. The numbers of neutrophils, macrophages, and lymphocytes are shown in (A ), (B ), and (C ) respectively. Data are presented as mean ± SE of n = 6 to 8 observations.

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Figure 5.64.8 Role of P2X7 receptor in cigarette smoke (CS)‐induced airway inflammation. Male C57BL/6 mice were challenged for 3 days with CS (500 ml/min, 1 hr, twice daily, 4 hr apart) or ambient air. Bronchoalveolar lavage fluid (BALF) samples were collected 24 hr after the final challenge. (A ) ATP levels in the BALF (ATPlite Luminescence Assay System, PerkinElmer). (B ) Amount of IL‐1β in the BALF (mouse ELISA, R&D Systems). (C ) Numbers of neutrophils in the BALF (differential counts of stained cytospin preparations). Data are presented as mean ± SE of n = 6 observations. # = statistical significance between air and CS challenge, Mann‐Whitney, P <0.05, * = statistical significance between wild‐type and P2X7 ‐/‐ mice, Mann‐Whitney, P <0.05.

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Literature Cited

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