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. Author manuscript; available in PMC: 2026 May 30.
Published in final edited form as: Methods Mol Biol. 2018;1809:379–394. doi: 10.1007/978-1-4939-8570-8_25

Mouse Models of COPD

Karina A Serban 1, Irina Petrache 1
PMCID: PMC13220970  NIHMSID: NIHMS2172039  PMID: 29987802

Abstract

Elastase and chronic cigarette smoke exposure animal models are commonly used to study lung morphologic and functional changes associated with emphysema-like airspace enlargement in various animal species. This chapter describes the rationale for using these two models to study mechanisms of COPD pathogenesis and provides protocols for their implementation. E-cigarettes are an emerging health concern and may also contribute to lung disease. Accordingly, approaches to study e-cigarette vapors are provided. This chapter also includes methods and tools necessary to assess lung morphologic and functional changes in animals with emphysema-like airspace enlargement.

Keywords: COPD, Mouse, Emphysema, Cigarette smoke, Elastase, Mean linear intercept, E-cigarette

1. Introduction

Animal models of COPD are instrumental for interrogating molecular mechanisms of disease suggested by findings in patients or cell culture models of cigarette smoke (CS) exposure. From the classical discovery that instillation of porcine pancreatic elastase or human neutrophil elastase into the lungs of mice and hamsters culminated in the development of emphysema-like alveolar destruction [1, 2], to the airspace enlargement induced by chronic CS exposure in mice, guinea pigs, and rats [3, 4], animal models have enabled a better understanding of the mechanisms that underlie COPD pathogenesis.

In humans, COPD comprises several anatomic lesions within the lung: emphysema, small airway remodeling, vascular remodeling, and large airway epithelial injury (bronchitis). Moreover, there are significant systemic effects associated with human COPD, including acute infectious exacerbations, weight loss that may culminate in cachexia, myopathy, bone marrow suppression, and cardiovascular disease. Whether animal models can fully recapitulate the anatomical and functional changes of the human COPD is still an open question. Comparative anatomy studies have demonstrated differences in lung development, maturation, and responses to injury between animal species and strains. These differences explain and account for variability between different animal models of COPD pathobiology and can be leveraged to select the best model for a particular research question. In this chapter we will focus on two well-established and commonly used animal models of COPD, the elastase and CS exposure models.

Elastase-induced emphysema is a relatively fast, low-cost rodent model, since a single administration may induce histological changes similar to panacinar emphysema over a period of 2–3 weeks. Moreover, the severity of lung and systemic changes (e.g., inflammation, oxidative stress, apoptosis, body weight loss, reduced endurance) can be easily modulated by titrating the enzyme dose. Due to perceived highest relevance to human COPD (which is most frequently caused by exposure to CS), murine models of emphysema caused by chronic CS exposure are considered the gold standard. Other models of COPD include 1. the tight skin and 2. the pallid mouse in which emphysema develops spontaneously and 3. various pharmacological or transgenic manipulations (e.g., VEGF receptor blockade, IL-13 overexpression, or TERT deletion, among many others) that induce airspace enlargement. While these models have provided unique and valuable discoveries in the field, they also have notable limitations. The elastase-induced model is limited by the artificial nature of enzyme exposure, a sudden onset of severe inflammatory injury which does not model human COPD, and the relatively limited initiation of pathways of lung injury and systemic involvement. In comparison, the CS model is a more accurate mimic of human exposure, but is cumbersome, requires extensive duration of experimentation (e.g. 6 months of exposure to CS), and produces only a mild emphysema phenotype with minimal large airway involvement, modest remodeling of the pulmonary microvasculature, few systemic manifestations, and lack of spontaneous disease exacerbations. Moreover shorter (minutes to days) CS or e-cigarette vape exposure allows us to investigate the molecular mechanisms responsible for the initiation and propagation of lung and systemic injury. Despite these concerns, the elastase and CS exposure models have informed our knowledge about the major mechanistic paradigms leading to COPD: inflammation, oxidative stress, protease/antiprotease balance, alveolar cell apoptosis, early senescence, and autophagy. Herein we describe the materials and methods used to induce emphysema-like changes in commonly used inbred mouse strains following exposure to elastase or CS. In addition, methods to assess lung function and histopathology are also provided.

2. Materials

2.1. Elastase and CS Exposures

  1. Porcine pancreatic elastase.

  2. Research-grade cigarettes. Use filtered research-grade cigarettes (3R4F, 0.73 mg nicotine/cigarette) or nicotine-free cigarettes (1R6F, 0.16 mg nicotine/cigarette) from the Kentucky Tobacco Research and Development Center (University of Kentucky).

  3. Nose-only smoke exposure device. The inExpose nose-only tower (SCIREQ, Scientific Respiratory Equipment) ensures uniform CS inhalation to individual animals and prevents rebreathing of air among animals.

  4. “Homemade” vacuum trap (see Subheading 3.4, step 2 below).

  5. Nebulizer unit (Aeroneb micropump, 2.5–4.0 VMD, Aerogen).

  6. Inhalation restraint devices for nose-only inhalation exposure.

  7. SoftRestraints (Scientific Respiratory Equipment) made from nylon-coated stainless steel wires, suitable for rodents from 15 to 30 g.

  8. Small acrylic platform at a 60° angle.

  9. Whole-body smoke exposure device. The two most commonly used devices are the TE-10 (Fig. 1) and SIU24 supplied by Teague (Woodland, California) and the Promech Lab Holding AB (Vintrie, Sweden). Inside the TE-10 smoking machine, the animal is exposed to variable mixtures of 89% sidestream and 11% mainstream smoke.

  10. Enzyme-linked immunosorbent assay kit for measurement of serum cotinine levels.

  11. For CO level monitoring, use the in-line gas analyzer for carbon monoxide and particulate matter provided by the supplier to measure real-time estimates of cigarette smoke intensity and ensure accurate and safe exposure of animals. Maintain CO concentrations of 190 ppm.

  12. Pallflex Air Monitoring Filters for TSP concentration monitoring (Emfab), 25 mm placed in the in-line gas analyzer.

  13. Animals. The most common inbred mouse strains used are C57Bl/6, DBA2/J, Balb/C, A/J, and AKR/J; however, strain susceptibility to CS injury should be considered (see Note 1).

Fig. 1.

Fig. 1

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Teague-10 smoking machine setup. The machine has four sections: cabinet, cigarette handling, chimney, and ash collection tray. This microprocessor-controlled machine generates sidestream and mainstream cigarette smoke from research-grade cigarettes automatically loaded into a wheel. The cigarettes are lit, puffed, and produce smoke at a constant rate. The smoke is mixed, diluted, and metered to the exposure cabinet at a constant rate. To maintain a target concentration of total suspended particulates (TSP), the level is monitored by an in-line gas analyzer and AEM dry gas meter

2.2. Sedatives and Anesthetics

  1. Ketamine (100 mg/kg) administered intraperitoneally.

  2. Xylazine (10 mg/kg) administered intraperitoneally.

2.3. Tracheostomy

  1. Intramedic polyethylene tubing (I.D. 0.045”, O.D. 0.062” Becton Dickinson, New Jersey).

  2. 18 gauge needle.

2.4. Tissue Processing and Analysis

  1. 0.25% agarose in 10% neutral-buffered formalin for fixation.

  2. Paraffin for embedding.

  3. Multi-fit glass syringe with Luer-Lock tip, 30 mL.

  4. Air balance (Hydrogen Balance air 103L, Air Liquide).

  5. Gas chromatograph.

  6. Glass syringe with extension tube and Luer-lock.

  7. Suture silk.

  8. Blunt-tipped scissors.

  9. Cold-buffered saline.

  10. Hematoxylin and eosin to stain tissue sections.

2.5. Devices for Functional and Morphological Assessment

  1. Plethysmograph (SCIREQ, Scientific Respiratory Equipment, Montreal) to detect cough.

  2. Flexivent (Fig. 2 SCIREQ, Scientific Respiratory Equipment, Montreal).

  3. Stereology for calculation of mean linear intercept.

  4. Micro-computed tomography (microCT).

Fig. 2.

Fig. 2

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Functional analysis of emphysema in mice. An anesthetized mouse is intubated and ventilated using the Flexivent. The computer screen enables real-time display of respiratory rate and airflow resistance, calibration parameters, and physiologic outputs such as lung compliance and elastance

3. Methods

3.1. Porcine Pancreatic Elastase (PPE) Administration

  1. Sedate the mouse using xylazine (10 mg/kg) and ketamine (100 mg/kg).

  2. After the animal has lost footpad-pinch response, place it face up on a small acrylic platform (60° angle) while the head is held stationary and in alignment with the body by a rubber band underneath the upper teeth.

  3. Pull the tongue aside to view the trachea, and use a pipette tip to cannulate the trachea as distally as possible.

  4. Administer elastase solution (30 μg in a total volume of less than 75 μL) or vehicle control intratracheally. Monitor the mouse as it breathes in the substance.

  5. Administer 100–200 μL of air. When no more liquid is visible, the animal is removed from the restraint board and monitored until it recovers.

  6. Measure physiologic parameters (see Subheadings 3.5 and 3.6), and isolate tissue for lung histology 21 days after PPE administration (see Note 2).

3.2. Chronic Nose-Only CS Exposure

  1. Inside the inExpose nose-only tower, the animals are exposed to firsthand CS that is similar to firsthand CS exposure that occurs in humans that smoke.

  2. Place animals in SoftRestraints or inhalation restraint device.

  3. Load 24 3R4F cigarettes into the carousel. The smoke is automatically generated and delivered as mainstream smoke into an individual inhalation restraint device.

  4. Expose the mice to four cigarettes, for 60 min, twice a day, 5 days/week for 6 months (see Note 3).

3.3. Chronic Whole-Body CS Exposure

  1. Transfer mice in standard cages to TE-10c smoking chamber.

  2. Load five 3R4F cigarettes into the two available hood ports, and push them into the “O” ring. The sidestream smoke is generated inside the TE-10c hood and pumped through the mixing and dilution chamber into the exposure chamber.

  3. Monitor carbon monoxide levels using the in-line gas analyzer provided by the supplier to obtain real-time estimates of cigarette smoke intensity, and ensure accurate and safe exposure of animals. Maintain CO concentration of 190 ppm or less (see Note 4). An adequately calibrated flow meter for the TE-10c chamber will ensure CO concentration within the recommended range.

  4. Monitor total suspended particulate (TSP) concentrations using Pallflex Air Monitoring Filters placed in the in-line gas analyzer (see Note 5). First, record dry filter weight. Then, run the sample and record the time, flow, readings on the AEM dry gas meter attached to the TE-10c setup, and the final filter weight. Finally, calculate TSP by dividing the difference in filter weight by the volume collected. Maintain TSP within concentrations of 90–110 mg/m3.

  5. Expose mice to CS for up to 5 h per day.

  6. Acclimate mice to normal air for an additional hour, and then return them to their normal cages in the animal facility.

  7. Serum and urine cotinine, a nicotine metabolite, or blood carboxyhemoglobin (COHgb) levels may be monitored during the experiment to ensure “physiologic” nicotine levels (<50 ng/mL). Use ELISA to measure serum or urine cotinine (expected ~5 ng/mL) and blood-gas analyzer to measure COHgb concentration (expected ~5%).

  8. Continuous CS exposure for 5 h/day, 5 days/week for 6 months will induce emphysematous changes in susceptible strains of mice (see Note 6). Alternatively, for acute smoke exposure studies, mice can be exposed to cigarette smoke from 5 min up to 10 days (5 days/week).

3.4. Acute e-Cigarette Exposure

  1. Acute cigarette exposure models are insufficient to cause immediate functional or morphological changes suggestive of emphysema/COPD, even in susceptible mouse strains. However, acute (minutes to days) cigarette smoke or e-ciga-rette vape exposure results in oxidative stress (lower reduced: oxidized glutathione ratio, increased level of inducible nitric oxide synthase or endothelial nitric oxide synthase), loss of epithelial and endothelial barriers, endothelial exosome shedding, and leukocyte adhesion to the lung microvasculature.

  2. Electronic cigarette (e-cigarette) vape is obtained from vaporization of commercially available electronic cigarette solution in a “homemade” vacuum trap (Fig. 3) and collection of the postvaporized condensate. Alternatively, one can use the e-cigarette extension designed for the inExpose (SCIREQ, Scientific Respiratory Equipment) CS exposure unit that offers automated activation and custom puff profiles of the e-cigarette cartridge.

  3. Pour the e-cigarette vape (~2 μg nicotine/200 μL) into the nebulizer unit (Aeroneb micropump, 2.5–4.0 VMD, Aerogen).

  4. Connect the nebulizer unit to the control module (Fig. 4), and follow the manufacturer’s instructions to turn on the system.

  5. Restrain the mouse by grabbing the scruff of the neck and tail.

  6. Place the animal’s head inside the tube extension that is connected to the nebulizer unit. Turn on the nebulizer unit. The nebulization procedure takes 1–2 min.

  7. Return the animal to the cage.

Fig. 3.

Fig. 3

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Custom-designed smoke trap to collect cigarette smoke or vape condensate. The in-line flowmeter is set at 1.5 cc/min. It will burn research cigarettes at a rate of one cigarette/min to 0.5 cm above the filter. The smoke is trapped inside the glass jar and bubbled into 20 mL sterile PBS. We burn two cigarettes (the filtered end inside the glass Pasteur pipette tip) per final 20 mL aqueous cigarette smoke solution. Alternatively, the mouthpiece of a commercially available vaporizer is connected in-line, and the e-cigarette vape is trapped and condensed directly into the glass jar

Fig. 4.

Fig. 4

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Nebulization setup. Electronic cigarette vape loaded in the nebulizer unit (a) is nebulized to the animal as demonstrated in the right panel (b)

3.5. Physiologic Assessment of Emphysema

  1. Perform functional assessment of emphysema on mice 21 days after elastase administration or chronic exposure to CS. Although emphysematous changes can be detected at earlier time points (e.g., D10), measurements at D21 after elastase administration provide a more consistent increase in lung compliance.

  2. Sedate the mouse using xylazine (10 mg/kg) and ketamine (100 mg/kg).

  3. Verify depth of sedation using footpad-pinch response. If needed, animals can be given a supplemental dose of ketamine (10 mg/kg) and xylazine (1 mg/kg).

  4. Place the mouse face up on a small acrylic platform. Position a rubber band underneath the upper teeth to hold the head stationary and in alignment with the body.

  5. Spray the anterior neck with 100% ethanol, to wet and clean the fur for easier access to the skin overlaying the trachea.

  6. Use sterile scissors to make an anterior cervical incision. Cut vertically to expose the underlying soft tissue. Spread the soft tissue laterally to expose the trachea.

  7. Carefully cut a small puncture in the anterior trachea.

  8. Insert a cannula made from a truncated 18 gauge needle into the hole and connect. Be careful not to transect or perforate the posterior tracheal wall.

  9. Intubate the trachea with a cannula fabricated from a 2.5–3 cm long piece of intramedic polyethylene tubing attached to the end of the truncated 18 gauge needle.

  10. Connect the cannula to the Flexivent (Fig. 2). Parameters for the Flexivent ventilation are a positive end-expiratory pressure (PEEP) of 2.5–5 cm H2O, 200 breaths per minute, and tidal volume of 0.3 mL.

  11. The respiratory machine also contains an in-line (or sidestream) nebulizer connected to the Y-tubing by which drugs or inhaled anesthetics can be administered directly to the unconscious animal’s lungs without release to the surrounding environment.

  12. One can use the Flexivent to measure resistance, impedance, elastance, or pressure-volume curves. Changes in resistance and respiratory impedance are measurements of airway obstruction, whereas a decrease in elastance or a shift in the pressure-volume curves indicates increased compliance consistent with emphysematous changes. Please see Chap. 19 for additional details about using Flexivent to monitor lung function.

  13. When this is a terminal procedure, the mice are then euthanized by bilateral pneumothorax and exsanguination, and the lungs are processed for morphologic and morphometric measurements.

3.6. Measurement of Diffusion Factor for Carbon Monoxide (DFCO)

  1. The DFCO measurement involves inhaling a gas mixture containing a small amount of carbon monoxide and an inert gas (e.g., neon in mice) [5].

  2. Sedate and intubate the mouse using the steps described in Subheading 3.5.

  3. Use a 3 mL syringe to draw 0.8 mL gas mixture from the tank containing the gas mixture, 0.3% Ne, 0.3% CO, in an air balance (hydrogen balance air103L, Air Liquide).

  4. Connect the tracheostomy cannula to the syringe containing the gas mixture.

  5. Quickly inflate the lungs with 0.8 mL of gas mixture.

  6. Hold inflation for 9 s, and then withdraw 0.8 mL from the lungs. Dilute the returning gas mixture with 2 mL room air inside the 3 mL syringe.

  7. Immediately use a gas chromatograph to measure the change in gas concentrations. Alternatively, one may use polypropylene gas sample bags to store the gas sample for delayed measurements.

  8. Compare uptake of CO to dilution of Ne. Use the following formula: 1 – (CO9/COc)/(Ne9/Nec) to define DFCO (c, the calibration gas, and 9, the gas from the 9 s measurement time). A value of 1 for DFCO reflects 100% CO uptake and normal diffusion factor for CO.

  9. This is a terminal procedure; the mice are euthanized post DLCO measurement (see Note 7).

3.7. Preparation of Lung Tissue for Histology

  1. Heat the 0.25% agarose in 10% formalin solution for 10 min and until it is melted and has clear appearance.

  2. Prepare a glass syringe by wrapping a heating cord evenly around it. This will keep the syringe warm and prevent the agarose from cooling and thickening.

  3. Attach the syringe to a ring holder. Attach an extension tube to the syringe using a Luer-lock.

  4. Set the heat box at 37 °C (Fig. 5), and center a heat lamp on the syringe to prevent agarose from hardening.

  5. Fill syringe with agarose-formalin solution to the top. Adjust the height of the fluid column so that it is 20 cm above the chest of the animal. This will ensure that the lungs inflate at a constant pressure of 20 cm H2O (Fig. 6).

  6. Open the stopcock to allow air to purge from the tubing, and then connect the extension tube to the tracheostomy cannula. Make sure the solution is still liquid before you let the lung fill. Keep the tubing system straight to promote flow of the agarose solution, and work quickly through all steps to prevent the agarose from cooling.

  7. Put suture silk around the lung. Tighten knot around the lung.

  8. Using blunt-tipped scissors carefully, cut the lung away from the chest. Avoid puncturing the lung.

  9. Dip the lung into cold-buffered saline until the agarose-formalin solution hardens.

  10. Measure lung volume (V) using the water displacement method. Perform three volume measurements; make sure you dry the lung between the three measurements.

  11. Place the dry lung in a histology cassette, and submerge it in a 10% formalin with 0.1% glutaraldehyde solution.

Fig. 5.

Fig. 5

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Custom-designed setup for lung inflation and fixation. The melted agarose-formalin solution is poured in the glass syringe that is warmed at 37 °C by the heating cord and heat lamp. The syringe is filled with fixation solution. An extension tube with Luer-lock connectors is attached to the distal end of the syringe

Fig. 6.

Fig. 6

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Lung inflation method. (a) The syringe plunger is set at 10 cm from the counter to ensure a constant pressure of 20 cm water during lung inflation. In the next step (b) the mouse tracheostomy tube is connected to the extension tube. The mouse lungs require less than 3 mL warm fixation solution (~1.5 mL for one lung) to be fully inflated at constant pressure. Next we use the suture silk to tighten a knot around the trachea or left main bronchus (if only one lung is inflated and fixed). The dissected lung is dipped into cold-buffered saline until the fixation solution hardens

3.8. Morphologic and Morphometric Analysis of Emphysema

Stereology can be used to obtain unbiased estimates of alveolar number, alveolar surface, and lung volume [6, 7]. For systematic uniform random sampling, use a tissue slicer to cut the lung into slabs of equal thickness.

3.8.1. Stereology

3.8.2. Cavalieri Method

  1. Use the Cavalieri method to calculate the total lung volume.

  2. Begin “bread loafing” the tissue by sectioning. Start the sectioning outside the tissue margins (i.e., cut through paraffin to get close to the lung surface). Once the lung surface has been reached, take every second section (or third or fourth, as decided a priori by your laboratory) for further processing.

  3. Place a point grid with a known area randomly over the slabs (cut face up). Count all grid points that are within the cut surface of the tissue.

  4. Calculate total lung volume by applying the formula Vlung = a(p) × T × ΣP where a(p) is the area per point, T is the tissue slab thickness, and ΣP is the number of points hitting the tissue cut face of each particular slab.

3.8.3. Calculation of Total Alveolar Surface Area Per Lung

  1. To calculate the total alveolar surface area per lung, use surface estimation by intersection counting.

  2. Section the tissue (5 μm sections) using a microtome.

  3. Stain tissue sections with hematoxylin and eosin.

  4. Image 20 fields at 10× to 20×, and take random pictures from random lung sections.

  5. Superimpose randomly over each picture a test system of two test lines of known length (50 μm), each associated with two points.

  6. Count the number of points that hit the alveolar surface. This can be done manually or with automated software packages such as STEPanizer or Metamorph.

  7. Calculate the mean linear intercept (Lm) using the following formula. Lm = d × Pair/Ialv where d is the length of single test line, Pair is the number of points hitting airspaces inside alveoli and ducts, and Ialv is the number intercepts with alveolar septal walls.

  8. The total alveolar surface area, S, can be quantified using the following formula: S = 4V × p/Lm. One can use the lung volume (V) measured via water displacement (Subheading 3.7, step 10) or Cavalieri (Subheading 3.8.2) methods.

3.9. Micro-computed Tomography (microCT)

  1. MicroCT allows recurrent volumetric measurements in a sedated and ventilated mouse.

  2. Images at end-inspiration and end-expiration are used to compute total lung volumes [8].

  3. Emphysema assessment using this method should be performed in collaboration with the radiology research core at your institution as the image acquisition, processing, and analysis protocols require qualified expertise.

Acknowledgments

Funding sources are RO1HL077328 (IP) and 2014 Alpha-1 Foundation Gordon L. Snider Scholar Award (KAS).

4 Notes

1.

The AKR/J mouse strain is the most susceptible to the development of emphysema; the C57BL/6/J, A/J, DBA2/J, Balb/C, and SJ/L strains are mildly susceptible when exposed to CS [9]. The earliest time point when AKR/J mice develop airspace enlargement after CS exposure is 6–7 weeks [10]. In common inbred strains of mice, such as C57Bl/6 or DBA2/J, chronic (6 months) CS exposure causes airspace enlargement associated with various levels of inflammation, oxidative stress, increased proteolysis, parenchyma cell apoptosis, markers of cellular stress, elevated lung static compliance, weight loss, and decreased exercise endurance [11]. However, the degree of large airway involvement is relatively mild; therefore, if CS-induced alteration in bronchial glands and epithelial cell metaplasia are the main end points of your study, other rodent models should be considered (e.g., rats, guinea pig, hamsters, rabbits, or ferrets) [12, 13]. One might consider guinea pig or rat models of CS exposure to study vascular remodeling and secondary pulmonary hypertension, because the common mouse strains do not develop marked increase in mean pulmonary arterial pressure despite significant alterations and endothelial cell apoptosis within the lung microvasculature [14].

2.

Compared to the CS exposure mouse model, the elastase model is of shorter duration and less labor intensive. Following elastase instillation, the airspace destruction resembles panacinar emphysema associated with the genetic form of emphysema due to alpha-1 antitrypsin deficiency [15]. The more commonly used elastase in this model is the porcine pancreatic elastase (PPE), which is cleared from the lungs within 24 h of administration. However, the airspace enlargement continues for 10 up to 21 days. Lesion severity is dependent on the PPE dose, and the progression of emphysema is related to the degree of inflammatory response generated by PPE administration [2, 16]. Repeated PPE instillations (5 PPE administrations 1 week apart) induce more severe airspace enlargement, body weight loss, increased right ventricular mass, and dia phragmatic dysfunction, all systemic manifestation of severe human COPD. These changes are perpetuated for up to 6 months in the elastase mouse model of emphysema [17].

3.

One needs to monitor smoking chamber atmosphere for nicotine, carbon monoxide (CO), and total suspended particulates (TSP).

4.

Carbon monoxide (CO) levels in the whole-body exposure chambers (150–400 ppm) are 40–400% higher than the levels experienced by individuals exposed to secondhand CS. Active smokers are exposed to CO levels of 10–26 ppm, while secondhand CS-exposed individuals experience CO levels of 5–20 ppm [18] that lead to COHgb levels of 4.43% [19]. Furthermore, animals may reach COHgb saturations of ~10.5% [20] that are typically higher than even those of actively smoking individuals (5% on average), while secondhand CS-exposed individuals may reach 0.5–2% COHgb saturations. Monitor animals carefully to avoid CO poisoning; watch for symptoms like uncoordinated movements, lethargy, seizures, and coma.

5.

Plasma nicotine concentrations in the CS exposure mouse models range from 38.5 to 188 ng/mL. By comparison, actively smoking individuals maintain nicotine blood levels between 20 and 50 ng/mL [21], and those with moderate secondhand smoke exposure have plasma nicotine concentrations of ~6 ng/mL [22, 23].

6.

There is a synergistic effect of co-exposure to CS and viral pathogen-associated molecular patterns (PAMPs). C57BL/6J or BALB/c mice exposed to CS for 2 weeks and four doses of 15–50 μg of polyinosine-polycytidylic acid [poly(I:C)] develop airspace enlargement, enhanced parenchymal inflammation and apoptosis, and airway fibrosis [24].

7.

DFCO analysis can be done without sacrificing the mouse if the animal is maintained under continuous anesthesia, and intubation is performed under direct fiber-optic visualization using a 0.5 mm fiber-optic cable (Edmund Optics) to intubate the trachea with a 20 gauge IV cannula (BD Insyte) [25]. After DLCO measurements, the animal is extubated and monitored until recovery from anesthesia.

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