While we know that cigarette smoke exposure will induce pathologic effects in the lungs of smokers, the mechanisms by which cigarette smoke mediates these changes remain incompletely understood. Studies in humans frequently utilize biological materials from patients who are already in the advanced stages of the disease. Although helpful, these studies may not provide insights into the processes by which cigarette smoke promotes the development of the disease state in the lung. Performing prospective studies in humans could address these limitations; however, such studies would be time-consuming and expensive as COPD is an insidious disease that evolves over many years.50 Over the long term, it would be difficult to perform these analyses in a rigorous scientific manner that would limit the confounding effects of other dietary, genetic and environmental variables. Moreover, obtaining biological specimens over this length of time would be costly and require years to answer scientific questions. For this reason, animal models of COPD are needed to better understand the underlying disease mechanisms. After Laurell and Erickson's observation that A1AT (alpha-1-anti-trypsin) deficiency resulted in the premature development of emphysema,51,52 the role of elastase in this disease became center focus. A1AT protein is an abundant circulating anti-protease that binds and neutralizes neutrophil elastase within the lung.53 Thus, it was postulated that the unopposed action of elastase would degrade elastin, which is abundant in the alveolar wall, thereby causing the destruction of lung tissue.54 This theory was further supported by the finding that intratracheal elastase induced the development of emphysema in rats.55 This was one of the first animal models of the disease and it had a tremendous influence on the research direction of the pulmonary field for the subsequent 30 years. After this discovery, emphysema was primarily regarded as a disease of elastin degradation that resulted from an elastase/anti-elastase imbalance in the lung. Although this model provided important insights into the disease, it had numerous shortcomings that limited its applicability to the human disease. For one, emphysema in this animal model develops within 3 weeks of exposure.56 This rapid onset of development does not mimic the human disease, which often takes decades to develop. Thus, it is likely that distinct biological processes are responsible for the pathologic changes that are required for disease initiation and progression to occur. Secondly, by its design, the elastase animal model cannot address the impact of cigarette smoke exposure on the lung. Although cigarette smoke induces expression, release and activation of lung elastases,57,58 it also triggers a myriad of other effects that will not be replicated by an elastase disease model. Thirdly, the elastase model reproduces the alveolar destruction that occurs in emphysema but it does not replicate other disease features such as mucus plugging, bronchiolitis and altered lung inflammation.59 Indeed, the hallmark features of human COPD such as chronic lung inflammation, impaired lung function, emphysema, mucus hypersecretion, vascular injury, and small airway remodeling are not well represented in this model. Thus, researchers have sought to utilize smoke exposure models to gain more relevant insights into the disease.
Developing a smoke exposure model that would generate emphysematous changes was a laborious process. The early attempts to develop smoke exposure models were complicated by the long exposure time required to develop the disease and the variable effects of cigarette smoke in the exposed animals. Indeed, some of the first studies reported cellular proliferation and mucus metaplasia but not the classical alveolar destruction seen in the human disease.60,61,62 Hautamaki et al were the first group to successfully overcome these challenges in a mouse exposure model.63 In their model, they used a pump to circulate the cigarette smoke generated by the burning of 2 cigarettes to a Plexiglas chamber containing the mice. Carbon monoxide (CO) measurements showed that the mice had CO levels that were comparable to human smokers. By exposing mice in this manner daily for several months they successfully generated alveolar destruction within the lungs of the exposed mice. Moreover, they demonstrated that this destruction was dependent on the expression of an elastase, matrix metalloproteinase-12 (MMP-12). Establishing an emphysema model that successfully generated alveolar destruction was one of the most important accomplishments in the pulmonary field in the past 2 decades. Today, investigators are actively using variations of this model to obtain important new insights into the mechanisms of this disease.
The 2 most common smoke exposure models that have been utilized are the nose only and the whole body exposure apparatus.
The nose only system requires restraining the mouse so that their nose is inserted into a cone where they inhale the cigarette smoke.64 This generates a uniform exposure that produces emphysematous changes. However, the prolonged periods of restraint are stressful for the mice and the machine can usually accommodate only a limited number of mice (eg, the Jaeger system has 18 ports which is depicted in Figure 57A–1A). In contrast, whole body exposure systems expose mice to a mixture of both passive and mainstream smoke released from a burning cigarette and mainstream smoke, which is actually smoke aspirated through the cigarette using a pump.65 The passive and mainstream smoke streams are mixed and then propelled by a fan to a chamber containing the mice that are housed within their cages. The advantage of this system is that the mice freely move about and have access to food and water (Figure 57A–1B). Thus, mice in this system can be exposed for longer periods of time. In addition, the whole body exposure system allows for the exposure of large groups of mice. Some systems allow the exposure of up to 120 mice at a time enabling researchers to use large numbers of mice and to perform multiple experiments simultaneously. An important note about whole body exposure systems is that the exposure intensity needs to be monitored very carefully. Cigarette smoke is removed from cages via exhaust valves that are typically maintained with a small aperture in order to allow smoke levels to build up within the chambers. Cigarette smoke releases a large amount of tar that can clog these valves and minimize the flow of cigarette smoke, which can allow for smoke levels to build up to toxic levels. If the tar obstruction of the valve becomes too great, all smoke/air flow stops and the mice receive no smoke exposure. Both of these outcomes can ruin a carefully planned experiment. Thus, the mice have to be closely monitored to ensure that airflow is circulating properly and that toxic levels of cigarette smoke are not building up.
Typical mouse models for smoke exposure. (A) A nose only cigarette smoke exposure system requires animals to be restrained while they inhale the cigarette smoke. (B) The passive and mainstream smoke streams cigarette smoke exposure system allows animals access to food and water without restraint.
This is accomplished by measuring total particulate matter (TPM) concentration within the chamber and carbon monoxide levels in the mice. Typically, the TPM is maintained at 80 to 100 mg/m3, which will produce CO levels in the mice of 10% to 12%.65 These levels are well tolerated by the mice and produce the alveolar lung tissue destruction.63,65 TPM can be monitored using a filter sample unit fitted with a diaphragm pump and a timer. Utilizing sampling air to measure particulate matter in a specific volume sampled over time allows accurate TPM measurements and consistent exposure to animals.
Although many animals have been used in COPD studies, mice offer very clear advantages, which have lead to their becoming the dominant animal model for this disease. For one, mice are small in size and the costs of feeding and housing these animals are far less than dogs, sheep, and large rodents. Also, the mouse genome has been extensively characterized and there is a plethora of antibody, molecular probes and equipment modified for mouse anatomy available for studies in these animals. Likewise, it is far more cost-effective to generate genetically manipulated mice than it is to do so in other species. The gestational period for a mouse is 21 days compared to a time period of up to 74 days in guinea pigs. This allows investigators to generate large numbers of genetically altered mice in a relatively short period of time. Exposing mice to smoke for 1 year represents approximately 50% of the animal's lifetime, thereby allowing a better representation of lifetime smoke exposure. These cost advantages allow for large numbers of animals to be utilized for smoke exposure studies. This is extremely important when studying this disease, as the effect of cigarette smoke on the lung is highly variable. In humans, some estimates state that only 15% of smokers will develop emphysematous changes in the lung66 and the susceptibility of mice to cigarette smoke is also quite variable.65,67 Thus, exposing large numbers of mice increases the power of a study to detect significant differences in emphysema between control and genetically altered mice. The typical enlargement of airways observed after 6 months of smoke exposure is depicted in Figure 57A–2.
Histologic comparison of mouse lung after smoke exposure. H&E-stained lung sections from age-matched room air littermates (C57BL/6J strain) and mice exposed to cigarette smoke for 6 months. Images are at 10× magnification.
Mice provide the best opportunity to investigate the mechanisms of this disease. However, as noted previously, not all strains of mice are equally responsive to cigarette smoke.65,67 Some strains of mice, such as C57Bl/6J, are relatively resistant to cigarette smoke and develop increases in their mean linear intercept (MLI), a parameter of airspace enlargement, which range from 15% to 20% after 6 to 12 months of exposure.67 Because of this, smoke exposure studies using C57Bl/6J mice require large numbers of mice to be exposed for long periods of time. This greatly increases the time and expense needed to carry out these exposure studies. In addition, the C57Bl/6J mice do not develop increased lung compliance following chronic cigarette smoke exposure.65 Numerous mouse strains have been utilized for smoke exposure studies. However, A/J mice are the most sensitive to cigarette smoke and develop increases in their MLI in the range of 20% to 30% after only 2 months of exposure,65 as depicted in Figure 57A–3.
Demonstrating the typical MLI, observed in age-matched C57Bl/6J and A/J mice after 6 months of cigarette smoke exposure or their room air controls.
Because of this, the A/J mouse is becoming the preferred strain for smoke exposure studies in mice.68 Unfortunately, many genetic mouse models are developed in a C57Bl/6J background.
Crossing these mice into a more sensitive A/J background requires several generations of backcrossing. This is a costly and time-consuming endeavor. If the primary endpoint of one's research is emphysema, it may be worthwhile to cross into an A/J background. These mice need less smoke exposure time and develop greater emphysema potentially making it easier to detect changes between groups. On the other hand, if one's focus is on inflammation, apoptosis, and protease expression then there is no evidence that A/J mice are superior to C57Bl/6J for examining these parameters.