Eligibility criteria and study selection

The study question of this systematic literature review (SLR) was specified using the population, intervention, comparison, outcomes, and study (PICOS) (design framework). Articles were retrieved using a set of search terms specific to animal models of chronic obstructive pulmonary disease, and they were then further filtered to find those that met the coding criteria. Eligible articles were those that met the following criteria:

1. Type of participants: All animals with successfully established COPD models were included, regardless of species, sex, or age. Animals without COPD (such as those with other diseases or health issues) were excluded.

2. Type of design: There were no restrictions on the types of studies. However, case reports, reviews, conference abstracts, mechanism discussions, and empirical summaries were excluded.

3. Type of interventions: Studies that created animal models of COPD using the following five methods were included: smoking, smoking combined with LPS, smoking combined with bacteria, smoking combined with PM2.5, and smoking combined with protease. Other types of COPD animal modelling methods not mentioned in the inclusion criteria were excluded.

4. Type of comparators: Control or comparison conditions included animals that did not have an established COPD model, such as those living under normal conditions without any intervention. Studies on the establishment of COPD animal models using other methods were excluded.

5. Type of outcomes: The main outcome indicators were the pathological changes in the lungs of COPD animal models, as well as lung function indicators and the number of inflammatory cells produced.

6. Other criteria: There were no limitations on the year of publication, language, or publication status. However, articles with repeated publications, incomplete data, or unclear outcome indicators were excluded.

Literature screening and data extraction

All authors of this study had experience in completing systematic reviews. Each citation retrieved was reviewed by two independent reviewers against the eligibility criteria. We imported the retrieved articles into EndNote 20.2.1 (Build 15,749, Clarivate), first performing automatic rearrangement through the software and then manual rearrangement based on basic information, such as article title and author. After excluding duplicates, the two reviewers assessed the titles and abstracts according to the inclusion and exclusion criteria. The articles that could not be identified were evaluated through a full-text reading. During this process, any disagreements between the reviewers were resolved through discussion and consultation with a third reviewer.

Quality assessment

Two researchers independently assessed each study, and a consensus discussion was used to resolve any disagreements. During the study, we evaluated the quality of the experimental animals in strict accordance with the seven items listed in the Stroke Therapy Academic Industry Roundtable (STAIR) checklist. The STAIR checklist was used to evaluate the quality of the animal experiments, and its seven items served as the criteria for assessing this quality:

1. Sample size calculation.

2. Inclusion and exclusion criteria.

3. Random sequence generation.

4. Concealment of grouping protocol for laboratory animals.

5. Reasons for excluding animals from the analysis.

6. Blind evaluation of outcomes.

7. Declaration of potential conflicts of interest and research funding.

Characteristics and classification of the included studies

The publication dates of the literature included in this systematic review ranged mainly from 2009 to 2022, as shown in Figure 2. A total of eight different animals were involved in establishing animal models of COPD in the included studies. The most commonly used animal was the rat, which was used in 389 studies (85.9%), followed by the mouse, which was used in 53 studies (11.7%). The guinea pig, hamster, cat, fruit fly, rabbit, and ferret were modelled in 4 (0.9%), 3 (0.7%), 1 (0.2%), 1 (0.2%), 1 (0.2%), and 1 (0.2%) studies, respectively. Young mice and rats with a median age of 8weeks were predominantly used in the studies included in this review, although the ages of the mice and rats ranged from 3 to 54weeks and 5 to 14weeks, respectively. The animal characteristics are presented in Table 2.

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Figure 2

The number of the included studies sorted by the year of publication.

Table 2

Frequency distribution of experimental species in a model of COPD.

Species	Frequency	Percentage	Body weight	Sex	Advantage	Disadvantage
Rats	389	85.9	Mainly (200±20) g	Male (321/389)	Large body size, easy to measure lung function. The cost effective in feeding and drug usage.	Rats are resistant to the development of COPD. Rats had no goblet cells and had significantly fewer mucosal glands than large animals.
Mice	53	11.7	Mainly (20±2) g	Male (31/53)	Fast breeding. Easy manoeuvre. Cost effective.	Difficult to obtain alveolar lavage fluid due to small airway. Smoking pattern is different from human due to different anatomy from human.
Guinea pig	4	0.9	Mainly (370±2) g	Male (3/4)	Similar inflammatory responses with human. Similar histological structure with human in airway.	Most of the lung injury is axonal reflex. Relatively cost expensive. Difficult to perform airway manoeuvre.
Hamsters	3	0.7	Mainly (100±20) g	Male (3/3)	Have abundant non-ciliated bronchiole cells and goblet cells, closer to human in anatomy, physiology, genetics and biochemistry.	Difficult to manoeuvre due to more aggressive.
Rabbits	1	0.2	Mainly (1,500±200) g	Female (1/1)	Large in size and long-life span for long-term studies.	Susceptible to bacterial and viral infections during sterility tests.
Cat	1	0.2	Mainly (2000±500) g	Male (1/1)	Enable parallel study in vivo and in vitro. Pulmonary inflammation is similar to humans.	Cost expensive.
Fruit fly	1	0.2	Not described		Its respiratory system is similar to the mammalian. Early damage to airway can be detected.	Difficult in handling due to small in size.
Ferret	1	0.2	Not described		The model relates to the complex clinical features of human COPD. Could be used for long-term research.	Costs is substantially high, as well as difficult in handling.

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The modelling methods used in different studies varied, and this article incorporated the most used modelling methods up to this point. The commonly used methods for COPD animal models were smoking (n=140), smoking combined with LPS (n=275), smoking combined with protease infusion (10 articles), smoking combined with bacterial respiratory infection (n=23), and smoking combined with particulate matter (PM2.5) (n=5). The findings from the literature review showed that smoking combined with LPS was the most common method (60.7%) used to establish animal models of COPD. At the same time, the data showed that the use of cigarettes in combination with other methods required less time to establish COPD animal models and produced more pronounced pathological changes in the lungs. The pros and cons of the different methods used for establishing animal models of COPD and the frequency of their use are shown in Table 3.

Most studies were performed in experimental animals of the male sex. Among the studies performed in rats, mice, and guinea pigs, 321 studies (82.5%) used male rats, 31 studies (58.5%) used male mice, and 3 studies (75.0%) used male guinea pigs, respectively. Male animals were also chosen for the hamster and cat studies, while female animals were chosen for the rabbit study. The number of articles that used either female animals or a 50/50 split between male and female animals in their studies was very small. Of the included literature, 92.5% described changes in the lung histopathology and inflammatory factors in the animals after modelling, and 29.1% of the studies included both lung function tests and observations of lung histopathology and inflammatory factors in the animal models of COPD.

COPD animal modelling methods

A variety of methods have been used to establish animal models of COPD over the past few decades, but there is no perfect modelling method that allows animal models to accurately reproduce the pathological and physiological changes in human COPD. Each model has its own advantages and disadvantages.

The elastase model of COPD has been commonly used for research. Recently, smoking alone has increasingly been used to establish COPD animal models; however, this method also has certain limitations (64). With the further deepening of the understanding of COPD in recent years, to shorten the modelling time and, at the same time, to present a better modelling effect, a combined method is now often used to establish COPD animal models.

When the smoke exposure method is used to establish COPD models, the duration, frequency, number, and type of exposures vary, as do the lung pathological changes in the animal models (65). There are two methods of smoke exposure: whole-body smoke exposure and smoke exposure through the mouth or nasal passages only. Different smoking methods make the smoke concentration inhaled by experimental animals different, which is emphasized as a potential source of phenotypic variation in mice (65, 66). The literature shows that animal models of COPD established by nasal exposure alone produce phenotypic changes that are more similar to those seen in humans (67). Animal models of COPD established by short-term smoking (≤ 7d) can be used to characterize the disease in the early stage, but they have the disadvantage of the histopathological changes in the animal models being not stable (68–70). Therefore, animal models of COPD induced by long-term smoking (≥ 3months) are more useful for studying the pathological mechanisms of COPD in the stable phase (71, 72). This leads to the conclusion that the selection of the stimuli and/or time is based on the establishment of the animal model as acute and/or chronic.

In addition, the combined modelling method of smoking combined with LPS or PPE is more preferred by researchers. Smoking combined with LPS to establish COPD animal models is the most commonly used method (73). Long-term exposure to LPS causes alterations in the lungs of experimental mice. Inflammatory factors increase rapidly, causing typical symptoms of COPD, such as decreased lung function, emphysema, airway hyper responsiveness, inflammatory cell infiltration, excessive mucus secretion, and pulmonary fibrosis, which in turn lead to increased destruction of the extracellular matrix, thus triggering COPD (74–80). Combined stimuli provide the benefit of combined stimulation in the development of COPD animal models. The establishment of COPD animal models by smoking combined with PPE infusion is more suitable for the preliminary evaluation of alveolar injury mechanisms (81, 82). However, care must be taken to keep the dose of protease within a reasonable range for application (77, 83, 84). Such findings indicate that researchers should select different biological stimuli based on the purpose of the clinical severity, pathological damage, or pathogenesis of COPD. Depending on the purpose of the study, smoke combined with bacteria or particulate matter (PM2.5) can also be used to establish animal models of COPD (85–87). In addition, animal models of acute exacerbation of COPD (AECOPD) can be established. However, the bacteria can damage other organs in the animals, which could affect the results of subsequent studies (88). Thus, such models offer the opportunity to determine the pathogenesis of bacterial and/or pollutants-induced COPD during an acute attack, especially by allowing for the control of the timing and dosage.

Outcome measurement

The current systematic evaluation found that most researchers used male animals to establish animal models of COPD; the reason for the choice of male animals may be twofold: first, male animals do not have the potential to interfere with the experimental kinetic cycle and second, it helps avoid the effects of female estrogen secretion on the modelling (89). In 1999, the Committee on Understanding the Biology of Gender and Sex Differences, established by the Institute of Medicine (IOM) of the National Academy of Sciences, US proposed that (90) “Sex matters, because women have 4 levels of ovarian cycles, leading to increased experimental costs” (91). Therefore, it is recommended that male animals be used for initial studies of drug efficacy and that both sexes be used for more detailed studies of the characteristics and mechanisms of drug action.

Histopathology is mainly used to determine the extent of pathological changes in the lungs and provide information on the inflammatory cell infiltration and lung interval size. A simultaneous observation of the lung histopathology, lung function test, and inflammatory factor infiltration was performed in 29.1% of the studies on animal models of COPD. The use of a variety of outcome tests helps researchers look at the likelihood of changes in the outcome indicators from different perspectives. This information can also help researchers evaluate the pathophysiological mechanisms of COPD. In addition, data can be obtained at the immunopathological level (92), e.g., the accumulation and over-activation of neutrophils lead to the overproduction of mucus from the cupped cells, which in turn triggers an inflammatory response; fibroblasts secrete connective tissue growth factor, leading to emphysema; and CD8⁺ cells are associated with extracellular matrix degradation and subsequent tissue remodelling associated with emphysema (93–95). Therefore, we can detect the relevant inflammatory factors and understand the pathophysiological mechanisms behind COPD through their signaling pathways, as well as improve the current therapeutic approaches and guide the further development of future research.