Resultados en el modo oscilatorio con amplitud de deformación

The data provided by a clinical sample demonstrating a specific result or particular association with a disease or its progression is considered crucial for validating a particular target for drug discovery purposes. However, in order to obtain powerful evidence that highlights a specific cause-effect relationship between a target and disease-like phenotype animal models have historically proven to be the best tool. For decades animal have played an instrumental role in broadening our understanding of disease mechanisms and pathogenesis. Various approaches have been attempted in order to replicate the phenotype of COPD in animal models, including exposure of animals to CS, inflammatory stimuli (e.g.

62 LPS) or instilling proteolytic enzymes into the airways and studying the effects of specific KO animals (Mahadeva & Shapiro, 2002; Groneburg & Chung, 2004; Stevenson & Birrell, 2010). However, as the studies in this thesis will be utilising CS, the primary etiological factor for COPD, background will be focused around animal models of CS exposure.

Furthermore, the inflammation seen in this model has also been shown to be insensitive to glucocorticoid inhibition both rats and mice (Marwick et al., 2004; Wan et al., 2010), providing the most accurate model of disease phenotype in animals. The second model used in this thesis is driven by lipopolysaccharide (LPS), a model previously shown to be sensitive to glucocorticoid inhibition (Birrell et al., 2005), that will facilitate paralleling experiments using a stimulus of the normal innate defence system that induces airway neutrophilia.

1.5.2.1 Models of cigarette smoke exposure

A wide range of CS exposure models are commercially available, whilst some groups also create or adapt their own. Although models vary predominantly around the way animals are exposed to CS, either nose-only or whole body, other variations include the concentration of smoke, duration and frequency of the exposures. The length of the CS exposure protocol is typically believed to replicate different aspects of the disease; acute (3 day) exposures typically produce a neutrophilic inflammatory response (Stevenson et al., 2005), whereas chronic (>6 month) exposure are required to bring about emphysematous changes in the lung (Churg et al., 2004).

The concentration of CS used to induce inflammation in the lungs of laboratory animals varies from model to model in the literature. Although most report a value using the TSP (total suspended particulate) standard, these figures vary significantly based on the type of

63 cigarette used and the amount of CS being drawn/pumped into the chamber at any given time.

The duration and frequency of exposures is also variable, however, typically a 1 hour exposure twice daily seems to be the standard used by most research groups.

1.5.2.2 Choice of animal species

Various species have been used to investigate CS-induced inflammation, including guinea pigs, rats and mice (Churg et al., 2008). It has been demonstrated that chronic CS exposure in guinea pigs led to progressive emphysematous changes that were associated with changes in lung function consistent to those observed in human emphysema patients (Wright & Churg, 1990). Whilst there are many advantages to working with guinea pigs, the disadvantages working with this species outweigh using others. Guinea pigs are expensive, and there is a serious lack of tools and antibodies commercially available that cross react with guinea pig proteins. Therefore most laboratories use rodents in order to develop CS-driven models of inflammation.

Most of the complex features of COPD have been replicated in mice, rats and guinea pigs including chronic inflammation, emphysema and small airway remodelling (Churg et al., 2004; Wright et al., 2007). However, other aspects of the disease such as mucus hypersecretion have been difficult to reproduce, although they have been reported in rats (Zheng et al., 2009). The literature affirms that most models of CS exposure have preferentially selected mice over rats as mouse models offer many advantages. These include, cost effectiveness, extensive gene and protein sequences and a wide range of biological tools/antibodies available. More importantly, the ability to produce mice with specific gene

64 modifications provides major insight into the underlying mechanisms driving the pathogenesis of COPD (Wright et al., 2008).

1.5.2.3 Limitations of CS models

One of the limitations of the CS exposure as an in vivo model is that there is currently no standardised method or protocol by which animals are exposed. There are many variations in how different groups expose animals, these include differences in strains or species used, different cigarettes used to generate smoke (commercial vs. research cigarettes), differences in the component of the smoke animals are exposed to (mainstream vs. sidestream), different delivery systems (whole body vs. nose-only) and most significantly the dose of smoke that is delivered to the animals. Variations in the dose of smoke delivered to the animals can be attributed to many design or mechanical factors in the various cigarette smoke exposure apparatus, however, the puff profile of each system may also vary. Many groups however, attempt to replicate the FTC (Federal Trade Commission) puff profile which suggests that smokers draw smoke for 2 seconds. Variations in the length of time air is drawn through a cigarette can affect the composition of the smoke generated as the temperature of the tip may vary. These differences make comparisons between findings of different research groups extremely difficult, although many groups have used different systems and reported similar findings (Vlahos et al., 2006; Morris et al., 2008). The protocol and dosing regimen developed here is similar to that of Morris et al., and produced similar results with 3 days of acute cigarette smoke exposure in C57BL/6 mice causing an increase in neutrophils peaking at 24 hours after challenge, followed later by macrophages (Morris et al, 2008).

65 Another limitation observed with CS models of inflammation is that the disease phenotype produced is typically mild, corresponding to GOLD I or II of human COPD (Hogg et al., 2004). Furthermore, most in vivo studies have examined a single aspect of COPD, such as inflammation, emphysema or mucus hypersecretion, but not the entire disease with all its features. This has the effect of making it difficult to model AECOPD as there is a lack of a comprehensive model of COPD. To address this problem, studies have begun to focus on factors that are associated with both COPD and AECOPD, and incorporate them in combination into experimental models (Gaschler et al., 2009; Kang et al., 2009)

Approximately 25% of smokers develop COPD, whilst 80% of COPD patients are smokers.

Therefore there is a portion of COPD patients who get COPD independently of smoking.

This model of COPD does not take into account patients who have a genetic predisposition to COPD development, or suffer from the disease as a result of alternative causative agents such as pollution. As models of CS exposure aim to replicate a complex disease that manifests itself over a long period of time, it is imperative to carefully interpret the data in order to provide insight into the mechanisms behind a very complex disease.