**2. Technical aspects of inhalation**

involving only the colon. Its primary clinical symptom is bloody diarrhoea [2, 4–7]. As UC is an inflammatory disease, the state of the immune system is a fundamental aspect of the disorder, with an atypical T helper cell (Th)2 response, mediated by natural killer T cells that secrete interleukin (IL)-13 [1, 8, 9]. CD is a relapsing, transmural inflammatory disease that may affect the entire gastrointestinal tract. Its major clinical symptom is abdominal pain or nonspecific abdominal symptoms and bloody diarrhoea is rare. The T cell profile in CD is different from that of UC and, in fact, a Th1 cytokine profile is dominant in patients with CD [4, 7, 10, 11]. Notably, innate immune responses are similarly activated in both CD and UC [12]. Several studies suggested that IBD pathologies result from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host, with consequent altera-

During IBD development, the paracellular space in the intestinal epithelium becomes more permeable, impacting defensive strategies naturally activated by specialized epithelial cells, including goblet and Paneth cells [13–16]. This process primes a positive feedback loop, with increased exposure to the intestinal microbiota, leading to amplification of the inflammatory response. Observations in patients or animal models show that host-microbiome interactions and microbiome fluctuations play prominent roles in such inflammatory processes [17, 18]. However, whether these alterations contribute to the disease, or simply reflect secondary

Indeed, the basic aetiology of IBD is still unclear and the potential factors contributing to the pathogenesis of the disease, such as dysbiosis, epithelial and/or immune system dysfunctions and oxidative stress, represent the major research topics in the IBD field. Moreover, new area of interest arose from the necessity of understanding the potential environmental causes

Among the environmental factors associated with IBDs, the most significant causes are cigarette smoke (CS) and nicotine, and these inversely affect the risk and course of UC and CD. The relationship between smoking and IBD has been known for many years, with the first report of a negative correlation between IBD and smoking, in a cohort of UC patients, published 40 years ago [19]. Since then, there have been numerous epidemiological, clinical and pre-clinical studies describing the dual effects of active smoking in the two forms of IBD [20, 21]. CS is associated with a higher risk for developing CD and a worse outcome in CD patients. In contrast, UC is considered a non-smokers' disease, with a significantly lower risk of disease development in current smokers. Despite the considerable research on smoking and IBD, the molecular mechanisms for CS-induced impacts on IBD development, as well as

To better understand the different aetiological factors in the onset of IBD, a variety of disease models were developed. Human and *in vitro* studies have historical limitations because of design complexity, duration and cost or, for *in vitro* studies, the lack of translational applicability. Therefore, animal models are advantageous by allowing *in vivo* experiments to be conducted under more easily controlled conditions than those in human studies, while providing the organism complexity lacking in *in vitro* systems. Increased knowledge of mucosal immunity and host-microbiome interactions and dynamic, as well as the availability of new

tion of the intestinal epithelium.

behind the disease onset.

changes caused by the inflammation, is still under debate.

182 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

the specific CS components responsible, are not well understood [22].

#### **2.1. Methods of acute and chronic pulmonary delivery of aerosols to rodents**

The technical means for pulmonary delivery of aerosols (either small molecules, proteins or mixtures) may employ either direct intratracheal administration or, alternatively, inhalation exposure, the latter often requiring restraint of animals.

For acute pulmonary delivery of an agent, intratracheal administration may be ideal. Its main advantages are that it requires little infrastructure or equipment and can be performed in a basic *in vivo* lab environment [25]. In addition, dose delivery can be accurately and reproducibly estimated [26]. However, this method also has several shortcomings, such as need for anaesthesia, inability to administer volatile agents or gases and unequal distribution in the lungs, resulting in minimal exposure to the alveoli. Overall, such concerns make intratracheal administration a less suitable method for subchronic or chronic pulmonary delivery.

For subchronic or chronic administration of aerosols to rodents, repeated inhalation exposure systems are preferred. Thus, animals are exposed to aerosols within a confined environment for a fixed daily duration. In the field of toxicology, testing guidelines for repeated dose exposure for toxicological assessments, such as the OECD TG413 guideline, recommend up to 6 h per day exposure for a 90 day exposure period. However, for therapeutic or disease modelling purposes, the exposure period must be determined empirically, based on the effective dose and the time needed for the target biological effect to occur. Importantly, exposure systems must enable consistent delivery of aerosols, at concentrations that are stable during the exposure period, and with appropriate aerosol properties to enable efficient inhalation and uptake [27].

Principally, two types of exposure chambers are routinely used to administer aerosols to rodents, whole body or nose-only exposure chambers, each with its own advantages and disadvantages [27]. Whole body exposure systems are restraint free, as the animals are placed into an exposure chamber, either in a cage or on a mesh or grid surface, depending on the specific system. Both chambers are technically simple, assuming sufficient infrastructure (aerosol generation and functional chambers). Both also enable exposure of large numbers of animals, for example, chambers of >700 L may each accommodate approximately 200 mice. The freedom of movement of animals during exposure results in minimal stress, although the animals require training to adjust to grid-caging systems and food is typically withdrawn to minimise oral uptake of aerosol constituents. One criticism of whole body exposures is that there is a high potential for compound uptake through non-inhalation routes because animals have surface contact with aerosol deposits on the cage surfaces and on their fur. In historical studies, up to 60% of aerosol constituents on the fur (pelt burden) were ingested following whole body exposures [28] and transdermal uptake may also be significant for some compounds. Because the skin is an effective barrier for drug transport, only potent drugs with appropriate physicochemical properties (low molecular weight and adequate solubility in aqueous and non-aqueous solvents) are suitable candidates for transdermal delivery [29–31]. Such mixed uptake mechanisms potentially occurring in whole body exposure systems complicate both dose estimations and require deconvolution of uptake amounts through oral/transdermal and inhaled routes.

Nose-only exposure chambers require restraint of the animals to permit only the head (nose) to be exposed to the test aerosol. This has the major advantage of decreasing deposition of aerosol constituents on the pelts, resulting in less oral uptake from grooming behaviour [32]. However, there are also disadvantages with this system, including technical asphyxiation (animal movements in the exposure tube may cut off their air supply); therefore, constant monitoring during the exposure period is required. In addition, because of stress associated with restraint in nose-only exposure systems, training is required to adapt animals to the technical procedures. Vehicle or fresh air exposures are also needed to help distinguish such stress-related effects from treatment effects [33]. The daily execution of nose-only exposures requires that animals be individually inserted into the exposure tubes, a technical aspect that may limit the numbers of animals that can be used in the experiments.

#### **2.2. Dose translatability**

Measurement of dosages in an *in vivo* inhalation experiment is dependent upon many parameters, including deposition of the agent to the lungs (which itself is dependent upon aerosol droplet size), respiratory minute volume and body weight of the animal. This relationship is generally described by the following formula [34]:

DD <sup>=</sup> <sup>C</sup> <sup>×</sup> <sup>R</sup>M<sup>V</sup> <sup>×</sup> <sup>D</sup> <sup>×</sup> IF \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Body weight (kg) (1)

performed when nose-only exposure tubes are used (head-out plethysmography measurements), is preferable as it would enable the researcher to control any effects of test item on the

Animal Inhalation Models to Investigate Modulation of Inflammatory Bowel Diseases

http://dx.doi.org/10.5772/intechopen.69569

Important for *in vivo* disease modelling is the translation of the animal models to human therapeutics or treatment regimen. This will require an estimation of human equivalent dose (HED), based on the animal data. The most commonly used method to convert to HED is with a body surface area conversion factor [37]. Alternatively, a mg/kg conversion factor may be applied, though this typically will result in a lower safety margin and higher HED values, compared with the body surface area conversion. HED is generally described by the follow-

where *K*m is the correction factor reflecting the relationship between body weight and body

The various types of animal models developed to study IBD may be divided into several categories depending on: the method of inducing the pathology (*chemically induced, bacteriainduced or genetically engineered*); the IBD subtype modelled in the animal (*UC or CD*); the site of inflammation (*colon, ileum, both sites or systemic*); and, in genetically engineered models, the gene modification strategy (*conventional transgenic* (*Tg*) *or knockout* (*KO*), *cell-specific conditional Tg or KO*, *inducible KO, knock-in, innate, mutagen-induced or spontaneous models*) [23, 38, 39]. The total number of IBD mouse models is growing, especially because of current genetic engineering approaches that accelerate development of new strains, so far, over 74 genetically engineered mouse models were reported to spontaneously develop intestinal inflammation [38]. The full description of all IBD models is beyond the scope of this chapter. However, **Table 1** summarises the most significant IBD murine models, highlighting their methods of pathology induction, IBD subtypes, sites of inflammation and mechanism of action (**Figure 1**). More detailed reviews of the different mouse models of IBD are available (e.g. see Refs. [23, 40, 41]). There is a close agreement in many pathological findings among experimental IBD models and human disease. These include the molecular pathways and histological features of tissue injury, dysfunction of the immune system (including impact of the microbiome), genetic heterogeneity and primary defects in mucosal barrier function. All pathologies have been well established in several experimental models of colitis; therefore, these models closely resemble aspects of the human diseases. These common features enable exploration of specific pathological mechanisms, facilitating development of new therapeutic approaches. However, none of these models fully reflects human IBD, with each representing rather a small tile of a mosaic. This hinders a generalised view of the systemic consequences of IBD, often masking

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ human *<sup>K</sup>*<sup>m</sup>

(2)

185

minute volume, when calculating the estimated dosage.

<sup>H</sup>ED <sup>=</sup> animal dose (mg/kg) <sup>×</sup> animal *<sup>K</sup>*<sup>m</sup>

**3. Overview of animal IBD models**

possible extra-intestinal implications [42].

surface area (e.g. human *K*<sup>m</sup> = 37; mouse *K*<sup>m</sup> = 3; rat *K*<sup>m</sup> = 6 and dog *K*<sup>m</sup> = 20).

ing formula [37]:

where DD is the delivered dose (mg/kg); C is the concentration of substance (mg/L); RMV is the respiratory minute volume (L/min) and IF is the inhalable fraction.

Among these parameters, the respiratory minute volume is important to determine the availability of compound for deposition and exchange in the lungs. This parameter may be calculated using allometric formulae relating body weights to minute volumes in laboratory animals [35, 36]. The alternative, direct measurement of the minute volume, as can be performed when nose-only exposure tubes are used (head-out plethysmography measurements), is preferable as it would enable the researcher to control any effects of test item on the minute volume, when calculating the estimated dosage.

Important for *in vivo* disease modelling is the translation of the animal models to human therapeutics or treatment regimen. This will require an estimation of human equivalent dose (HED), based on the animal data. The most commonly used method to convert to HED is with a body surface area conversion factor [37]. Alternatively, a mg/kg conversion factor may be applied, though this typically will result in a lower safety margin and higher HED values, compared with the body surface area conversion. HED is generally described by the following formula [37]:

ing formula [37]:

$$\text{HED} = \frac{\text{annual dose (mg/kg)} \times \text{axial } K\_m}{\text{human } K\_m} \tag{2}$$

where *K*m is the correction factor reflecting the relationship between body weight and body surface area (e.g. human *K*<sup>m</sup> = 37; mouse *K*<sup>m</sup> = 3; rat *K*<sup>m</sup> = 6 and dog *K*<sup>m</sup> = 20).
