Influencia del factor de riesgo individual en el nivel de reincidencia

Animals were exposed to either aerosolised 1 mg/ml LPS in saline, or control (sterile saline) for 30 minutes. Wild-type and knock-out mice were challenged concurrently, to ensure identical exposure conditions. Mice were returned to their IVCs after exposure, with water and food available ad libitum. For further details of the LPS exposure system, please refer to section 2.3.2.

6 hours after the challenge, the animals were terminally anaesthetised by inter-peritoneal injection of sodium pentobarbitone (200 mg/kg). BALF and lung tissue were subsequently collected as described in sections 2.4.1 and 2.4.2 and processed to establish total and differential cell counts. Cytokines were also measured in the BALF as detailed in section 2.4.4. Collection and processing of samples was performed in an identical fashion to the acute smoke model.

5.2.2.1 Characterisation of the LPS Exposure Model

The LPS exposure model was previously characterised by Respiratory Pharmacology, and is published in Eltom et al. 2011. As with the acute smoke exposure model, a sub-maximal dose of 1 mg/ml LPS in saline was established after a dose-response, by measuring BALF neutrophilia. A time-course study was subsequently performed to identify the relevant time-points for key inflammatory mediators, especially cellular recruitment in the BALF and

128 lung tissue, as well as cytokine release. LPS induced inflammation was found to share many phenotypic similarities with 3-day smoke induced inflammation in mice, including neutrophilia, KC release and IL-1β release (Figure 5.1).

There are however a number of differences as well, notably that neutrophil levels are far higher after LPS than they are after smoke, and these higher levels also peak at 24 hours after exposure rather than 48 hours. It is also worth noting that while in the acute smoke model neutrophils were the only cell type significantly increased in the BALF, increased levels of eosinophils (peaking at 2 hours), macrophages and lymphocytes (24 – 96 hours) are observed after LPS exposure. The increases in these cell-types after LPS exposure can also be measured in the lung tissue (Figure 5.2), whereas changes are limited to BALF after the acute smoke model.

The cytokine levels similarly peak at earlier time-points after LPS exposure compared to after acute smoke exposure (Figures 5.1 and 4.1). Levels of KC and IL-1β both peak at or before 2 hours (the earliest time point measured), compared to 24 hours in the acute smoke model. A number of other cytokines are also significantly increased after LPS, including IL-6, TNF-α and IL-18.

From these data the 6 hour time-point was chosen to examine the role of TRP channels, as this is a sub-maximal time-point for neutrophilia, IL-1β and KC levels, which were the main end-points measured in the acute smoke model.

5.2.3 Statistical analysis

Data are expressed as mean ± S.E.M in all graphs. All experiments were performed with an n of at least 6 (please refer to individual figure legends). Statistical significance of differences between KO and wild-type groups was determined by a Mann-Whitney U test for non-parametric data. P values < 0.05 were taken as significant, and are denoted with a * on each graph.

129

Figure 5.1 Temporal characterisation of inflammation in BALF after LPS exposure model. Wild-type (C57BL/6) mice were exposed to LPS (grey bars), or Vehicle (Saline, white bars), for 30 minutes. BALF samples were collected at 2, 6, 24, 48, 72, 96 and 168 hours after the final exposure for analysis of inflammatory markers, including neutrophilia and cytokine release. Data are represented as mean ± SEM for n=6 animals in each group. Statistical significance was determined using Mann-Whitney U test. * = P<0.05, denoting a significant difference between the smoke exposed group and the time-matched air exposed control.

130 Neutrophilia in lung tissue

2 2 6 6 24 24 48 48 72 72 96 96 168 168

0 5000 10000 15000 20000

Vehicle LPS

Time after challenge (h)

*

*

*

* Neutrophils (103 /ml)

Figure 5.2 Temporal characterisation of inflammation in tissue after LPS exposure model. Wild-type (C57BL/6) mice were exposed to LPS (grey bars), or Vehicle (Saline, white bars), for 30 minutes. BALF samples were collected at 2, 6, 24, 48, 72, 96 and 168 hours after the final exposure for analysis of neutrophilia. Data are represented as mean ± SEM for n=6 animals in each group. * = P<0.05, denoting a significant difference between the smoke exposed group and the time-matched air exposed control.

131 5.3 Results

Knock-out and wild-type control animals were exposed to LPS concurrently, before being returned to their own cages. Six hours later the animals were terminally anaesthetised before collection of tail-tips for genotyping, then BALF and lung tissue for analysis of inflammation. The main end-points measured in this model were neutrophil recruitment in the BALF, as well as cytokines including IL-1β and KC. These end-points are common to both the acute smoke exposure model and inflammatory markers measured in COPD patients.

In all of the following experiments, LPS exposed wild-type mice exhibited significantly increased levels of neutrophilia and cytokine release in BALF when examined at the 6 hour time-point. Cellular recruitment was also measured in the lung tissue for the TRPA1, TRPM2 and TRPV1 knock-out mice in conjunction with the relevant wild-types. All cellular data from BALF and lung tissue is presented in tables in the appendix. Below the BALF neutrophil and cytokine data is presented for each of the channels, as these are the end-points which are in common with the acute CS model, and for which the 6 hour time-point was chosen. Lung tissue cellular infiltration data is also presented for the TRPM2 experiment, as this was the only channel where a significant effect was observed.

As in the previous chapter, all strains of knock-out mice were viable, fertile and largely indistinguishable from their wild-type controls. Tail-tips were collected from all animals for DNA extraction and genotyping, to confirm their genetic integrity.