Bartlett, Laura Grace
(2024)
Defining the pathophysiological impact of cigarette smoke extract in mice as a model for Chronic Obstructive Pulmonary Disease (COPD).
PhD thesis, University of Nottingham.
Abstract
INTRODUCTION:
Chronic obstructive pulmonary disease (COPD) is a progressive, long-term group of respiratory diseases that encompasses chronic bronchitis and emphysema, and is typically caused by chronic cigarette smoke (CS) exposure. Although it is currently listed as the third leading cause of mortality worldwide by the WHO, current understanding of COPD pathophysiology is limited and as such the disease is frequently underdiagnosed or misdiagnosed. One of the hallmark characteristics of COPD is chronic and persistent inflammation, and much of our current understanding of the immunopathogenic mechanisms of COPD comes from the use of whole-body or nose-or-head only CS-exposure mouse models. Despite the existence of such models, these are not permitted for use under UK laws protecting the use of animals in science, as the model systems are considered to place mice under significant unnecessary stress and harm, and phenotypic outcomes do not accurately mimic human disease. Therefore, this project aims to develop a novel CS-exposure murine model of COPD that encompasses the three Rs of animal research (replacement, refinement and reduction) to improve model design in regards to both animal welfare and applicability to human disease.
METHODS:
Prior to model development, a 24-colour spectral flow cytometry was developed and optimised for the characterisation of major innate and adaptive immune cells. In addition to this, a reverse phase protein microarray (RPPA) panel to assess proteomics of the lungs was tested for cross-reactivity to mouse samples, though after no cross-reactivity was detected an RT-qPCR panel was developed to assess changes in gene expression in tissues. Mice were exposed intranasally to an aqueous cigarette smoke extract (CSE) to minimise stress of restraint and handling, and provide an exposure methodology more reflective of human exposure methods. Once per week, microsampling of tail vein blood was obtained to assess impact of CSE exposure on peripheral immune cells via flow cytometry. Following 12 weeks of CSE exposure, mice were culled and tissues were isolated for RT-qPCR and histological analysis.
RESULTS:
Due to low cell numbers in peripheral blood as a result of microsampling restrictions, no differences were detected in peripheral immune cell profiles between control groups and two disease groups (light-smoker and heavy-smoker). Despite this, both conventional gating strategies and unbiased clustering analysis displayed comparable cell profiles, demonstrating the effectiveness of the comprehensive flow panel. Although no differences in systemic immunopathophysiology were observed, significant differences in gene expression, neutrophilia and airway remodelling were observed in the lungs, dependent on either CSE dose or sex. In particular, whilst most inflammatory and immune signalling pathway genes were upregulated in males, these were downregulated in females. Additionally, pulmonary neutrophilia and alveolar diameter were significantly increased in both male and female heavy-smoker mice in comparison to control groups, and these findings were more pronounced in females in comparison to males.
CONCLUSIONS:
Overall, we have developed the foundations of a novel murine model of COPD that better encompasses the 3Rs and mimics the pulmonary manifestations of COPD. Whilst no differences in systemic immune cell profiles were observed, marked alterations to lung structure were observed akin to results reported in both humans and mice with COPD, and these appeared to be exacerbated in females. In order to fully assess systemic inflammation, blood samples could be taken less frequently in order to increase blood volume permitted to be withdrawn, thus increasing the number of cells available for analysis and allowing differences to be better visualised. This model system provides a toolkit for researchers in the UK to further our current understanding of COPD immunopathophysiology, and may potentially be used in the future to assess pathophysiological differences dependent on other causative factors, such as environmental and household air pollutants and e-cigarette smoke.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Fairclough, Lucy
Watkins, Adam
Tighe, Paddy |
| Keywords: |
Chronic obstructive pulmonary disease; Pathophysiology; Animal models; Cigarette smoke exposure; Immunopathophysiology |
| Subjects: |
R Medicine > RC Internal medicine |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
78375 |
| Depositing User: |
Bartlett, Laura Grace
|
| Date Deposited: |
11 Dec 2024 04:40 |
| Last Modified: |
11 Dec 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/78375 |
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