Introduction
Ventilation (V) and perfusion (Q) are fundamental physiological processes within the lung contributing to gas exchange, and the relationship between these processes is dysfunctional in patients with chronic obstructive pulmonary disease (COPD) [
1]. Cigarette smoke (CS) is a primary risk factor for the disease, prolonged exposure to which can lead to airway inflammation, airspace enlargement, and several other pathologies [
2], ultimately resulting in irreversible airflow limitation [
3,
4]. Cessation of cigarette smoking is capable of slowing the progression of COPD but years of cessation are often necessary before improvements to airflow limitation, inflammatory state, infection risk, and cardiovascular comorbidities are seen [
5,
6]. To better understand the benefits and limitations of smoking cessation, the impact on V/Q requires investigation in the context of the pathologies associated with cigarette smoke exposure.
V/Q relationships in the lung can be measured by several non-invasive methods but these techniques are not commonly used in clinical practice, aside from the diagnosis of pulmonary embolism; quantification of results, even in clinical research, is rare. Rodríguez-Roisin
et al.[
7] made use of the multiple inert gas elimination technique (MIGET) to quantitatively demonstrate that V/Q is a sensitive measure of the earliest stages of COPD. A more widely available methodology for V/Q can be performed in nuclear medicine departments utilising single photon emission computed tomography (SPECT) to provide three-dimensional maps of ventilation and perfusion [
8,
9]. Jogi
et al.[
10] have shown the ability of this technique to identify early disease and stage disease severity in COPD patients. Further, Suga
et al.[
11] have quantified the impact of emphysema on V/Q in the lungs of COPD patients.
Modelling aspects of COPD in mice provides the means by which to investigate the individual pathologies that make up this heterogeneous disease [
12]. Using a methodology adapted from clinical V/Q SPECT to a murine model, our laboratory has confirmed the utility of this technique in measuring changes in V/Q with age [
13] and in the context of prolonged cigarette smoke exposure [
14].
Understanding the mechanisms behind V/Q mismatch in COPD-associated pathologies is an important step in furthering the ability to diagnose and treat this widespread and burdensome disease. In the current study, the impact of smoking cessation on V/Q mismatching has been investigated. In addition, the contributions of inflammation and airspace enlargement have been examined, by employing simple models for these pathologies, to provide insight into the dysfunction associated with cigarette smoke exposure. We have sought to explore the V/Q relationships associated with cigarette smoke exposure using these models alongside cellular and structural assessments, using both traditional and non-invasive methods.
Materials and methods
Animals
Specific pathogen-free 10–12 week old female BALB/c mice were purchased from Charles River Laboratories (Senneville, QC, Canada). The studies were approved by McMaster University’s Animal Research Ethics Board in accordance with the Canadian Council on Animal Care guidelines.
Cigarette smoke exposure protocol
Mice were exposed to cigarette smoke 5 days/week using a SIU48 whole body exposure system (Promech Lab, Vintrie, Sweden). Details of the exposure protocol have been reported previously [
15]. Control animals were exposed to room air only. Following 24 weeks of smoke exposure, mice were divided into two groups, continued smoke exposure and smoke cessation, and studied for 16 weeks. Controls continued to receive room air.
Lipopolysaccharide exposure protocol
To model neutrophilic lung inflammation, mice intranasally received either 10.5 μg of LPS (Sigma-Aldrich, Oakville, ON, Canada) in 35 μL of sterile phosphate-buffered saline (PBS) or PBS only. Animals were imaged 24 hours post LPS exposure and sacrificed immediately after imaging.
Porcine pancreatic elastase exposure protocol
To model emphysema, mice intranasally received 4 units of PPE (EPC Inc., Owensville, MO, USA) in 30 μL of sterile PBS or PBS only. After exposure mice were left for a period of 45 days prior to acquisition of data.
Imaging protocol and per-voxel image analysis
Imaging was performed as previously described [
13] with minor modifications. SPECT scans were acquired on an X-SPECT system (Gamma Medica, Northridge, CA, USA). Technegas™ and
99mTc-macroaggregated albumin were used to provide the distributions of V and Q, respectively. CT images were acquired for both SPECT scans, also on the X-SPECT. SPECT and CT images were reconstructed, fused, and co-registered as previously described [
13]. A ‘Lung’ region of interest (ROI) was produced for the ventilation CT images using Amira 5.1 software (Visage Imaging, Andover, MA, USA) and used during co-registration and analysis. V/Q ratios were calculated using normalised V and Q frequencies. To assess emphysema in CT images, volumes of low attenuation (VLA) were calculated by summing the percentage of lung volume less than -400HU. Additional details regarding this threshold and other specifics of the methods used are provided in the Additional file
1.
Collection and measurement of specimens
Mice were sacrificed at the experimental endpoints indicated in the results. Bronchoalveolar lavage (BAL) was collected and measures of airspace enlargement were made (Pneumometrics software V.1, Hamilton, Ontario, Canada) from haemotoxylin and eosin (H&E) stained lung histology sections as described previously [
14]. BAL cell counts and the histological data for animals at 24 weeks CS have been reported previously [
14]. Additional detail regarding lung fixation, histological assessment, and BAL quantification is provided in the Additional file
1.
Data analysis
Data were expressed as the mean ± SEM. Statistical significance was determined by an unpaired, two-tailed t-test in Prism (Graphpad Software Inc, La Jolla, CA, USA) when comparing age-matched experimental groups. For cessation-related data, a one-way ANOVA with Tukey post-hoc test was performed. p < 0.05 was considered statistically significant for all statistical tests. The number of mice studied is described in Table one, found in the Additional file
1.
Discussion
Ventilation and perfusion of the lung can be compromised in COPD, and the capability of matching these processes dysregulated. The objective of the current study was to investigate the V/Q perturbations associated with two of the major pathologies associated with COPD using mouse models of neutrophilic inflammation and emphysema. Further, the impact of smoking cessation was described and evidence gathered regarding the relative roles of inflammation and airspace enlargement in V/Q mismatching.
The mouse models employed in the studies presented were relatively simple in nature to allow for effective interpretation of results. These models, utilising LPS, PPE, and cigarette smoke, are all well-established and the impact of these exposures on resistance to airflow, the immune system, and other aspects of the lung have been reviewed previously [
12]. Investigation of V/Q relationships in these models adds to the understanding of the consequences of pathological disruption on the potential for gas exchange. The LPS and PPE models, causing inflammation and airspace enlargement, respectively, were used as examples of severe pathology, so the V/Q mismatching observed was not surprising. It is important to note that the extent and distribution of pathology associated with these administered reagents does not necessarily reflect the pathologies found in COPD, but demonstrate that each causes V/Q mismatching. Cigarette smoke, on the other hand, caused less pronounced inflammation and only subtle airspace enlargement but nevertheless caused V/Q mismatching similar to that observed in the other models employed.
A seminal article by Wright and Sun [
16] investigated long-term smoking cessation in guinea pigs and found that airspace enlargement persists in ex-smokers while pulmonary function increases over their smoking counterparts. Similarly, we found that smoking cessation resulted in a return to normal lung function, as measured by V/Q relationships, and decreased inflammation. However, it has been established that other pathological markers, such as bronchial-associated lymphoid tissue, remain after smoking cessation [
17]. Likewise, the airspace enlargement present after 24 weeks of cigarette smoke exposure persisted after smoking cessation, but these emphysematous lesions caused by cigarette smoke exposure were mild compared to elastase induced airspace enlargement and likely were not sufficient enough to contribute to V/Q mismatch. It is possible that the resolution of the imaging methodology was unable to detect the mismatch caused by these small, persistent structural changes; however, if airspace enlargement were to continue, V/Q mismatch and impaired gas exchange would eventually ensue, indicating that smoking cessation in human patients is critical before emphysematous lesions are present; at these early stages of disease the V/Q mismatch from inflammation could resolve leaving the gas exchange capabilities of the lung largely intact.
Work by Suga
et al.[
11] has begun to explore the V/Q relationships in COPD patients with advanced emphysema but our results suggest that changes in V/Q may not be apparent, due to airspace enlargement alone, until this pathology has progressed substantially as evidenced by the lack of V/Q mismatching in the presence of mild emphysema in cessation mice. Also of interest, the relationship between volumes of low x-ray attenuation and V/Q was not apparent in the comparison of log(V/Q) images to CT images in PPE-exposed animals. It is possible that regions neighbouring emphysematous volumes are unable to function properly, leading to the V/Q mismatch observed. This warrants further investigation and V/Q SPECT/CT provides the necessary tools to address this concern. It is now understood that emphysema progression continues after smoking cessation [
18,
19], so development of methods that can be used clinically to track and understand this pathology are paramount.
The impact of inflammation on V/Q status is also an important topic that is not yet well understood. While LPS caused a greater inflammatory reaction than cigarette smoke, as observed in both BAL measurements and CT images, it did not elicit a V/Q disturbance greater than that of cigarette smoke. It is likely that the distribution of this inflammation is an important factor, especially as it pertains to the small airways; constriction of the small airways is undoubtedly heterogeneous and would lead to heterogeneous ventilation patterns. As airflow resistance is inversely proportionate to the radius of the airway to the fourth power, as described by Poiseuille’s equation, even slight changes in the lumen of small airways could alter the distribution of ventilation and impact V/Q relationships. Investigations by Gaschler
et al.[
20] demonstrated that mucus secretion is not present within the small airways after 8 weeks of cigarette smoke exposure, but there is thickening of the epithelial layer [
21]. V/Q mismatching is present after 8 weeks smoke exposure in this model [
14], but further investigation into the mechanisms by which inflammation could affect airflow in this manner is required. While LPS-derived inflammation caused V/Q mismatch, likely through airflow obstruction, it is important to note that cigarette smoke contains additional components, such as nitric oxide, that could interfere with vascular mechanisms, such as hypoxic vasoconstriction, leading to inadequate matching of perfusion to ventilation [
22,
23]. Thus, the V/Q mismatch observed in this model of cigarette smoke exposure is likely dependent on both inflammation and an alteration in perfusion, though greater investigation is still necessary.
In comparison to clinical findings, our data are consistent with those previously reported by Rodríguez-Roisin
et al.[
7] using MIGET where V/Q was shown to be sensitive to GOLD stage I and that mismatching increased with GOLD staging severity. The authors also provided evidence that the V/Q abnormalities seen in GOLD stage I were associated with smaller airways, alveolar airspaces, and blood vessels. Our data suggests that inflammation could play a large role in the V/Q mismatching observed in early COPD, while other pathologies, such as emphysema and small airway fibrosis, become a principal cause of V/Q mismatching in the later stages of COPD. SPECT V/Q has previously been shown, through work by Petersson
et al.[
24], to closely parallel MIGET results such as those described above but it is important to note that our protocol did not contain a measurement of total cardiac output. As such, this preclinical technique approximates overall V/Q distribution. Nevertheless, due to the consistent attributes inherent in an experimental model, as compared to human subjects, we believe that our V/Q results are representative of the state of the lungs in the contexts described.
The pulmonary processes of ventilation and perfusion are both affected by long-term exposure to cigarette smoke. Cessation of cigarette smoking results in a return of V/Q assessed lung function to normal, but the pathological consequences of continued exposure eventually lead to structural damage and functional impairment. It is possible that these pathologies could be detected early with the aid of V/Q methods and cessation initiated before major damage permanently alters pulmonary lung function.
Acknowledgements
The authors gratefully thank Dr. T.H. Farncombe and C. Saab for their expertise and contributions towards imaging and J. Kasinska for expert technical support; Dr. K. Gulenchyn and the McMaster Department of Nuclear Medicine, Hamilton Health Sciences (Hamilton, ON, CAN) for use of the Technegas™ system; and the Firestone Institute for Respiratory Health (St. Joseph’s Healthcare, Hamilton, ON, CAN) for tuition support (BNJ).
Competing interests
Financial Support: Firestone Institute of Respiratory Health – AstraZeneca Collaboration Unrestricted Grant; The Canadian Institutes of Health Research; N.R. Labiris holds an internal Department of Medicine Career Award.
Authors’ contributions
BNJ was involved in concept and design, experimentation and collection of biological and imaging data, analysis, drafting and review of the manuscript. CAJRM was involved in experimentation and collection of biological and imaging data. MCM was involved in experimentation and collection of biological data as well as review of the manuscript. RGR was involved in collection and analysis of imaging data. MRS contributed to concept and design and review of the manuscript. NRL contributed to concept and design and review/editing of the manuscript. All authors read and approved the final manuscript.