Dynamic Changes in Lung Function and Blood Flow During Lung Adenocarcinoma Progression in Mice by Whole-body Plethysmography and Doppler Ultrasound

Summary

This protocol uses mouse pulmonary function and Doppler ultrasound as investigative techniques, offering a non-invasive, simple, and efficient method for studying lung adenocarcinoma.

Abstract

Lung adenocarcinoma (LUAD) is a common malignant pulmonary tumor, and its progression leads to alterations in ventilation and blood flow, highlighting the distinct functional characteristics of the lung under pathological conditions. The non-invasive whole-body plethysmography (WBP) system enables real-time monitoring of lung function and airway responsiveness in awake mice, avoiding interference from tracheotomy and anesthesia. Small animal Doppler ultrasound (DU), utilizing high-frequency imaging technology, non-invasively captures microvascular structures and changes in pulmonary blood flow velocity, offering crucial support for investigating ventilation and perfusion abnormalities associated with LUAD. In this study, a LUAD model was established in A/J mice using 100 mg/kg of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). WBP combined with DU was employed to monitor pulmonary function and hemodynamic parameters in mice. The results revealed a significant decline (p < 0.05) in key metrics, including 50% expiratory flow (EF50), minute ventilation (MV), peak expiratory flow (PEF), peak inspiratory flow (PIF), enhanced pause (Penh), and tidal volume (TV), as well as a marked reduction in pulmonary arterial blood flow velocity (p < 0.05) in the model group during tumor progression. This combined approach effectively captures the functional disruptions occurring during the pathological development of LUAD in mice, providing a non-invasive and efficient method for advancing research into respiratory diseases.

Introduction

Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer¹. With increasing environmental pollution, tobacco use, and aging populations, the global prevalence of LUAD has been rising annually^2,3,4. Current studies indicate that patients experience progressive declines in pulmonary function and hemodynamic disturbances as LUAD advances^5,6,7,8. Developing non-invasive, efficient, and cost-effective methods to track and evaluate pulmonary function and pulmonary artery blood flow fluctuations can provide critical evidence for clinical LUAD management.

Existing research demonstrates that LUAD tumor growth progressively obstructs airways, reduces lung capacity, and ventilation⁹, and triggers inflammatory cytokine secretion that exacerbates airway stenosis through localized chronic inflammation¹⁰. Consequently, decreased pulmonary function leads to aggravated hypoxia and blood flow abnormalities^11,12. These pathological changes often manifest clinically as progressive dyspnea and chest tightness. While CT imaging fails to capture real-time pulmonary function parameter abnormalities or hemodynamic alterations induced by LUAD progression, repeated short-term CT scans also increase radiation exposure¹³. In contrast, whole-body plethysmography (WBP) and Doppler ultrasound flowmetry serve as non-invasive, operator-friendly, and reproducible complementary approaches for dynamic monitoring. Integrating these techniques to monitor pulmonary function and hemodynamics could improve disease progression assessment and therapeutic decision-making.

In this study, we utilized 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice to model LUAD initiation and progression. Pulmonary function and hemodynamic changes were longitudinally monitored via WBP and Doppler ultrasound (DU) at weeks 4, 10, and 20. This approach aims to provide novel insights into LUAD pathophysiology and therapeutic strategies.

Protocol

All animal experiments described in this study were approved by the Ethics Committee on Animal Welfare of Chengdu University of Traditional Chinese Medicine and were conducted in compliance with relevant laws and standards for animal research ethics (20240616IV). Female inbred A/JGpt mice (7-8 weeks old) were maintained at 20-24 °C and a relative humidity of 40%-70%. Throughout the 12 h light-dark cycle, the mice were provided with standard animal feed and purified water. The animals were acclimated to this environment 7 days before beginning the experiments.

1. Creation and preparation of the animal model

1. Dissolve NNK in normal saline to prepare a stock solution with a concentration of 10 mg/mL.
2. Measure pulmonary function using WBP at weeks 4 (day 28 after modeling), 10 (day 70 after modeling), and 20 (day 140 after modeling). Pulmonary arterial blood flow velocity was measured using DU at week 20 (day 140 after modeling). Before each measurement, place the mice in an appropriate environment to minimize stress.

2. Non-invasive pulmonary function testing

NOTE: The WBP system measures respiratory function in awake, freely moving animals using a specially designed measurement chamber. The chamber is fitted with pneumotach screens, which allow air to pass in and out of the chamber. The air resistance created by the pneumotach screens causes small pressure changes in the chamber relative to the ambient air. A sensitive pressure transducer is connected to measure these pressure changes inside the chamber, from which flow can be derived (called box flow). The software provides respiratory parameters such as flow and volume, along with real-time reporting.

1. Obtain the following main components: WBP controller, controller power transformer, console bar, long console bar cable and short console bar cable, calibrator column, WBP plethysmograph, flow transducer, temperature and humidity probe, nebulizer head (purple), bias flow filter and tubing, PC.
2. Assemble the hardware as described below.
   1. Perform rear panel connection as described below.
      1. Connect the controller unit to the computer using a USB 2.0 cable and make sure the connection is stable.
      2. Fill the column with tap water to the water fill line, connect to the port on the back of the controller, and make sure the line is dry.
      3. Plug the power transformer into the back of the controller, making sure it is well connected.
      4. Depending on where the console strip is placed, choose a long or short console strip cable to connect to the back of the controller.
      5. Push the controller unit into place and arrange the calibration column and power transformer.
   2. Perform front panel connection as described below.
      1. Plug each flow sensor into the port labeled Sensor. Plug each bias flow filter and tubing into the controller port labeled Bias Flow on the front panel. Connect a bias flow isolation resistor to the open end of the bias flow tube.
   3. Perform chamber connection as described below
      1. Place the chamber in front of the controller unit, ensuring that it is placed on a solid, stable surface that is free of vibration.
      2. Connect the flow sensor to the chamber and orient the sensor so that the DSI sign is facing up.
      3. Connect the bias flow tubing (with isolation resistor) to the bottom port on the plethysmograph.
      4. Remove the plug on the top of the plethysmograph and replace it with a nebulizer head (purple).
3. Set up the software as described below.
   1. Launch the control program software and log in. On the Controller homepage, click Hardware Configuration. Launch the configuration wizard by clicking New Configuration, then select WBP (Serial Number: DEMO-061) and click Next.
   2. In the Species dropdown menu, select Mouse. Set the flow rate to the default values. Select 4 sites (each site corresponds to one chamber, with a maximum of 4 chambers per session). Ensure that the bias flow rate for all active sites is consistent. Match the sites in the software with the actual ports on the WBP controller. Finally, click Finish to complete the configuration.
4. Data collection
   1. Open the main program and calibrate the chambers, as this step is critical. Hover the mouse over the hardware to display a yellow tool icon. Click the Icon to enter the calibration interface, then click All to calibrate the chambers for all four animals simultaneously.
   2. Create the required study protocol by selecting the Dose Response Study in Create Study Options. Set the GLP configuration to not follow GLP.
   3. To configure measurement parameters and task sequences, click New Parameter to open the Parameter Builder and add the parameters to be measured. Use the default settings for the task sequence.
   4. To start data collection, navigate to the My Laboratory page in the workstation, then drag and drop the prepared study protocol into WBP for mouse.
   5. Click the red triangle button for each site to begin data acquisition. To start all four sites simultaneously, click Session > Acknowledge all sites. After starting the acquisition, workstation will open the Configure Acquisition page, and use this to edit the information for each mouse in the acquisition sites.
   6. Sequentially click Session > Acknowledge all Sites to begin data collection. To ensure that the experimental results are true and reliable, stabilize the experimental mice in a quiet room for 30 min beforehand.
   7. Place the quiet mice in the completely sealed test jar. Click the Start option in the system to collect data. The system collects data every 2 s, so collect at least 10 min. Intercept 4 min of data in the middle. Here, five animals were tested in each group, and the test was repeated 3x.
   8. Click Create Report on the Study page and select the report type. Save the experimental data in spreadsheet format. In this experiment, peak expiratory flow rate (PEF), peak inspiratory flow rate (PIF), enhanced pause (Penh), and tidal volume (TV) were used to evaluate the lung function of mice.

3. Pulmonary arterial blood flow velocity measurement

1. Animal preparation
   1. Place the mouse in an anesthesia induction chamber and anesthetize it with 5% isoflurane (flow rate: 1 L/min). Secure the mouse on an animal handling platform, maintaining the platform temperature at 35-37 °C to preserve body temperature.
   2. Position the mouse's mouth and nose into an anesthesia mask. Maintain anesthesia with 1%-2% isoflurane (flow rate: 0.6-1 L/min). When the mouse maintains a stable supine position with stable heart rate and regular respiratory rhythm, complete muscle relaxation, absence of limb movement, no vibrissae twitch response, disappearance of pedal withdrawal reflex, pink mucous membranes, and no pupillary dilation, it is considered to have reached an appropriate anesthesia depth.
   3. At this stage, apply 2-3 mg of vet ointment to the lower conjunctival sac using a sterile cotton swab. Gently stroke the upper and lower eyelids to promote closure without compressing the eyeball, ensuring uniform distribution of the ointment across the corneal surface.
2. Imaging area preparation
   1. Remove chest hair using depilatory cream and a depilatory device. Clean the treated area with a damp gauze or wet tissue, then apply the coupling agent pre-warmed to 37°C evenly with a cotton swab to achieve a uniform thickness of 0.3 mm, ensuring the absence of visible air bubbles.
3. Doppler ultrasound imaging
   1. Use a 40 MHz Doppler ultrasound probe to measure pulmonary arterial blood flow velocity from the parasternal long-axis view. Tilt the probe 45° to the left and the operating table 15° to the right. Lower the probe from the right side of the chest and fine-tune the X and Y axes to locate the right ventricle and pulmonary artery, guided by their anatomical spatial relationships, morphological features, and hemodynamic flow direction.
   2. Press the CF button to switch to Doppler mode. Visualize the blue pulmonary arterial blood flow signal. Position the sampling line on the pulmonary artery and press the PW button to display the pulmonary arterial blood flow spectrum.
   3. Press the Freeze button to freeze the image, then press the Measure button and select the Velocity option on the touchscreen. Place the measurement line at the peak of the spectrum to obtain the maximum pulmonary arterial blood flow velocity.
4. Data acquisition
   1. Use the offline analysis software to select pulmonary arterial pulsed Doppler data. Access the Cardiac Package measurement pack, select RV and PV Function, and output pulmonary arterial blood flow velocity and saved images.

4. Data analysis

1. Use R4.4.2 and GraphPad Prism 9.5.1 for data analysis and plot the data. Inter-group comparisons of pulmonary function data were performed using independent samples t-test, while intra-group comparisons utilized repeated measures ANOVA. Pulmonary arterial blood flow velocity comparisons between groups were analyzed with independent samples t-test, with statistical significance set at p < 0.05.

Results

To validate the feasibility, accuracy, and reproducibility of the pulmonary function and pulmonary arterial blood flow assessment methodology, we plotted the minute-by-minute mean curves of PIF, PEF, and expiratory flow at 50% of vital capacity (EF50, 10x magnified) during the 4th-week monitoring period in the blank group (Figure 1A). As shown in this panel, the three corresponding curves exhibit a high degree of conformity in shape. Despite the minimal absolute changes, particularly in EF50...

Discussion

In this study, NNK combined with A/J mice was selected to make a lung adenocarcinoma model according to previous reports. Relevant studies have shown that NNK combined with A/J mice can start to have minimal tumor formation in the 10^th week, and stable tumor formation in the 16^th to 20^th week^18,19. Therefore, we selected the test at the 4^th week after injection to observe the changes of lung function and pulmonary arter...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
A/J mice	GemPharmatech LLC.	N000018
0.5ml EDTA tubes	Labshark	130201070
1-Butanone,4-(methylnitrosoamino)-1-(3-pyridinyl)	Gu Shi Gong Yuan Medical Equipment Co.	N589770
75% ethanol	ChengDu Chron Chemicals Co,.Ltd	2023052901
Anesthesia induction chamber	Shanghai Tawans Intelligent Technology Co., Ltd.	MZ-MR
Depilatory cream	Shanghai Yuyan Instruments Co., Ltd.	20230623
Depilatory device	Shanghai Yuyan Instruments Co., Ltd.	3180
Isoflurane	Shanghai Duma Biotechnology Co., Ltd.	DM-M9462
Medical tape	Beijing Lanbokangs Technology Co., Ltd.	19-5023
medical tricorder	MedChemexpress	69652
PBS buffer solution	Shanghai Fusheng Co., Ltd.	A-RY5256
Saline (medicine)	Beijing Biolabs Technology Co.	GL1736‌
Ultrasound coupling agent	Qisheng (Shanghai) Medical Equipment Co., Ltd.	20212060273