Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Summary

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Abstract

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Introduction

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder with elevation in pulmonary artery pressure (PAP) and right ventricular pressure (RVP) that is caused by cellular proliferation and fibrosis of small pulmonary arteries¹. Pulmonary artery catheters, also called Swan-Ganz catheters², are commonly used in the clinical monitoring of RVP and PAP. Furthermore, a wireless PAP monitoring system has been used clinically^3,4,5. To mimic the disease for study in mice, a hypoxic environment is used to simulate human clinical manifestations of PAH⁶. In the evaluation of PAP in animals, large animals are relatively easy to monitor through pulmonary artery catheters using the same technique as for human subjects, but small animals such as rats and mice are difficult to assess because of their small body size. Hemodynamic measurement of the right ventricular system in mice is possible with an ultrasmall size 1 Fr catheter⁷. A method for measuring RVP and PAP in mice has been reported in the literature^8,9, but the methodology lacks a detailed description. RVP and PAP are more challenging to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems.

To get both PAP and RVP parameters in the same mouse, we describe an open-chest surgery-based approach for right heart hemodynamic measurements, its validation with healthy and PAH mice, and how to avoid generating artificial data during the complicated open-chest surgery. Although this technique is best performed by a well-trained surgeon, it has the advantage of being able to assess PAP and RVP in the same mouse.

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Protocol

The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Fuwai Hospital, Chinese Academy of Medical Science, Peking Union Medical College (NO.0000287). The experimental animals were housed and fed according to the guidelines of animal welfare in China.

NOTE: Eight- to 12-week-old male C57BL mice were housed in an environment with a 12 h dark/ 12 h light cycle. The PAH mice were housed for 4 weeks under an oxygen concentration of 10%, maintained by an oxygen-controlled hypoxia chamber to induce pulmonary hypertension, and control mice were housed in room air (21% oxygen) under identical conditions. RVP and PAP measurements were performed at the end of the 4 weeks of hypoxia challenge.

1. Preoperative preparation

1. Soak the pressure transducer catheter (size: 1 Fr) in 0.9% saline at room temperature for at least 30 min before the hemodynamic experiment.
2. Filter 2,2,2-Tribromoethanol solution with 0.22 μm filter and store in 4-degree refrigerator.
3. Prepare cleaned surgery tools and supplies such as gloves for surgery.
4. Prepare 10 mL of 1.0% digestive enzyme solution for catheter cleaning.
5. Connect the pressure transducer catheter to a pressure-volume system.
6. Calibrate the pressure transducer prior to obtaining pressure measurements for each mouse.
   1. Turn the calibration knob to 0 mmHg and 25 mmHg to send a verification pressure signal to the data acquisition software and configure the calibration setting in the software.
   2. Turn the knob to Transducer and adjust the Balance knob to zero baseline.
7. Set up a standard stereomicroscope and a temperature-controlled small animal surgical table for body temperature maintenance during the surgery.
8. Set up a light illumination system for microsurgery to provide enough light over the surgical area.

2. Open-Chest surgery and hemodynamic measurement

1. Anesthetize mice with 250 mg/kg of 2,2,2-Tribromoethanol via intraperitoneal (i.p.) injection. If needed, repeat supplemental doses at 1/3 to 1/2 of the original dose during the procedure.
2. Remove chest and neck fur using a shaver and hair removal lotion (Figure 1A, 2A).
3. Secure each mouse in the supine position on a temperature-controlled small animal surgical table to help maintain body temperature (37 °C) during surgery.
4. Clean the surgical site with 70% ethanal.
5. Once anesthesia is in effect, confirm adequate anesthesia induction using a toe pinch.
6. Make a midline incision on the neck skin (Figure 1A).
7. Dissect the skeletal muscle using curved forceps and expose the trachea (Figure 1B, 1C).
8. Perform intubation through the mouth using a modified 22 G intravenous sheath catheter. Confirm that the tubing is in the trachea using forceps (Figure 1D).
9. Connect the tubing to a small animal ventilator. Calculate and set respiration rate and tidal volume based on body weight according to the ventilator user manual¹⁰. For example, set respiration rate to 133/min and tidal volume to 180 μL for a 30 g mouse based on the described calculation.
10. Secure the tubing for ventilation using tape.
11. Confirm adequate anesthesia induction using a toe pinch.
12. Make a midline incision on the chest skin and carefully dissect the chest muscles using a cautery tool (Figure 2B, 2C).
13. Cut the sternum using scissors across the middle and expose the thoracic cavity (Figure 2D).
14. Prevent any bleeding using the cautery tool during the open-chest surgery procedure.
15. Expose the right ventricle with retractors (Figure 2E).
16. Insert the saline-soaked pressure transducer catheter through a small tunnel created with a 25 G needle into the right ventricle to measure RVP (Figure 2F and Figure 3A, 3C).
17. Hold the catheter cable and cross the pulmonary valve in a coaxial manner with the pulmonary artery. Observe the pressure waveform and obtain a stable PAP signal (Figure 3B, 3D).
18. Record hemodynamic data using the data acquisition system and software.
19. After the final measurements, euthanize mice humanely through i.p. injection of an excess dose of 2,2,2-Tribromoethanol solution.
20. Carefully remove catheter from the right heart system and place into a 1 mL syringe containing 1% digestive enzyme solution.
21. Use distilled water to continuously flush the catheter carefully and store it in the original box.

3. Data analysis for hemodynamics

NOTE: The hemodynamic data were recorded and analyzed using analysis software¹¹ (Table of Materials).

1. For each mouse, select at least 10 continuous and stable heartbeat cycles without noise to obtain the average data of RVP or PAP data for each parameter.
2. Use Student’s t-test to compare the normal air control and hypoxia groups. NOTE: p < 0.05 was considered statistically significant. Data are presented as the mean ± SD.

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Results

The pressure transducer catheter was inserted into the right ventricle (Figure 3A) through a tunnel expanded by a 25 G needle, and a typical RVP waveform (Figure 3C) was obtained. The catheter was continually adjusted and slowly advanced and kept in the same axis as the pulmonary artery while passing through the pulmonary valve (Figure 3B). When the pressure sensor was successfully ...

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Discussion

Tracheal intubation is the first important step for open-chest surgeries. The classic method of tracheal intubation for small animals, such as rats or mice, involves making a T-shaped incision on the trachea and directly inserting Y-type tracheal tubing into the trachea. In practice, we find that this method is not easy during operation. The Y-type tracheal tubing is too large for small animals and forms an angle with the trachea. Thus, it is difficult to fix the tubing in place. Additionally, once the intubation tubing ...

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Materials

Name	Company	Catalog Number	Comments
2,2,2-Tribromoethanol	Sigma-Aldrich	T48402-5G	For anesthesia
Animal temperature controller	Physitemp Instruments, Inc.	TCAT-2LV	For temperature control
Dissection forceps	Fine Science Tools, Inc.	11274-20	For surgery
Gemini Cautery System	Gemini	GEM 5917	For surgery
Intravenous catheter (22G)	BD angiocath	381123	For intubation
LabChart 7.3	ADInstruments		For data analysis
Light illumination system	Olympus		For surgery
Mikro-Tip catheter	Millar Instruments, Houston, TX	SPR-1000	For pressure measurement
Millar Pressure-Volume Systems	Millar Instruments, Houston, TX	MVPS-300	For pressure measurement
O2 Controller and Hypoxia chamber	Biospherix	ProOx 110	For chronic hypoxia
PowerLab Data Acquisition System	ADInstruments	PowerLab 16/30	For data recording
Scissors	Fine Science Tools, Inc.	14084-08	For surgery
Small animal ventilator	Harvard Apparatus	Mini-Vent 845	For surgery
Stereomicroscope	Olympus	SZ61	For surgery
Surgery tape	3M		For surgery
Terg-a-zyme enzyme	Sigma-Aldrich	Z273287-1EA	For catheter cleaning