Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

Summary

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

Abstract

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

Introduction

Pulmonary artery hypertension (PAH) is defined by a mean pulmonary artery systolic pressure greater than 20 mmHg at rest^1,2. It is a progressive and fatal disease characterized by a sustained elevation in pulmonary arterial pressure, leading to right ventricle overload and ultimately death due to right ventricular failure¹. Currently, there is no cure for PAH.

The use of animal models of pulmonary hypertension is important for testing the effectiveness of experimental PAH therapies. Among those models, the mouse hypoxia-induced PAH model has provided key insi....

Protocol

All animal procedures were performed under protocols approved by Yale University Institutional Animal Care and Use Committees.

1. Preparation of animals, tools, blood pressure measuring equipment, and hypoxia chamber

1. Animal acclimation.
   NOTE: Experimental animals used for this study were male, 8-week-old C57BL/6 mice weighing 25-27 g. Several factors should be considered when estimating the number of animals required for the experiment, including surgery-associated mortality, unexpected surgical complications, and sudden unexpected death. Use at least 10 mice per group to reach statistical power and avoid....

Results

Anesthesia often reduces blood pressure. Therefore, a minimum dose of anesthesia was used to abolish the movements in response to a noxious stimulus. Successful right ventricular chamber access can be visualized as the hemodynamic waveform changes in different regions of venous systems (Figure 8).

In this study, mice were randomly assigned to the normoxic (21% O₂) group (n = 10), hypoxia (10% O₂) group (n = 10), or hypoxia + 7C1/let-7 treatme.......

Discussion

Several pulmonary hypertension animal models have been established to mimic the elevated pulmonary vascular resistance events in human subjects. Among them, the mouse hypoxia-induced PAH model has been widely used for evaluating the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds to the mice. In comparison with other published intravenous (IV) injection and invasive hemodynamic assessment protocols, this method provides both visual illustration.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-0 prolene suture pack	Ethicon	8698G	for incision closure
8-0 nylon suture pack	AROSurgical Instruments	T06A08N14-13	for ligation
Anesthesia induction chamber	VETEQUIP	#941444	Holds the animal during anesthesia exposure
Catheter Interface Cable PEC-4D	Millar		for connecting Millar Mikro-Tip catheter to PCU-2000
Charcoal canister filters	VETEQUIP	#931401	to help remove waste anesthetic gases
Cotton swabs	McKesson	24-106	for applying pressure to the injection site to prevent bleeding
Fine scissors	Fine Science Tools	14059-11	Surgical tools
Insulin syringe 28 G	EXEL	26027	for jugular vein IV injection
Isoflurane	COVETRUS	#029405	for mouse anesthesia
LabChart 8 Software	ADInstruments		for data analysis
Mikro-Tip Pressure Catheter SPR-1000 (1.0 F)	Millar		for invasive blood pressure measurement
Needle-25 G	BD	305124	for making a samll hole in a vessel
Oxygen controller ProOx Oxygen Sensor	BioSpherix	E702	for oxygen concentration monitoring
PCU-2000 Pressure Control Unit	Millar		for connecting Millar Mikro-Tip catheter to PowerLab 4/35
PowerLab 4/35	ADInstruments		for Data Acquisition. Investigator needs to connect the PowerLab 4/35 to a personal laptop containing LabChart 8 software for operation.
Prism 8	GraphPad		for statistics and scientific graphing
Semisealable hypoxia chamber	BioSpherix		an artificial environment that simulates high-altitude conditions for animals
Spring Scissors	Fine Science Tools	15021-15	Surgical tools
Tweezer Style 4	Electron Microscopy Sciences	0302-4-PO	Surgical tools
VasoRx compound 7C1/let-7 miRNA	VasoRx, Inc.	Lot# B2-L-16Apr	IV injection compound
VIP 3000 Veterinary Vaporizer	COLONIAL MEDICAL SUPPLY CO., INC.		for accurate anesthesia delivery