Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Summary

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

Abstract

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

Introduction

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial (PA) pressure exceeding 25 mm Hg at rest, as assessed by right heart catheterization^1,2. There is a variety of diseases that can lead to PH. In an attempt to organize the PH-associated conditions, several classification systems have been developed. The current clinical classification categorizes the multiple PH-associated diseases in 5 different groups¹. This distinction is of importance since various groups of patients have diseases that differ in their clinical presentation, pathol....

Protocol

Prior to any animal experimentation obtain the local institutional animal care committee authorization. The current experiments were performed after approval by the Institutional Animal Care and Use Committee (IACUC) at the Icahn School of Medicine at Mount Sinai.

1. PH induction

1. Preparation
   1. Before beginning the study, carefully plan the experimental design. Ensure that mice are subjected to hypoxia at the same time point as the first SU5416 injection. An example of the experimental design for inducing PH using the Hypoxia/SU5416 method is shown in Figure 1A. Control mic....

Results

In this protocol, we describe in detail the creation of the Hypoxia/SU5416 model for inducing PH in mice. Furthermore, we detail all the needed steps for performing pulmonary vascular and cardiac evaluation at the end of the observation period.

An overview of the experimental design for this model is shown in Figure 1A^13,14. Mice are subjected to normobaric hypoxia (10% O₂

Discussion

This protocol describes how to model PH in mice by combining two pathological stimuli: chronic hypoxia and SU5416 injection (Hypoxia/SU5416)¹⁸. In an attempt to correlate this mouse model with the human PH condition, one inevitably must look at the current PH classification, shown in Table 1. PH in almost all forms is characterized by pulmonary vasoconstriction and aberrant proliferation of endothelial and smooth muscle cells. This leads to elevated pressure in the pulmonary arter.......

Materials

Name	Company	Catalog Number	Comments
Acetic acid glacial	Roth	3738.1
Acetone, Histology Grade	The Lab Depot	VT110D
ADVantage Pressure-Volume System	Transonic	ADV500
Bouin's solution	Sigma	Ht10132
Cautery System	Fine Science Tools	18000-00
Connection tubing and valves
Cotton-Tipped Applicators	Covidien	8884541300
Coverslips, 24 x50 mm	Roth	1871
Data Acquisition and Analysis	Emka	iox2
Direct Red 80	Sigma	365548-5G
DMSO (Dimethyl Sulfoxide)	Sigma Aldrich	276855
Dry ice
Dumont # 5 forceps	Fine Science Tools	11251-10
Dumont # 7 Fine Forceps	Fine Science Tools	11274-20
Embedding molds	Sigma Aldrich	E-6032
Eosin Solution Aqueous	Sigma	HT110216
Ethanol, laboratory Grade	Carolina Biological Supply Company	861285
Fast Green FCF	Sigma	F7252-5G
Fine scissors	Fine Science Tools	14090-09
Goat Serum	invitrogen	16210-064
Heating pad	Gaymar	T/Pump
Hematoxylin 2	Thermo Scientific	7231
Hypoxic chamber	Biospherix	A30274P
Induction chamber	DRE Veterinary	12570
Intubation catheter (i.v. catheter SurFlash (20 G x 1") )	Terumo	SR*FF2025
Iris scissors	Fine Science Tools	14084-08
Isoflurane	Baxter	NDC-10019-360-40
Isoflurane vaporizer	DRE Veterinary	12432
Mice (C57BL/6)	Charles River
Needles 25 G x 5/8"	BD	305122
OCT	Tissue Tek	4583
PBS (Phosphate Buffered Saline)	Corning	21-031-CV
Piric Acid- Saturated Solution 1.3 %	Sigma	P6744-1GA
Pressure volume catheter	Transonic	FTH-1212B-4018
Retractor	Kent Scientific	SURGI-5001
Static oxygen Controller ProOx 360	Biospherix	P360
SU 5416	Sigma Aldrich	S8442
Surgical Suture, black braided silk, 5.0	Surgical Specialties Corp.	SP116
Surgical tape	3M	1527-1
Syringe 10 ml	BD	303134
Syringes with needle 1 ml	BD	309626
Sytox Green Nuclein Acid Stain	Thermo Scientific	S7020
Tenotomy scissors	Pricon	60-521
Toluol	Roth	9558.3
Ventilator	CWE	SAR-830/P
WGA Alexa Fluor	Thermo Scientific	W11261
Xylene	Roth