Evaluation of Right Ventricular Function in Experimental Models of Pulmonary Arterial Hypertension

Summary

The presentation of detailed methods for the evaluation of right ventricle function will enhance the quality and reliability of pulmonary hypertension research, offering a robust framework for future studies and enhancing reproducibility across different laboratories.

Abstract

Pulmonary hypertension (PH) is a chronic condition associated with reduced survival, making it a critical area of research. Experimental models of PH have significantly improved our understanding of its pathophysiology and facilitated the design of small observational and clinical trials. However, it is crucial to establish standardized methodologies to ensure comparability across different experimental studies and research groups. Standardization not only enhances reproducibility but also fosters collaboration among research teams.

In this video, we outline the methodological steps required to analyze the functional parameters of PH accurately. These include echocardiographic assessment, invasive right ventricle (RV) puncture, and calculation of the Fulton's index. Concerning echocardiography, measurement of the pulmonary arterial acceleration time and the pulmonary ejection time ratio, as well as the calculation of RV output, are presented in detail. Regarding invasive measurements, essential procedures, including anesthesia protocols, optimal animal positioning, mechanical ventilation setup, precise chest wall opening, visualization of the RV, and RV puncture techniques, are described.

The methodology for ventricular dissection (Fulton's index) is explained in detail, as well as the calculation of the RV/left ventricle plus septum ratio, corrected for the animal's body weight. We believe that providing a detailed presentation of these methods will enhance the quality and reliability of PH research, offering a robust framework for future studies and enhancing reproducibility across different laboratories.

Introduction

Adequate functional measurement of the right ventricle (RV) function in small animals has become essential since the increase in studies using animal models in respiratory and cardiovascular sciences. There are several animal models of pulmonary hypertension (PH), using monocrotaline¹, hypoxia², or Sugen + hypoxia³. They have served well to unravel new pathophysiological pathways^1,4 and study new potential pharmacological⁵ and non-pharmacological⁶ treatments for PH. Nevertheless, despite the ....

Protocol

The study protocol was approved by the Ethics Committee on the Use of Animals (CEUA) of the Federal University of Rio de Janeiro under process number 043/22. All procedures complied with the Principles of Laboratory Animal Care and the US National Academy of Sciences Guide for the Care and Use of Laboratory Animals. The study followed the ARRIVE guidelines¹¹.

1. Animal preparation and experimental protocol

1. Calculate the number of animals as reported previously¹².
   NOTE: Taking into account that the differences in RVSP between CTRL and PAH achieved an effect siz....

Results

For the statistical analyses, each variable was tested for normality and variance using the Shapiro-Wilk test. Data are presented as means ± standard deviation. The unpaired t test was used to compare particle concentrations in the control (CTRL) and pulmonary arterial hypertension (PAH) groups (p<0.05).

Echocardiographic alterations in the animal model of PAH
Figure 4 shows that the PAT/PET ratio was lower in animals with PAH (0.4.......

Discussion

PAH is a progressive disease characterized by excessive vascular remodeling, which promotes an increase in pulmonary vascular resistance, resulting in augmented RV afterload¹⁴. Accordingly, the RVSP represents a functional variable that directly correlates with the severity of PAH¹⁵. Over time, the adaptive response of the RV is compensatory concentric hypertrophy (measured by Fulton's index)¹⁶ and ventricular dilation (measured by RV outflow dia.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Alcohol 70%	Montenegro	ALC70-001
Conductive gel	Mercor	CG-002
Saline solution (NaCl 0,9%)	BioPharma	SAL-009
Instruments
19 G scalp vein set	Descarpack	SVS-19G
Curved kelly forceps	ABC instrumentos cirurgicos	CKF-003
Kelly forceps	ABC instrumentos cirurgicos	KF-004
Needles (18, 22 and 26 G)	Descarpack	NDL-MIX
Petri dish	GlassLab	PD-150
Razor blade	ABC instrumentos cirurgicos	RB-005
Scalpel	ABC instrumentos cirurgicos	SC-006
Scissors	ABC instrumentos cirurgicos	SCIS-007
Suture line	ABC instrumentos cirurgicos	SUT-008
Syringes (1, 3, and 5 mL)	Descarpack	SYR-135
Tracheostomy cannula	MedTech	TC-010
Tweezer	ABC instrumentos cirurgicos	TWZ-011
Drugs
Dextroketamine hydrochloride 5%	Cristália	DXT-005	87.5 mg/kg intraperitoneal
Isoflurane 1 mL/mL 240 mL	Cristália	ISO-240	2.5% inhalation solution
Lidocaine hydrochloride 1%	Cristália	LID-001	400 µL subcutaneous locally
Monocrotaline	Sigma-Aldrich	C2401	60 mg/kg intraperitoneal
Pancuronium bromide 2 mg/mL	Cristália	PAN-002	2 mg/kg intravenous
Sodium heparin 5000 IU/mL	Cristália	HEP-5000	diluted 1:4 in 1 mL
Xylazine hydrochloride 2%	Syntec	XYL-002	12.5 mg/kg intraperitoneal
Equipment
GraphPad Prism 9	GraphPad Software, Inc	GP9
Networked Multiparameter Veterinary Monitor LifeWindow 9x	Digicare Animal Health	LW9x
LabView software	National Instruments, Corp.	LV-2019
MATLAB R2019a (Version) software	The MathWorks, Inc.	MATLAB-2019
Monitor and signal transducer	MedTech	MST-015
Precision scale	Sartorius	PS-020
R540 Mice&Rat Animal Anesthesia Machine	RWD Life Schience Co.,LTD	R540-ANA
Scale	Ohaus	SCL-025
Servo-i mechanical ventilator	Getinge AB	SERVO-I	FiO2 = 21%; tidal volume = 8 ml/kg, respiratory rate = 70 bpm, and PEEP = 3 cmH2O
SonoView software	SONOTEC Ultraschallsensorik Halle GmbH	SV-2020
UGEO HM70A system	Samsung Eletrônica da Amazônia LTDA	HM70A
G*Power 3.1.9.7	University of Düsseldorf, Germany	gpower