Background

Pulmonary hypertension (PH) encompasses a group of pulmonary vascular disorders with varying etiologies. Depending on the cause and site of the pathogenesis, PH is classified into five groups and many subgroups (Sahay, 2019; Sarah et al., 2021). Despite the differences in the pathological basis of various groups of PH, the disease shares a common definition: a pathological condition in which the mean pulmonary arterial pressure (mPAP) is greater than 25 mmHg at rest or 30 mmHg during exercise (Galiè et al., 2016; Simonneau et al., 2019). Of the five groups of PH, pulmonary arterial hypertension (PAH) and its various subgroups fall in Group I (Wijeratne et al., 2018). In the case of PAH, pulmonary arteries and arterioles undergo pathological changes and thus become thicker and stiffer, resulting in elevated mPAP (Lai et al., 2014; Lan et al., 2018). In other forms of PH (Groups II–V), lung or heart diseases or other conditions increase mPAP (Galiè et al., 2016; Simonneau and Hoeper, 2019).

Because the chief manifestation of PH is increased mPAP, animal models for PH are developed to recapitulate elevated mPAP (Liu and Yan, 2022; Wu et al., 2022). Of the various animal models for PH, Sugen/hypoxia and monocrotaline-induced rat models of PH are two commonly used models (Stenmark et al., 2009; Vitali et al., 2014; Sztuka and Jasinska-Stroschein, 2017) (Figure 1). To develop hypoxia-based models, animals are first treated with a subcutaneous injection of 20 mg/kg Sugen 5416, a vascular endothelial growth factor receptor antagonist. Then, they are housed in 10% oxygen for 3–4 weeks (Bhat et al., 2017; Honda et al., 2020). A right heart catheterization (RHC) is performed to measure mPAP soon after the removal of the animals from the hypoxic environment or after three weeks of hypoxia followed by one week of normoxia (V. Gupta et al., 2013; Nahar et al., 2014; N. Gupta et al., 2015 and 2017; Nahar et al., 2016; Rashid et al., 2017, 2018a, 2018b and 2019; Keshavarz et al., 2019; Al-Hilal et al., 2021). In the case of monocrotaline-based models, animals receive a single subcutaneous injection of 30–80 mg/kg monocrotaline (Lachant et al., 2018; Jing et al., 2020), an alkaloid that causes PH by injuring pulmonary arterial endothelium (Xiao et al., 2017). The mPAP is measured 3–4 weeks after injection (Gomez-Arroyo et al., 2012; N. Gupta et al., 2017). In both models, mPAP rises to as high as 40–60 mmHg.

Figure 1. Sugen-5416/Hypoxia-induced pulmonary arterial hypertension (PAH) model and monocrotaline (MCT) model. For the development of Sugen/hypoxia-based models, animals are first treated with a subcutaneous injection of 20 mg/kg Sugen 5416 and then are housed in 10% oxygen for 3–4 weeks. A right heart catheterization (RHC) is performed to measure mean pulmonary arterial pressure (mPAP) soon after the removal of the animals from the hypoxic environment or after three weeks of hypoxia followed by one week of normoxia. In case of monocrotaline-based models, animals receive a single subcutaneous injection of 30–80 mg/kg monocrotaline. The mPAP is measured 3–4 weeks after MCT injection.

In fact, over the past several decades, these models have been used for studying PH pathophysiology and investigating the efficacy of drugs in reducing mPAP (V. Gupta et al., 2013; Maarman et al., 2013; Nahar et al., 2014; N. Gupta et al., 2015 and 2017; Nahar et al., 2016; Rashid et al., 2017, 2018a, 2018b and 2019; Keshavarz et al., 2019; Al-Hilal et al., 2021; Dignam et al., 2022). For mechanistic studies or to evaluate drugs’ efficacy in ameliorating pathological alterations, pulmonary arteries/arterioles are collected, and various histopathological studies are performed. Further, PH is treated with vasodilators to reduce elevated mPAP (Rose-Jones and McLaughlin, 2015). Reduced mPAP increases survival by reducing right ventricular hypertrophy, the chief cause of death in PH patients (Ryan et al., 2015; Zhu et al., 2019; Benza et al., 2022). However, vasodilator therapy also reduces blood pressure, resulting in peripheral hypotension–induced side effects (Packer, 1985; Rich, 2009; Humbert et al., 2014; Chida-Nagai et al., 2020). Thus, for studying the efficacy of investigational and commercially available drugs in animal models of PH, mPAP is measured as a functional endpoint for therapeutic outcomes (V. Gupta et al., 2013; Maarman et al., 2013; Nahar et al., 2014; N. Gupta et al., 2015 and 2017; Nahar et al., 2016; Rashid et al., 2017, 2018a, 2018b and 2019; Keshavarz et al., 2019; Suen et al., 2019; Al-Hilal et al., 2021; Beckmann et al., 2022; Dignam et al., 2022). Mean system arterial pressure (mSAP) is also measured to evaluate the influence of the drug on peripheral blood pressure or to assess whether the drug or formulation has pulmonary selectivity, by comparing the extent of reduction in mPAP and mSAP in the same rats. In a series of studies, we have shown that pulmonary selectivity can be evaluated by comparing mPAP and mSAP upon administration of various drug therapies to PH rats (V. Gupta et al., 2013; Maarman et al., 2013; Nahar et al., 2014; N. Gupta et al., 2015 and 2017; Nahar et al., 2016; Rashid et al., 2017, 2018a, 2018b and 2019; Keshavarz et al., 2019; Al-Hilal et al., 2021; Dignam et al., 2022).

To measure mPAP, an RHC is performed to place the catheter in the pulmonary artery. Likewise, for mSAP, a catheter is placed in the carotid artery of the same rats that underwent RHC. In our published studies, we inserted polyethylene (PE) catheters in both pulmonary and carotid arteries (N. Gupta et al., 2014; Nahar et al., 2014; Rashid et al., 2018b). However, insertion of PE catheter in pulmonary arteries is challenging because catheters require a specialized curvature for maneuvering from the jugular vein through the right atrium and right ventricle and finally to the pulmonary artery. Further, exposure to the jugular vein for RHC and carotid artery requires microsurgical procedures and specialized tools. Similarly, recording mPAP and mSAP entails using a specialized instrument. PowerLab, a system connected to bridge amplifiers and equipped with Lab Chart Pro software, is universally used to record mPAP and mSAP (Doggett et al., 2018). However, this system must be calibrated and set up accurately.

While we and others have been performing RHC to measure mPAP and mSAP using PowerLab systems for many years now (Rey et al., 2012; V. Gupta et al., 2013; Nahar et al., 2014; N. Gupta et al., 2015 and 2017; Nahar et al., 2016; Rashid et al., 2017, 2018a, 2018b and 2019; Keshavarz et al., 2019; Al-Hilal et al., 2021), there are no published protocols that list the surgical tools, explain the steps for preparation and insertion of catheters, and describe the setup and calibration of PowerLab lab data acquisition systems. In the absence of such a protocol, a new investigator may take months to establish the methodologies for RHC in rats and recording of mPAP and mSAP using the PowerLab system. Importantly, the unavailability of a thorough protocol is a major reason for significant delays in training new lab personnel in RHC or the transfer of methods from one investigator to another in the same lab. Thus, there is an important need for a detailed protocol for performing RHC in rats and measuring such; in this protocol paper, we put together all steps and processes involved in RHC and measurement of mPAP and mSAP, so that investigators with no training can perform the surgery and record mPAP and mSAP. This protocol can also be deployed in performing RHC in animal models for heart failure, congenital heart disease, heart valve disorders, and heart transplantation.