Primary pulmonary arterial hypertension: Protocol to assess comprehensively in the rat the response to pharmacologic treatments

Abstract

The identification of new treatments for primary pulmonary arterial hypertension (PAH) is a critical unmet need since there is no a definitive cure for this disease yet. Due to the complexity of PAH, a wide set of methods are necessary to assess the response to a pharmacological intervention. Thus, a rigorous protocol is crucial when experimental studies are designed. In the present experimental protocol, a stepwise approach was followed in a monocrotaline-induced PAH model in the rat, moving from the dose finding study of treatment compounds to the recognition of the onset of disease manifestation, in order to identify when to start a curative treatment. A complete multidimensional evaluation of treatment effects represented the last step. The primary study endpoint was the change in right ventricular systolic pressure after 14 days of treatment; echocardiographic and biohumoral markers together with heart and pulmonary arterial morphometric parameters were considered as secondary efficacy and/or safety endpoints and for the evaluation of the biologic coherence in the different results.

Keywords: Alanine transaminase; Blood pressure; Cardiac biomarkers; Creatinine; Echocardiography; Heart histology; Monocrotaline; Morphometric analysis of pulmonary arteries; Randomization; Right ventricular systolic pressure; Sample size.

Graphical abstract

Fig. 1

Two-dimensional (2D) and M-mode parasternal long-axis view showing in particular the increased end-diastolic thickness of the right ventricular free wall (RVThd) in a control (A), CTRL = 0.3 mm, and in a monocrotaline treated rat (B), MCT = 1 mm.

Fig. 2

Four-apical chamber view: The dotted line correspond to the right ventricle end-diastolic diameter (RVEDd) in a control (CTRL) (A) and a monocrotaline (MCT) treated rat (C). A yellow spline delineate the endocardial end-diastolic (A and C) and end-systolic (B and D) right ventricular borders for the calculation of the respective right ventricle areas (CTRL: RVAd = 0.31 cm², RVAs = 0.07 cm²; MCT: RVAd = 0.62 cm², RVAs = 0.39 cm²). The dotted white line indicate the right ventricle diastolic diameter (CTRL: RVD = 3 mm; MCT = 5 mm).

Fig. 3

The tricuspid annulus plane systolic excursion (TAPSE) in a control (A), CTRL = 4.5 mm, and in a monocrotaline treated rat (B), MCT = 1.3 mm, expression of an impaired right ventricular systolic function in the last.

Fig. 4

Pulmonary artery acceleration time in control (CTRL) (A). In normal conditions as in CTRL rats the pattern of systolic flow is symmetrical (PAAT = 37 ms); in case of a moderate and severe increase in RV systolic pressure as in monocrotaline (MCT) treated rats, the peak of the Doppler flow shifts toward the early systole resulting in a mid-systolic “notch” (arrow in B); a marked increase in RV systolic pressure is reflected by a reduction in echo signal with an asymmetric wave (PAAT = 18 ms).

Fig. 5

Left ventricular (LV) volumes, end-diastolic volume EDV (A), end-systolic volume ESV (B) and LV ejection fraction (EF) were calculated by the modified simple plane Simpson’s rule from the parasternal long-axis view.

Fig. 6

Right ventricle systolic pressure (RVSP) measured with a tip-transducer catheter (Millar) in a control (A), CTRL = 25 mmHg, and in a monocrotaline treated rat (B), MCT = 100 mmHg.

Fig. 7

Representative images of intra-acinar pulmonary arterioles, 15–60 μm in diameter, showing typical non-muscularized (N), partially muscularized (P) and muscularized (M) morphology (x200).

Fig. 8

Right ventricular histology in control (CTRL) rats and in rats with pulmonary artery hypertension two and five weeks after monocrotaline injection (MCT).