Single intraperitoneal injection of monocrotaline as a novel large animal model of chronic pulmonary hypertension in Tibet minipigs

Abstract

Objective: The purpose of this study was to establish an animal model of chronic pulmonary hypertension with a single-dose intraperitoneal injection of monocrotaline (MCT) in young Tibet minipigs, so as to enable both invasive and noninvasive measurements and hence facilitate future studies.

Methods: Twenty-four minipigs (8-week-old) were randomized to receive single-dose injection of 12.0 mg/kg MCT (MCT group, n = 12) or placebo (control group, n = 12 each). On day 42, all animals were evaluated for pulmonary hypertension with conventional transthoracic echocardiography, right heart catheterization (RHC), and pathological changes. Findings of these studies were compared between the two groups.

Results: At echocardiography, the MCT group showed significantly higher pulmonary arterial mean pressure (PAMP) compared with the controls (P<0.001). The pulmonary valve curve showed v-shaped signals with reduction of a-waves in minipigs treated with MCT. In addition, the MCT group had longer pulmonary artery pre-ejection phases, and shorter acceleration time and ejection time. RHC revealed higher mean pulmonary arterial pressure (mPAP) in the MCT group than in the control group (P<0.01). A significant and positive correlation between the mPAP values and the PAMP values (R = 0.974, P<0.0001), and a negative correlation between the mPAP and ejection time (R = 0.680, P<0.0001) was noted. Pathology demonstrated evidence of pulmonary vascular remodeling and higer index of right ventricular hypertrophy in MCT-treated minipigs.

Conclusion: A chronic pulmonary hypertension model can be successfully established in young minipigs at six weeks after MCT injection. These minipig models exhibited features of pulmonary arterial hypertension that can be evaluated by both invasive (RHC) and noninvasive (echocardiography) measurements, and may be used as an easy and stable tool for future studies on pulmonary hypertension.

Figure 1. X-ray visualization for positioning the catheter tip in the main pulmonary artery of a minipig.

Figure 2. M-mode imaging in all minipigs at 6 weeks after MCT or placebo injection.

Panel A: a-wave in the control group. Panel B: The V-shaped signals with no a-wave in the MCT group (arrows).

Figure 3. Measurements of the velocity and pressure gradient of the tricuspid regurgitation (Panel A) and pulmonary valve regurgitation (Panel B) using continuous-wave Doppler in a minipig at 6 weeks after placebo injection in the control group.

Figure 4. Measurements of pulmonary flow using pulsed-wave Doppler in a minipig at week 6 of the study.

Panel A: pulmonary artery valve pre-ejection phase (PEP, time from the onset of QRS wave to the onset of pulmonary valve flow on Doppler echocardiogram) in MCT group; Panel B: pulmonary artery valve flow acceleration time (PAAT, time from the onset of pulmonary valve flow to peak velocity by pulsed-wave Doppler recording) in MCT group; Panel C: pulmonary artery valve ejection time (ET, time interval between the onset and end of the systolic flow velocity) in MCT group and Panel D: PAAT in the control group, measured based on the QRS wave.

Figure 5. Linear regression plot showing a negative correlation between the mPAP values measured by invasive right heart catheter and the ET values estimated by echocardiography (R = 0.680, P<0.0001).

mPAP = mean pulmonary arterial pressure; ET = pulmonary artery valve ejection time. •,the control group;▴,MCT group.

Figure 6. Typical microscopic views of pulmonary hypertension (PH) in a 14-week-old pig at 6 weeks after injection of monocrotaline; Panel A, control group; Panels B-F, MCT group.

Panel A: Typical microscopic view of a small muscular pulmonary artery (arrows) in a 14-week-old minipig from the control group. The lumen of normal pulmonary artery is surrounded by a uniform thin wall with no fibrosis or inflammatory cell infiltration (Hematoxylin and Eosin staining; original magnification:×200; scale bar = 100 µm); Panel B: The vessel lumen is narrowed, occluded, twisted or deformed with formation of plexiform lesions (arrows) as a result of proliferating and hypertropic endothelial and smooth muscle cells, intimal fibrosis and accompanying hyalinization (Hematoxylin and Eosin staining; original magnification:×400; scale bar = 50 µm). Panel C: Pulmonary vascular remodeling in the lung tissue. There was intimal thickening accompanied with plexiform lesions (arrows) (Hematoxylin and Eosin staining; original magnification:×100; scale bar = 200 µm). Panel D: Uneven thickening of the pulmonary arterial wall (arrows). (Hematoxylin and Eosin staining; original magnification:×100; scale bar = 200 µm); Panel E: The presence of large amount of mucus (arrows) in bronchioles led to luminal obstruction and lymphocyte infiltration. In the periphery of lungs, congestion of capillaries, thickening of interstitium and focal lymphocyte infiltration were observed (Hematoxylin and Eosin staining; original magnification:×100; scale bar = 200 µm). Panel F: Parabronchus arteries (arrows) were largely occluded owing to extensive neointimal proliferation and medial hypertrophy accompanied with hyalinization (Hematoxylin and Eosin staining; original magnification:×100; scale bar = 200 µm).