To the Editor:
Pulmonary hypertension (PH) is a disease characterised by progressive remodelling of the pulmonary vasculature eventually leading to right heart failure. Various animal models have been used to mimic the disease, involving pigs, dogs, rats and mice [1]. The most commonly used model is the monocrotaline (MCT) rat model. In this model MCT is injected subcutaneously and becomes metabolically activated, as a pyrrolizidine alkaloid, by hepatic cytochrome P450 3A [2, 3]. The active MCT pyrrole is pneumotoxic and damages the pulmonary artery endothelial cells (PAECs), which leads to a disturbed barrier function [4]. Other features of MCT-induced pulmonary vascular remodelling are arterial medial hyperplasia of axial arteries, interstitial oedema, adventitial inflammation, haemorrhage and, eventually, fibrosis [1, 2, 5, 6]. As a result, pulmonary vascular resistance (PVR) increases and the right ventricle compensates by hypertrophy and eventually fails [7, 8].
Besides the MCT PH rat model, chronic hypoxia with or without Sugen 5416 and pulmonary artery banding are used to study experimental PH [1]. The PH animal model of choice is mostly dependent on the research question that needs to be answered. Ideally, an animal model would recapitulate the progressive and irreversible pulmonary vascular remodelling, which is the hallmark of human PH [1, 4, 9]. However, none of the animal models fulfil this criterion. Concerns have been raised about the MCT rat model since many therapies were successful in MCT rats but not in humans with PH [4].
We, therefore, investigated the long-term progression and reversibility of MCT-induced PH in rats over 12 weeks, using a dose of 40 mg·kg−1 in a randomised placebo-controlled study design. Since it is known that a high dose of MCT (60 or 80 mg·kg−1) is fatal within 3–6 weeks, the only possible way to study the long-term effects of MCT was to use a lower dose [7, 8, 10, 11]. Alterations in the lungs and heart were measured 4, 8, and 12 weeks after administration of MCT or saline.
We randomly assigned 22 male Wistar rats (Harlan Laboratories, Horst, The Netherlands) to a subcutaneous injection of either 40 mg·kg−1 MCT or saline when their body weight was 175–200 g. Rats were sacrificed at either 4 weeks (n = 3 control, n = 5 MCT), 8 weeks (n = 3 control, n = 4 MCT), or 12 weeks (n = 3 control, n = 4 MCT). Echocardiography was performed 3 weeks after MCT injection, to determine baseline haemodynamics, and on the day of sacrifice. Before sacrifice, all animals underwent a right heart catheterisation to construct right ventricle pressure–volume loops. Internal organs were harvested for histology. All methods have been described in detail previously [12]. Data are presented as mean±sd. The study was approved by the local animal ethics committee.
At baseline echocardiography, 3 weeks after MCT injection, all MCT rats had a higher PVR index compared with controls (mean of the three MCT groups: 4.33±2.58 mmHg·mL−1·min−1·mg−1 versus controls: 0.50±0.13 mmHg·mL−1·min−1·mg−1; p<0.001) with decreased cardiac index (0.14±0.06 versus 0.29±0.04 mL·min−1·g−1; p<0.001) and lower tricuspid annular plane systolic excursion (TAPSE) (2.3±0.5 versus 3.6±0.3 mm; p<0.001). Body weight was significantly lower in the MCT rats sacrificed at 4 weeks (311±21 g) compared with controls (354±6 g; p<0.05). After 8 and 12 weeks body mass of the MCT rats (405±27 and 455±27 g, respectively) was similar to age-matched controls (408±36 and 496±46 g, respectively).
We observed a significantly higher PVR index in the MCT rats sacrificed at 4 weeks compared with controls (fig. 1a) with an increased right ventricle systolic pressure (57.8±10.3 versus 15.6±2.3 mmHg; p<0.05). Although stroke volume and heart rate were decreased, cardiac index was relatively preserved (fig. 1b–d). However, cardiac index was negatively correlated to estimated right ventricle systolic pressure in all MCT rats (r = -0.65; p = 0.02, fig. 1e) but not in control rats (r = 0.30; p = 0.46, data not shown). Diminished right ventricle function compared with controls was seen by means of lower TAPSE (2.0±0.6 versus 3.7±0.3 mm; p<0.05), a trend to increased right ventricle wall thickness (1.4±0.2 versus 1.0±0.1 mm; p = 0.07) and increased right ventricle end-diastolic diameter (6.2±1.3 versus 3.5±0.5 mm; p<0.05). In line with these findings, invasive haemodynamic measurements demonstrated high right ventricle afterload (Ea), increased right ventricle diastolic stiffness (Eed) and increased right ventricle systolic elastance (Ees, a measure of contractility) in MCT rats sacrificed at 4 weeks compared with age-matched controls (fig. 1f–h). Because Ea and Ees are increased to the same extent, ventriculo-arterial coupling, represented by Ees/Ea ratio, is preserved in all animals (fig. 1i). Interestingly, all aforementioned parameters are normalised at 8 and 12 weeks after MCT injection (fig. 1a–i).
Signs of right ventricle adaptation to high afterload after 4 weeks of MCT were also found at autopsy, since heart weight was increased compared with controls (1.54±0.02 versus 1.27±0.08 g; p<0.05). This was mainly due to an increase in right ventricle weight indicated by the high right ventricle/left ventricle+septal weight ratio (0.63±0.11 versus 0.28±0.06 in controls; p<0.05). Lung weight was greater in MCT rats sacrificed at 4 weeks (1.73±0.06 versus 1.36±0.19 g; p<0.05), but liver weight was reduced compared with controls (11.8±0.4 versus 14.8±1.1 g; p<0.05). In MCT rats sacrificed at 8 and 12 weeks, the right ventricle/left ventricle+septal weight ratio was decreased compared with MCT rats sacrificed at 4 weeks, but not completely normalised (8 weeks: 0.37±0.03 and 12 weeks: 0.40±0.07, both p<0.01 versus 4 weeks MCT). In addition, lung wet weight reduced to 1.74±0.17 g in MCT rats sacrificed at 8 weeks and 1.59±0.10 g in MCT rats sacrificed at 12 weeks. Liver weight increased to control values in MCT rats sacrificed at 8 and 12 weeks (14.0±0.67 and 15.2±1.4 g, respectively).
Lung histology showed increased media muscularisation in the smallest arterioles (<40 μm) after 4 weeks of MCT (32±4%; p<0.01 versus controls) which returned to control values (24±1%) after 8 weeks (25±6%) and 12 weeks after MCT administration (21±7%; p<0.05 versus 4 weeks MCT) (fig. 1j–l). Right ventricle cardiomyocyte cross-sectional area was considerably increased in the MCT rats sacrificed at 4 weeks (388±91 μm2) compared with controls (258±44 μm2; p<0.001) and remained at that level at 8 and 12 weeks (344±76 μm2 and 358±57 μm2, respectively). Although right ventricle cardiomyocyte size was greater in MCT rats, the number of capillaries was similar in all MCT groups (mean 1.2±0.5 capillaries per cardiomyocyte). Right ventricle fibrosis was similar at all time points in MCT animals and controls (0.6±0.3 versus 0.6±0.4% collagen; p = 0.88). In addition, right ventricle inflammation, expressed as the number of CD45 positive leukocyte cells per unit area was similar in all MCT animals (mean 25.8±15.4 CD45+ nuclei·mm−2) and similar to controls (mean 19.5±15.8 CD45+ nuclei·mm−2; p = 0.17), indicative of a compensated rather than a failing right ventricle.
The MCT model is considered, by some, to be a toxic model and it has been suggested that MCT rats die from hepatic veno-occlusive disease with liver failure instead of right ventricle failure [13]. In the current study, liver weight was lower in MCT rats sacrificed at 4 weeks compared with controls. However, right ventricle systolic pressure and PVR are largely increased in these rats and increased pulmonary arteriolar muscularisation is found, which cannot be explained by liver damage alone.
To conclude, we demonstrated that 40 mg·kg−1 MCT induces acute muscularisation of the smallest pulmonary arterioles, together with high PVR and right ventricle hypertrophy at 4 weeks after MCT administration. However, at 8 and 12 weeks after MCT administration, the pulmonary arteriolar abnormalities were restored accompanied by normalisation of right ventricular function, although cardiomyocyte hypertrophy was maintained. This shows that MCT induced PH is reversible after 4 weeks and does not resemble the progressive nature of human PH. Therefore, this model is not suitable for therapeutic studies after 4 weeks of a low dose of MCT.
Footnotes
Conflict of interest: None declared.
- Received January 21, 2013.
- Accepted March 24, 2013.
- ©ERS 2013