Animal model
This study was approved by the 2nd Local Ethical Committee in Cracow, Poland (No 60/2016), and was performed in accordance with European Union directives on the care and use of experimental animals. This study was performed using Wistar rats (Experimental Medicine Center of the Medical University of Bialystok, Poland). The animals were kept in standard, controlled conditions with a temperature of 22 ± 2 °C, a 12:12 h light-darkness cycle, and with free access to water and food. A total of 66 Wistar male rats (8 weeks old) were randomly assigned to two groups after being quarantined for 2 weeks, and the date of assignment was designated as day 0. In the study group (monocrotaline-induced PAH model, n = 48), animals were injected intraperitoneally with a single dose of 60 mg/kg of monocrotaline (Sigma Aldrich, Germany) with Dulbecco’s Phosphate Buffered Saline (3 mL/kg) medium (Sigma Aldrich, Germany) to induce PAH [11]. In the control group (non-PAH, n = 18), rats were injected intraperitoneally with Dulbecco’s Phosphate Buffered Saline (3 mL/kg) medium (Sigma Aldrich, Germany).
Experiment’s structure
Experiment’s structure is presented on Fig. 1. There were two endpoints in this study:
(1) early signs of PAH were defined as first morphological lesions of the right ventricle visible in in vivo echocardiography (RVFWTd > 0.7 mm) [12]. A total of 12 animals form the study group that first met this criterion were sacrificed.
(2) heart failure secondary to PAH (end-stage PAH) was defined as a clinical signs of right ventricular insufficiency up to end-stage circulatory and respiratory insufficiency, diagnosed when at least one of the following criteria occurred: [1] dyspnea, defined as increased respiratory effort and alternate respiratory motions of the rat’s thorax and the abdomen, [2] decreased temperature of the lower half of the body, the extremities and the tail, assessed subjectively during physical examination [3], cyanotic eyes [4], significantly decreased physical activity, lethargy. A total of 30 animals with heart failure were sacrificed.
The remaining rats from the study group that did not develop PAH (n = 4) or died in uncontrolled conditions (n = 2) were excluded from the study. As described in literature, the time of occurrence for hemodynamic (and next structural) signs of PAH after single monocrotaline administration in rats is diverse [13]. Due to this fact, rigid timeframe limiting maintenance of animals in the model was abandoned and only strong objective criteria (echocardiography and clinical signs) were used to allocate animals to specific study groups.
Echocardiographic examination
Regularly performed transthoracic echocardiographic examinations were used to assess the dynamics of changes in pulmonary circulation and morphometric cardiac parameters. Examinations were performed using a transducer dedicated for echocardiographic examination of small animals (Mindray M7 with P12-4s, 4.2–11.0 MHz transducer, Mindray Bio-Medical Electronics Co., Shenzhen China) in all animals from both groups on the following days: +5, +10, +15, +18, +20, and then every 3 days and on the day the rats were euthanized. The echocardiography was performed on conscious animals (without any drug administration) who were immobilized manually in a supine position on the dorsum. To ensure the cooperation of animals, rats were subjected to extensive handling. The examination was performed with blinding—that is, the researcher was not aware of whether the animal belonged to the study group or the control group. In addition, digital recordings of the examinations were blindly assessed by two independent researchers to minimize human bias. End-diastolic left ventricular free wall thickness (LVFWTd) was assessed in the long-axis parasternal view, and end-diastolic right ventricular free wall thickness (RVFWTd) was assessed in the apical 4-chamber projection at a 10.0 MHz frequency and a rate of 114 frames/s. In addition, tricuspid annular plane systolic excursion (TAPSE) and pulmonary artery acceleration time normalized to cycle length (PAAT/CL) were measured according to standard guidelines [12, 14].
Hemodynamic examination
All animals underwent invasive hemodynamic testing on the euthanasia day. The rats were placed in the dorsal position on the operating table and anesthetized with pentobarbital sodium (30 mg/kg body weight, Biowet, Poland), which was administered intraperitoneally. Animals were mechanically ventilated during the whole procedure using a pressure-controlled respirator and a mixture of air and oxygen. Anesthesia was maintained by additional bolus doses of pentobarbital sodium as needed. Lidocaine (20 mg/mL, B. Braun Melsungen AG, Germany) was used for local infiltration of the surgical sites. The chest cavity was opened by the left and right mini-thoracotomy in the 6th intercostal space. For the measurement of ventricular systolic and end-diastolic pressure, the heparinized 21G venous cannulas connected to a pressure recording system (Siemens SC 7000, Erlangen, Germany) were introduced simultaneously to the right and left ventricles via their apexes [13]. The pressure transducer was fixed to the operating table and set at the level of the animal’s heart. The values were registered from a stable signal with 300-s periods, and mean values were calculated as output values. After the procedure, animals were euthanized via an overdose of sodium pentobarbital (Biowet, Poland) which was administered intraperitoneally.
Animal dissection
Directly after declaring termination of vital functions, the chest cavity was opened. The inferior vena cava and descending aorta were cannulated, all blood was removed, and infusion of the body using large volumes of Ringer’s solution (Fresenius Kabi, Germany) was conducted in order to clean the organs, including the myocardium, from protein material originating from the vascular bed. Next, the heart, along with proximal parts of main vessels, was dissected, blotted dry, and weighted using an electronic laboratory scale (Ohaus PA224C, Switzerland). Using a stereoscopic microscope, the pulmonary trunk and ascending aorta were dissected, and the diameters of their lumen were measured. Then, the atria and main vessels were separated from ventricles, the mid-diameters of the ventricles were measured, and then the ventricles were weighed en bloc. Next, muscle tissue of the left and right ventricle free wall and interventricular septum were completely separated from each other and the remaining heart structures and were then weighed. The whole wall thicknesses of the left and right ventricle free wall and interventricular septum were measured in their middle sectors. Linear measurements were performed using 0.03-mm precision electronic calipers (YT-7201 YATO, Poland). Small tissue samples from the left ventricle were fixed in 10% buffered paraformaldehyde solution.
Histological analysis
It has been proven that monocrotaline, apart from its pneumotoxicity responsible for PAH induction, also presents a direct cardiotoxic effect expressed by myocarditis [15]. Therefore, paraformaldehyde-fixed left ventricle samples were used to assess the microscopic structure of the myocardium and signs of possible inflammation. Samples were dehydrated in a series of alcohols. Then, they were cleared in xylene and embedded in paraffin blocks. Next, samples were cut into 5-µm thick sections and stained with hematoxylin and eosin (Sigma Aldrich, Germany) according to standard protocols [16]. The infiltration of inflammatory cells was assessed semi-quantitatively (0 = lack, 1 = low, 2 = moderate, 3 = high, 4 = severe) (Fig. 2), and samples with high and severe infiltration were defined as significant myocarditis samples.
Statistical analysis
We performed statistical analyses using StatSoft STATISTICA 13.5 software for Windows (StatSoft Inc, Tulsa, OK). The data are presented as mean values with the corresponding standard deviations (SD) or percentages. All morphometric values were standardized to the rat’s body weight on the euthanasia day (morphometric value [in grams or mm]/body weight [in grams] * 0.01). The Shapiro–Wilk test was used to determine if the quantitative data were normally distributed. In order to compare values between groups, the analysis of variance (ANOVA) or non-parametric Kruskal–Wallis test was used. A detailed comparison was performed using Tukey’s post hoc analyses. An obtained p value of less than 0.05 was considered to be statistically significant.