Forty four male Wistar rats, weighing 180–220 g, were used. All study animals were used in compliance with local and institutional regulations. The study conformed to the Guide for the Care and Use of Laboratory Animals, US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and was approved by the local ethics committee (Second Warsaw Local Ethics Committee for Animal Experimentation). The manuscript follows the recommendations in the ARRIVE guidelines. ITPP was a kind gift from professor Jean-Marie Lehn, UNISTRA, University of Strasbourg to CK.
1. Study protocol
At the age of 4-5 weeks the rats received a subcutaneous injection of MCT (60 mg/kg, Sigma) to induce pulmonary hypertension (n=26) or saline (n=18) as a control group. Twenty four days later the animals from both control (n=13) and MCT (n=14) groups were anesthetized using inhaled 2% isoflurane, underwent echocardiographic examination, followed by hemodynamic evaluation and pO2 measurements. Thereafter they were euthanized and their lungs were processed for histological examination.
The remaining animals from the control (n=5) and MCT (n=5) groups underwent echocardiographic examination, received a single intraperitoneal dose of ITPP (1.5 g/kg b.wt.) and 1 hour later were re-anesthetized, underwent echocardiographic examination, followed by hemodynamic evaluation and pO2 measurements. Thereafter they were euthanized and their lungs were processed for histological examination. A total of 7 rats died in the MCT group, while there were no deaths in the control group.
2. Echocardiographic evaluation
Transthoracic echocardiography was performed using E-cube 15 Platinum (Alpinion Medical Systems) with 17 MHz linear transducer under a light isoflurane sedation. The rats were placed on a heating pad to sustain proper body temperature. Images of the parasternal short-axis view (SAX) at the papillary muscle level, parasternal long axis view (PLAX) and the apical 4-chamber view (4CH) were. LV and RV fractional shortening as well as LV and RV diastolic and systolic wall thickness were assessed using the M-mode in PLAX view. All measurements were obtained by one observer blinded to the study groups.
3. Hemodynamic evaluation
As reported previously , rats were put on a heating pad, anesthetized with 2% isoflurane, intubated and put on an animal ventilator. The upper abdominal cavity was opened and the heart was exposed through cutting of the diaphragm. The left ventricular and subsequently right ventricular apex was punctured with a 25G needle and a microtip pressure-volume (PV) catheter (SPR-838, Millar Instruments; Houston, TX) was inserted into the LV. Its position was established based on pressure and volume signals. After stabilization for 5 minutes, the signals were continuously recorded at sampling rate of 1000/s using an ARIA P-V conductance system (Millar Instruments) coupled to a PowerLab/4SP A/D converter (AD Instruments; Mountain View, CA) and a personal computer. To characterize cardiac function, first the inferior vena cava was compressed for 10 seconds and then released to achieve reduction and augmentation of venous return and cardiac preload, respectively. Heart rate, maximal LV and RV systolic pressure (ESP), end-diastolic pressure (EDP), maximal slope of systolic pressure increment (+dP/dt max) and diastolic pressure decrement (−dP/dt max), ejection fraction (EF), end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and cardiac output (CO) were computed using a cardiac P-V analysis program (PVAN3.2, Millar Instruments). Indexes of contractility and stiffness [slope of end-systolic and end-diastolic P-V relations (ESPVR and EDPVR)] were also calculated using PVAN3.2.
4. pO2 measurements
Myocardial tissue pO2 was measured using a fiberoptic oxygen-sensing device, the OxyLite Pro pO2 monitor (Oxford Optronics Ltd., Oxford, UK). This device measures pO2 by determining the oxygen-dependent fluorescent lifetime of ruthenium chloride. Probes are supplied precalibrated by the manufacturer. The tip of an optical fiber probe is covered with ruthenium chloride, which fluorescence lifetime is O2-dependent and is inversely proportional to the pO2 in the tissue. Following excitation by a flash of green light, the measured half-life of the phosphorescence signal can be quantitatively related to the oxygen tension.
The rats were anesthetized using inhaled isoflurane (2%) and connected to a ventilator. The heart was exposed by left-sided thoracotomy, and a 20-gauge needle was used to pierce the epicardium and guide the probe into the myocardium immediately beneath the epicardium. A 100-µm diameter sensing tip was inserted into three locations in the cardiac RV and LV as well as in the liver and spleen. The pO2 signal was recorded until the signal was stable. Subsequently data were averaged for each location.
Under baseline conditions the animals were mechanically ventilated with 30-40% inspiration oxygen, which is above the ambient air content of oxygen (21%) with spontaneously breathing. The higher inspiration oxygen levels are necessary to compensate for the known ventilation-perfusion defects caused by anesthesia and mechanical ventilation, especially in diseased animals. Mechanical ventilation with a FiO2 of 21% resulted in an arterial tension of approximately 80 mmHg, below the 100 mmHg normally obtained with spontaneous breathing, indicating that the use of ambient air oxygen levels with mechanical ventilation and anesthesia was indeed associated with insufficient blood oxygenation . Finally 100% oxygen was used to ventilate the rat lungs and the pO2 measurements were repeated.
5. Histological lung analysis
The lungs were inflated with OCT (Optimal Cutting Temperature) compound and then placed in plastic molds filled with OCT and snap-frozen in liquid nitrogen. All tissues were maintained at −80°C until further analysis.
The representative lung parts were cut into 5 µm-thick sections using Cryostat Microm HM550 (Thermo Fisher Scientific, Massachusetts, USA) and stained with the hematoxylin and eosin (H&E) stain for microscopic analysis. Arterioles with an external diameter of 30-90 µm were assessed and % wall thickness was expressed as (2×wall thickness / external vessel diameter).
6. Statistical analysis
Shapiro–Wilk test was used to test normality of data distribution. Homogeneity of variances was tested by Bartlett’s. Normally distributed data were expressed as means ± SEM. One-way ANOVA was used to test differences between groups. Tukey post hoc test was used to compare data pairs. Non-normally distributed data were presented as median ± inter-quartile ranges + outliers. Subsequently statistical analysis of differences was tested using non-parametric methods (Mann–Whitney test to compare two groups or Kruskal–Wallis ANOVA to compare three or more groups followed by Dunn's post hoc test). Pearson correlation analysis was used to analyze the correlations. Differences were considered significant when P < 0.05. Statistical analyses were performed using SigmaPlot version 14.