Urethane

To assess the pathobiological effects of urethane, all mice were subjected to once weekly, i.p. injections of urethane (Sigma) at 1 g/kg diluted in 1X PBS for a total of 7 weeks [19 (link)]. Body weight, physical condition, and behavior were assessed at least 3 days per week throughout the course of each study. Mice were harvested at 7 or 14 weeks post-urethane exposure or when moribund. On the day of harvest, mice were euthanized via CO₂ asphyxiation and approximately 1 ml of blood was collected by cardiac puncture. The lung, liver, and spleen were harvested from each animal. Portions of each tissue were placed either in formalin for histopathology or frozen and stored in the −80 for RNA/protein analysis. Samples of spleen were also prepared for flow cytometry.

One month after IR, or sham surgery, the animals were anesthetized (urethane, 1 g/kg, i.p., Sigma-Aldrich, St. Louis, USA) and had their cardiac contractility evaluated by measuring the left ventricle (LV) pressure under baseline conditions and also during the infusion of increasing doses of dobutamine. Following, an additional dose of urethane (1g/kg, i.p., Sigma-Aldrich, St. Louis, USA) was administered for the animal euthanasia and subsequently tissue and blood sampling for histological and molecular analysis were collected immediately.

Ninety-six BALB/C mice (4–6 weeks old, same number of females and males) were purchased from the Guangdong Provincial Experimental Animal Center. They were fed for 3 weeks and then divided into the negative control and urethane induction groups. Mice in the urethane induction group were intraperitoneally injected with 1g/kg urethane (Sigma-Aldrich, St. Louis, MO, USA) once a week for 4 weeks, and the control mice were given same injections of normal saline. The mice were then fed for another 6, 9, or 12 weeks. Sixteen mice in each group were sacrificed; serum and lung tissues were collected. LncRNA expression analyses were performed on serum and tissues (n=12 per group) and pathological examination was performed on lung tissues (n=4 per group).

Data were collected from 11 female Wistar rats (Rattus norvegicus, body mass = 254 ± 11 g), from the own breed of the animal facility of the Vrije Universiteit Amsterdam. All animals were housed in pairs with access to food and water ad libitum. Rats were anaesthetized by an i.p. injection of urethane (Sigma‐Aldrich) (initial dose: 1.2 ml/100 g body mass, 12.5% urethane solution; additional doses to maintain suppression of withdrawal reflexes: 0.2 ml). Animal core body temperature was maintained with heating pads and lamps. Subcutaneous injections of lactated Ringer solution (1 ml/h) were given to maintain fluid balance. At the end of the experiment, rats were killed with an overdose of intracardially injected pentobarbitone sodium (Euthasol 20%, Ast Farma B.V) followed by a double‐sided pneumothorax.

During the whole experiments, the rats were anesthetized. Anesthesia was induced with an intraperitoneal injection of alpha‐chloralose (50 mg/Kg b.w.) (Sigma‐Aldrich, St. Louis, MO) plus urethane (600 mg/Kg b.w.) (Sigma‐Aldrich) and maintained with following administration of urethane alone (100 mg/kg, i.v., every hour) through a catheter placed in the right femoral vein. As previously described (Lapi et al. 2013, 2014, 2017), rats were tracheotomized, intubated and mechanically ventilated with room air, and a supplemental mixture of O₂/CO₂ (end‐tidal CO₂ was continuously measured by a CO₂ analyzer and a respirator was adjusted to maintain end‐tidal CO₂ from 4.5% to 5.0% and to keep arterial blood gas tension within the normal range).
A polyethylene cannula was placed in the left femoral artery to measure Mean (intra) arterial blood pressure (MABP).
The rats were secured to a heating stereotaxic frame which allowed to maintain the body temperature at 37.0 ± 0.5°C measured with a rectal probe.