Monocrotaline

The protocol for PAH induction was performed in group M animals with injection of
one single intraperitoneal dose of monocrotaline (Sigma Chemical, St Louis, MO,
USA) at the proportion of 60 mg/kg in 1 mol/L in HClph buffer 7.0 with 1 mol/l
of NaOH.^{19 (link)}After receiving monocrotaline, the animals were separated into individual cages
to measure their daily consumption of food preparation. Group M animals were fed
ad libitum; however, their food preparation intake
decreased because of RV dysfunction. Therefore, group C animals received the
mean amount of food preparation consumed by group M animals.
Group C animals underwent an intraperitoneal saline solution injection (0.9%
NaCl), to ensure all study animals would undergo the same degree of stress.

All animal protocols were approved by the Laval University and the IUCPQ Biosafety and Ethics Committes. The Sugen-hypoxia (Su/Hx) and the monocrotaline (MCT) PAH rat models were used in the present study. Male 250–350 g Sprague-Dawley rats (Charles River Laboratories) were used for both animal models. For the MCT model, rats were injected subcutaneously with 60 mg/kg of monocrotaline (Sigma). For the Su/Hx model, rats were injected with 20 mg/kg of SU5416 (Sigma) and put in hypoxia (10% O₂) for 3 weeks. Once PAH was established (after 14 days for MCT and week 5 for Sugen and confirmed by echography), TubA (a specific HDAC6 inhibitor, 25 mg/kg) or vehicle (DMSO) was intraperitoneally administrated every day for 2 weeks. Similarly, Macitentan (30 mg/kg/day) and Tadalafil (10 mg/kg/day) treatments were started 5 weeks after Sugen injection (once PAH was established) and were administered via gavage alone or in combination with TubA for 2 weeks. Adult knock-out (Hdac6^-/Y) mice (aged 8 to 10 weeks) and their control groups (C57BL/6 mice) were exposed or not to chronic hypoxia (10% O₂) for 3 weeks. Mice were genotyped by PCR, as previously described^{41 (link)}.

The protocol was approved by the Animal Research Committee, Central South University, Hunan, China, and carried out in accordance with the Guidelines for Animal Experimentation of Central South University and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85‐23, revised 2011).
Male SD rats (180 g) were obtained from the Hunan SJA Laboratory Animal Co. Rats randomly received an intraperitoneal injection of normal saline (control, n = 12) or monocrotaline (MCT) (Sigma, 60 mg kg⁻¹·rat, n = 24) to induce PAH. The rats in control group were examined at the third week (day 21), and rats in MCT group were randomly examined at the second (day 14, n = 12) and third week (day 21, n = 12).