Sodium pentobarbital

Mice on a C57BL/6 background with a selective Atg7 gene deletion in VSMCs (Atg7^F/F SM22α-Cre⁺) (Michiels et al., 2015 (link)) and wild-type littermates without the Atg7 deletion but expressing SM22α-Cre (Atg7^+/+ SM22α-Cre⁺) were housed in the animal facility of the University of Antwerp in standard cages with a 12:12 h light-dark cycle and had access to regular chow and water ad libitum. At the age of 2 months, animals [both male (n = 15) and female (n = 3) mice] were euthanized by perforating the diaphragm after anesthesia with pentobarbital sodium (Sanofi, 250 mg kg-1, i.p.). The femoral artery was carefully isolated systematically and stripped from adherent tissue. Atg7^F/F SM22α-Cre⁺ mice and Atg7^+/+ SM22α-Cre⁺ mice were dissected in parallel. Vessel segments were immersed in Krebs Ringer (KR) solution containing (in mM) 118 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 KH₂PO₄, 1.2 MgSO₄, 25 NaHCO₃, 0.025 CaEDTA and 11.1 glucose; pH 7.4 at 37°C and continuously aerated with 95% O2/5% CO₂. High K⁺ solutions were prepared by replacing NaCl with equimolar KCl. 0Ca²⁺ solution was prepared by omitting Ca²⁺ from the KR solution and adding 1 mM EGTA (Sigma-Aldrich) to chelate remaining Ca²⁺ residues. All animal procedures were approved by the ethics committee of the University of Antwerp.

Male C57BL/6J mice (6 months old; Charles River Laboratories, France) were housed in the animal facility of the University of Antwerp in standard cages with 12 h–12 h light-dark cycles and had free access to regular chow and tap water. The animals were euthanized by perforating the diaphragm while under anesthesia [sodium pentobarbital (Sanofi, Belgium), 75 mg/kg i.p.]. The ascending, descending and infrarenal aorta were carefully removed and stripped of adherent tissue. The tissue was cut into segments of 2 mm. The segments were immersed in Krebs Ringer (KR) solution (37°C, 95% O2/5% CO2, pH 7.4) containing (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaEDTA 0.025 and glucose 11.1. The study was waived by the Ethics Committee for Animal Research at the University of Antwerp according to article 3 of the EU legislation (L 276/38, 2010).

30 min after coming out of the hyperbaric chamber, all the animals were anesthetized by induction with isoflurane (Bellamont, firstly at 5% then 2%), then by intraperitoneal injection (1 ml syringe, Omnican, B. Braun, Melsungen, Germany) of a mixture of ketamine (Imalgene 1000, 100 mg/kg, AstraZeneca, London, UK), acepromazine (Calmivet, 1,65 mg/kg, Vétoquinol S.A., Lure, France) and xylazine (Rompun 2%, 16 mg/kg, Bayer HealthCare, KVP, Kiel, Germany).
At the end of the experiment, rats were sacrificed by an injection of sodium pentobarbital (200 mg/kg IP; Sanofi, Paris, France).

Coming out of the hyperbaric chamber after 60 min, all the animals were anesthetized by induction with isoflurane (Bellamont, first at 5% and then at 2%), then by intraperitoneal injection (1-ml syringe, Omnican^®, B. Braun, Melsungen, Germany) of a mixture of ketamine (Imalgene 1000, 100 mg/kg, AstraZeneca, London, United Kingdom), acepromazine (Calmivet^®, 1.65 mg/kg, Vétoquinol S.A., Lure, France) and xylazine (Rompun^® 2%, 16 mg/kg, Bayer HealthCare, KVP, Kiel, Germany).
At the end of the experiment, rats were sacrificed by an injection of sodium pentobarbital (200 mg/Kg IP; Sanofi, Paris, France).

Mice were deeply anesthetized with sodium pentobarbital (i.p. 100 mg/kg, Sanofi) and decapitated. The brain was immediately removed, and the hippocampi were dissected out and rapidly frozen on dry ice. 2-AG was extracted from the hippocampus as previously described^{78 (link)}. Samples were weighed and placed into borosilicate glass culture tubes containing 2 ml of acetonitrile (ACN) with 186 pmol [²H₈] 2-AG. They were homogenized using IKA homogenizer and kept overnight at −20 °C to precipitate proteins and subsequently centrifuged at 1500 × g for 3 min. The supernatants were transferred to a new glass tube and evaporated to dryness under N₂ gas. The samples were resuspended in 500 µl of methanol to recapture any lipids adhering to the glass tube and dried again under N₂ gas. Dried lipid extracts were suspended in 50 µl of methanol and stored at −80 °C until analysis. The content of 2-AG was determined using isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry (LC-MS/MS)^{79 (link)} and the content of both 2-AG and 1(3)-AG isomers were pooled for quantification, given the potential isomerization between 1(3)-AG and 2-AG following ACN precipitation. All the analysis was performed blinded to experimental conditions.