Chirocaine

The ESB study medication will be 20 mL of levobupivacaine 0.25% (Chirocaine, AbbVie, Lake Bluff, IL, USA). The sham group received 20 mL of NaCl 0.9% (B. Braun, Melsungen, Germany).

Briefly, rabbits were administered induction anesthesia with IM ketamine (35 mg/kg Nimatek^®, Eurovet Animal Health BV, Bladel, The Netherlands) and xylazine (5 mg/kg XYL-M^® 2%, VMD, Arendonk, Belgium), antibiotic prophylaxis (10 mg/kg enrofloxacin, Baytril^® 2.5% SC, Bayer, Diegem, Belgium), tocolysis (10 mg/kg medroxyprogesterone, Depo-Provera^® SC, Pfizer, Puurs, Belgium), and analgesia (0.03 mg/kg buprenorphine, Vetergesic^® SC, Ceva Animal Health, Brussels, Belgium) prior to surgery. Anesthesia was maintained with a continuous IV infusion of ketamine (8–16 mg/kg/h) and xylazine (2.4–4.8 mg/kg/h) while monitoring vital signs. Following laparotomy, 33–50% of the vessels going to each placenta were ligated in one random horn with Vicryl^® 5–0 (Ethicon^®, Johnson & Johnson, Diegem, Belgium), leaving the contralateral horn as internal control. The abdomen was closed with Vicryl^® 2–0 and Monocryl^® 3–0 (Ethicon^®, Johnson & Johnson) for fascia and skin, respectively. The surgical wound was infiltrated with levobupivacaine (2 mg/kg Chirocaine^®, Abbvie, Wavre, Belgium) and sprayed with aluminum (Kela, Hoogstraten, Belgium).

All experimental protocols, procedures, and endpoints criteria were approved by the institutional animal committee of Paris University (French Ministry of Research number CEEALV/#9498).
A total of 64 Sprague-Dawley male rats (Janvier Labs) weighting 392 ± 5 g, were included in this protocol. They were acclimated in a room with a 12-h dark/light cycle for 5 days with free access to water and feeding until they were anesthetized for experiments. The anesthesia was carried out by spontaneous mask ventilation with isoflurane (Iso-Vet, Piramal Healthcare). For induction and during surgery, the FiO₂ in the mask was 33%; thereafter, it was reduced to 26%. A subcutaneous injection of buprenorphine (50 μg/kg) and 100 μL of local anesthetic, lidocaine hydrochloride (Xylocaine, Aguettant) 2.8 mg/mL + levobupivacaine chlorydrate (Chirocaine, Abbvie) 1.4 mg/mL, was performed on the incision site. These injections were repeated before the animals were awakened at the same dose. Animals were placed on a heating blanket (temperature 37 °C) in supine position. Both carotid artery and jugular vein were catheterized for bleeding the animals (controlled hemorrhage) and for administration of fluid resuscitation, treatment, and blood re-transfusion. After surgery, an injection of 300 μL heparin Choay was carried out (100 UI/mL, Sanofi).