Wound clips

Animals were anesthetized with 4% isoflurane and maintained at 2% isoflurane and 2% oxygen (at a flow-rate of 2 L/min). An incision was made along the dorsal midline of the skull, bregma and lambda identified and a small burr hole (50 µm) was drilled. The virus (AAV1/2-OXTp-hM4Dgi-mCherry, AAV1/2-OXTp-Venus, or AAV1/2-OXTp-mCherry) was loaded into a 20 μl NanoFil syringe fitted with a 33gauge needle (World Precision Instruments Inc, USA). 270 nl was injected into the PVH (A-P −1.7 mm, M-L ± 0.3 mm, D-V 8.0 mm) at a 10°. For CA2 (A-P −3.5 mm, M-L ± 4.2 mm, D-V 3.3 mm) and SuM (A-P −4.5 mm, M-L ± 4.2 mm, D-V 8.9 mm, 15°), 0.05 μl or 0.07 μl, respectively. Following injection, the syringe was left in place for 10 min before being withdrawn and wound closed using wound clips (Stoelting Inc, USA). Rats received intraoperative subcutaneous fluids for hydration (Thermo Fisher Scientific, USA) and buprenorphine (0.05 mg/kg) as analgesia for 24–72 h. For cannulation, a guide cannula (7 mm, P1 Technologies Inc, USA) was implanted at a 15° (A-P −4.6 mm, M-L – 0 mm). Two bone screws (Stoelting Inc, Wood Dale, IL, USA) were implanted on the skull and secured using dental cement (Stoelting Inc, USA). A dummy cannula (7 mm) was left in place and OXTR antagonist/saline was delivered using an infusion cannula (9 mm).

Female NCr nude mice (CrTac:NCr-Foxn1nu) aged 7 weeks were purchased from InVivos, Singapore. Mice were fed on a normal rodent diet and provided water ad libitum under humidity and the climate-controlled environment with a 12 h light-dark cycle. Mice were acclimatized to laboratory conditions for a minimum of 3 days prior to study initiation. All surgical procedures were performed under strict aseptic conditions according to the protocol approved by the SingHealth Institutional Animal Care and Use Committee (IACUC). Mice were anesthetized by exposure to 2.5% of isoflurane (IsoFlo, Abbott, Australia). A 1-cm incision was made at the abdominal mammary fat pad area. The growth of the tumor was induced by direct implantation of 3 × 106 luciferase-expressing MDA-MB-231 cells suspended in 30 µl of PBS/Matrigel (1:1) into the mammary fat pad tissues. The incision was closed with wound clips (Stoelting Co, USA). To relieve post-surgical pain and prevent dehydration following surgery, 1 ml of 2 mg/ml meloxicam (Troy Ilium, Australia), diluted in 0.9% saline (B. Braun, Germany) was administered subcutaneously at the right flank of mice after surgery. Mice were recovered on a heating pad until fully awake from anesthesia before being returned to cages.