Buprenorphine

Rats were anesthetized in a 3% isoflurane sleep box (Patterson Veterinary, Greenley, CO, USA), aseptically prepped with alternating betadine surgical scrub (Purdue Products, Stamford, CT, USA) and 70% isopropyl alcohol in triplicate, and transferred to a sterile field. During MCLT and MMT surgery, a 1–2 cm midline skin incision was made along the medial side of the knee. Skin was retracted and the MCL was exposed by blunt dissection, and transected. The knee was moved into a valgus orientation, allowing central aspects of the medial meniscus to be cut radially. After meniscus transection, absorbable 5–0 vicryl braided sutures (Ethicon, Somerville, NJ, USA) were used to internally close the surgical path; 4–0 ethilon nylon monofilament sutures (Ethicon, Somerville, NJ, USA) were used to close the skin. Anesthesia was maintained via mask inhalation of 2.5% isoflurane throughout all procedures. Rats recovered post-operatively in a warming box until ambulatory. For pain management, rats received buprenorphine (0.05 mg/kg) (Patterson Veterinary, Greeley, CO, USA) subcutaneously every 12 hours for 48 hours post-surgery.

A laparotomy was conducted under isoflurane (USP – Patterson Veterinary, St. Greeley, CO, USA) /oxygen anaesthesia (5% for induction and 1.5% for maintenance, Eagle Eye Anesthesia, Jacksonville, FL, USA) and in sterile conditions to allow the placement of a temperature telemetry emitter into the abdominal cavity (G2 E-Mitter; Starr Life Sciences, Oakmont, PA, USA) for measurements of core temperature (T_c) during the experimental protocol. After surgery, mice were given buprenorphine (Patterson Veterinary, St. Greeley, CO, USA) injections S.C. (0.1 mg/kg) every 12 h for 48 h and then monitored for a minimum of 2 weeks until fully healed. After this recovery period, mice were given in-cage running wheels (model 0297-0521; Columbus Instruments, Columbus, OH, USA) to allow for voluntary exercise training for 3 weeks. Animals were brought to the laboratory in the third week to begin familiarization with the set-up (e.g. chamber and forced wheel system) and training period. A forced running wheel (model 80840; Lafayette, Lafayette, IN, USA) powered by a DC power supply was used to train the animals. Four training sessions were performed, as described previously (Garcia et al., 2018 (link); King, Leon, Mustico, Haines, & Clanton, 2015 (link)). After the training sessions, mice were given 2 days of recovery, before initiation of the EHS protocol.

For ovariectomy (OVX) surgery, mice (~49 weeks old) were anesthetized and maintained under 2% isoflurane. A single 1 cm dorsal midline incision was made in the skin along the mid-lumbar region followed by two bilateral muscular incisions to expose the ovaries. OVX included removal of the whole ovary, ovarian bursa and part of the oviduct. After ovary removal, the skin incision was closed using skin sutures (4–0 Vicryl; Ethicon) and adhesive (3M VetBond Tissue Adhesive). Buprenorphine (Patterson Veterinary, Devens, MA) was administered subcutaneously at a dose of 0.05 mg/kg immediately after surgery and post-operatively as needed. OVX effectiveness was determined at the conclusion of the study via verification of uterine atrophy; uterine weights are provided herein for INT and OVX WT, αKO, and βKO.