Ketamine

Prior to the surgical procedures, the animals were sedated with an intramuscular (im) injection of ketamine (10 mg/kg; Vedco, Saint Joseph, MO, USA) and medetomidine (0.015 mg/kg; Zoetis, Kalamazoo, MI, USA). Tracheal intubation was performed and the animals were placed on inhaled isoflurane (1–4.5%; Piramal Healthcare, Nashville, TN, USA) in oxygen (delivered at 1.0 L/min). Homeostatic monitoring (respirations, vital signs) was performed according to locally established procedures. After completion of the surgical intervention, atipamezole hydrochloride (im, 0.15 mg/kg; Zoetis) was administered and the animals were extubated and returned to their home cages. The animals were visually monitored cage side at 15-min intervals until full recovery from anesthesia. For analgesia and post-operative care, animals received, at minimum, buprenorphine (im, 0.03 mg/kg twice daily for 1.5 days; Patterson Veterinary, Mendota Heights, MN, USA), carprofen (subcutaneously or orally, 2.2 mg/kg twice daily for 2 days; Zoetis), and ceftriaxone (im, 50 mg/kg once daily for 5 days; Patterson Veterinary). Following completion of the carprofen, all animals were given ketofen (im, 2 mg/kg once daily for 3 days; Zoetis).

About 6–8 week-old female Balb/c mice were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed in the Animal Facility of the Comparative Medicine Center at the University of Utah under standard conditions and all procedures were conducted following approved Institutional Animal Care and Use Committee (IACUC) protocols.
A total of 30 mice were used for E2 pellet implantation and randomly divided into six groups (n = 5). Animals total body weights were recorded before the surgical procedure and each mouse was anesthetized with a mixture of 1 mL of ketamine (Vedco Inc., Saint Joseph, MO), 0.1 mL of xylazine (LLOYD, Shenandoah, IA), and 8.9 mL of normal saline. Mice received intraperitoneal injections of ketamin–xylazine mixture combined with subcutaneous buprenorphine (Reckitt Benckiser Pharmaceuticals, Richmond, VA). Doses were adjusted accordingly to each animal body weight. A small incision, ∼2–3 mm in length, was made between the shoulder blades to form a subcutaneous pocket. One E2 pellet was inserted into the pockets of each animal. Incisions were closed using 4-0 polyglycolic acid suture with a taper needle (Ethicon, Somerville, NJ) and then disinfected with alcohol/iodine.

Following baseline PIT testing for subjects treated with muscimol, rats were anesthetized with a mixture of ketamine (100 mg/kg: Vedco) and xylazine (10 mg/kg) via intraperitoneal (IP) injection (0.1% bodyweight). Subjects were placed in a Kopf (David Kopf Instruments; Tujunga, CA) stereotaxic instrument, and an incision was made over the midsagittal line to reveal bregma and lambda on the surface of the skull. Stainless steel (22 gauge) guide cannula (Plastics One, Roanake VA) were fixed in place with jeweler’s screws and dental cement 1.5 mm above CeA at -2.5 mm posterior, 4 mm lateral to the midline, and 6 mm ventral from the surface of the skull. Subjects recovered in the home cage for one week following surgery and then underwent further PIT testing. Prior to testing, muscimol (0.3 μl of 1 ng/nl solution) or deionized water was infused through (28 gauge) injectors extending 1.5 mm beyond the guides bilaterally at a rate of 0.15 μl/min with subjects connected to the infusion lines for an additional min for dispersal. This was accomplished using 10 μl Hamilton syringes (Model 701N) controlled by a Harvard Apparatus pump (PHD 22/2000) via polyethylene tubing connected to injectors extending 1.5 mm beyond the tip of their guide cannula. Subjects were tested 15–20 min after treatment.

Mice were anesthetized with Ketamine (80 mg/kg; Vedco, St. Joseph, MO) and Xylazine (10 mg/kg; Akorn Inc., Decatur, IL) that was administered via intraperitoneal (ip) injection. Following a laminectomy at thoracic level 8 (T8), a severe contusion injury (70 kilodyne force) was induced using the Infinite Horizon Impactor (Precision Systems and Instrumentation, Lexington, KY). Uninjured mice were anesthetized, received superficial skin incisions, and received wound clips to ensure blinded evaluations. Since a laminectomy does not induce major inflammatory changes in the SC (Supplementary Fig. 9), a surgical control was not included in the current studies. Immediately postoperatively, all mice received subcutaneous injections of Lactated Ringers solution (1 ml; Baxter, Deerfield, IL), Baytril (0.02 ml; Bayer, Kansas City, KS), and Buprenorphine (0.05 ml; Hospira, Lake Forest, IL). Antibiotics, analgesics, and 1 ml of normal saline (Baxter) were administered to mice twice daily for 7 days thereafter. Bladders were manually expressed twice per day. On day 1 post-injury (pi), hind limb locomotor function was assessed with the Basso Mouse Scale (BMS)^{64 (link)} to confirm that all mice received an injury of equivalent severity (BMS score ≤ 2), and that each uninjured mouse exhibited normal baseline stepping and coordination (BMS score = 9).