Normal saline

The drugs used were Human Urotensin II (Servier, Australia), L-phenylephrine HCl (Sigma Company, Australia), and clonidine HCl (Boehringer Ingelheim Pty Ltd, Australia). The doses of drugs were expressed in μg of the base. Drugs administered into the lateral ventricle or fourth ventricle were dissolved in Ringer's (Baxter, Old Toongabbie, NSW, Australia) solution while drugs given i.v. were dissolved in normal saline (Baxter, Old Toongabbie, NSW, Australia).

An animal mechanical ventilation model was established and modified as previously described [10 (link)]. In detail, animals were anesthetized with sodium pentobarbital (40 mg/kg body weight, IP). After anesthesia, animals were placed on a recirculating heating blanket and fixed. Then, animals were tracheostomized and connected to a volume-driven small animal ventilator (VentElite, Harvard Apparatus; Cambridge, MA, USA). The tidal volume (TV) was set at 5 mL/kg body weight, and the respiratory rate (RR) was set at 55 to 60 breaths/min. Breathing air was humidified and enriched with oxygen. To avoid ventilator-induced systemic hypoxia, the ventilator parameters and oxygen flow were adjusted to maintain PaO₂ between 80 and 100 mmHg and PaCO₂ between 35 and 45 mmHg during the entire study. The values of PaO₂ and PaCO₂ were determined through arterial blood gas analysis, which was performed every 2 hours during the study. Blood pressure (BP) and heart rate (HR) were monitored at the tail using tail cuff plethysmography (BP-2010 Series Blood Pressure Meter, Softron, Japan). The right jugular vein was cannulated for continuous infusion of normal saline (Baxter, Deerfield, IL) at a rate of 1 mL/kg⁻¹/h⁻¹ and pentobarbital sodium (10 mg/kg⁻¹/h⁻¹) using an electric pump. Body temperature was maintained at 37°C during the experiment by external warming with a homeothermic blanket system.

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).