Ketaset

ET was performed by flank laparotomy in pseudo-pregnant MF1 recipients (7–8.5 weeks) obtained by mating with vasectomised MF1 males. Two-cell embryos and blastocysts were washed three times in M2 medium prior to ET into the oviduct and uteri, respectively, in minimal medium, as previously described (Velazquez et al., 2018 (link)). Recipients were anaesthetised by a single intraperitoneal injection of Ketamine (50 mg/kg, Ketaset, Pfizer, UK) and Xylazine (10 mg/kg, Rompun, Bayer, UK). Embryos were transferred (19.7 ± 6.05 per recipient) in equal numbers into both maternal tracts with separate recipients used for different treatments, as below. After transfer, exposed tracts were placed back into the abdominal cavity, the peritoneum was sutured, and the skin was closed with wound clips. Recipients were then kept individually in a clean cage in a warm room (28–30°C) to recover from anaesthesia. Females were then housed in a quiet room for the rest of their pregnancy and lactation. Litter size was adjusted to up to 8 per dam at birth with similar numbers of males and females.

Male Sprague–Dawley (Envigo, Dublin, VA, USA) rats weighing 225–250 mg were used for these studies. ARRIVE guidelines were followed and all the animal experiments were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee (IACUC) at the Medical University of South Carolina (MUSC), Charleston, SC (protocol ARC no. 2079). All animal studies were approved by the IACUC at the MUSC. Animals were anesthetized with ketamine (Ketaset at 80 mg/kg, Pfizer, New York, NY, USA) and xylazine (AnaSed at 10 mg/kg, Lloyd laboratories, Shenandoah, IA, USA) as described [14 (link)]. Laminectomies were performed at T10 and the spine immobilized with a stereotactic device, and SCI was induced by the method of Perot by dropping a 5 g weight from a height of 8 cm onto an impounder tip gently placed on the exposed spinal cord [36 (link),37 (link)]. Sham animals receiving laminectomy but no injury were used as controls (n = 4–10). PRM (100 µg/kg), E2 (10 µg/kg), and/or ICI (1 mg/kg) were given a bolus dose via tail vein injection at 15 min post-injury followed by a daily intraperitoneal injection up to day 7 (n = 4–10) (Figure 1). Animals were sacrificed by decapitation under ketamine/xylazine anesthesia at 7 days following injury.

The study was performed in accordance with University of Bristol animal care policies and with the authority of appropriate UK Home Office licenses. Adult male Wistar rats (225–270 g; Charles River) were anaesthetized with intraperitoneal ketamine (60 mg/kg; Ketaset, Pfizer Animal Health) and medetomidine (0.4 mg/kg; Dormitor, Pfizer), and placed in a stereotactic frame. Either 3.7 or 16 μg of rhCDNF in 5 µl of artificial CSF (Biovian) was infused bilaterally into the rat striatum (A/P, +0.75; M/L, ±3.0; D/V, −5.0) at a rate of 1 µl/min, using a custom-made catheter composed of fused silica. After the infusion, the catheter was left in situ for 5 min and then withdrawn at a speed of 1 mm/min. For immunohistochemistry (IHC) analysis, each rat received a high dose and a low dose of rhCDNF to the left and right striatum, respectively (n = 3 for each time point). For ELISA, the rats received injection with either the high or low dose of rhCDNF bilaterally into the striatum (n = 3 per dose for each time point).