Male Sprague–Dawley (SD) rats (aged 4–5 weeks and weighing 140–150 g) were purchased from the Experimental Animal Center, the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Housing was performed in individual cages under a 12h–12h light/dark cycle at ambient temperature with food and water at will, for 1 week before initiating the study. According to the International Association for the Study of Pain guidelines for pain research in animals, experiments involving animals were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University (No. 2014-002).
The effects of RvD1 were investigated by randomly dividing the rats into five groups (n = 6): sham group (Sham + Veh1), burn group (Burn + Veh1), small-dose (100 ng) RvD1 treatment group [Burn + R(S)], large-dose (300 ng) RvD1 treatment group [Burn + R(L)], and sham + large-dose RvD1 treatment group [Sham + R(L)]. The vehicle [Veh1, PBS containing 0.1% ethanol] or RvD1 (100 or 300 ng in 50 μL of Veh1) was administered intraperitoneally (ip) 30 min before burn injury and subsequently twice a day for 7 days. Less than 1 h before injection, the aqueous solution of RvD1 (Cayman Chemical, USA) was prepared by evaporating the stock RvD1 to dryness under a gentle steam of nitrogen and immediately adding the Veh1, while minimizing exposure to light.
The effects of p38 MAPK were investigated by randomly dividing the animals into five groups (n = 6): sham group (Sham + Veh2), burn group (Burn + Veh2), SB203580(Abcam, UK, which inhibits the enzymatic activity of p38 MAPK on its downstream targets) treatment group (Burn + SB203580), sham + SB203580 treatment group (Sham + SB203580), and large-dose RvD1 treatment group [Burn + R(L)]. SB203580 (10  μm/50 μL) or vehicle (Veh2, normal saline with 1% dimethyl sulfoxide) was injected intraspinally (lumbar L4/L5 space) 7 days after burn injury. The animals underwent anesthesia by isoflurane inhalation and lower back shaving. In the prone position, a lumbar puncture was performed in the L4–L5 intervertebral space. A slight tail or hind limb movement was taken as an evidence for successful lumbar puncture.
The effects of BDNF/TrkB were investigated by randomly dividing the rats into five groups (n = 6): sham group (Sham + Veh3), burn group (Burn + Veh3), TrkB-Fc (Abcam, UK, a specific BDNF inhibitor) 48 (link) treatment group (Burn + TrkB-Fc), sham + TrkB-Fc treatment group (Sham + TrkB-Fc), and large-dose RvD1 treatment group [Burn + R(L)]. TrkB-Fc (100 μg, 10 μL) or vehicle (Veh3, 0.01 m PBS, 10 μL) was administered intrathecally, followed by flushing with Veh3. Treatment was initiated 1 h before burn injury and continued daily (10:00 a.m.) for 1 week post-burn injury. In the Sham group (sham + Veh3) without bilateral paralysis of hind limbs, insertion failed following intrathecal administration of lidocaine in one rat, which was removed from the study; therefore, n = 5 in this group.
In these experiments, TrkB-Fc and RvD1 doses were based on Zhou et al. 48 (link) and Lima-Garcia et al.,49 (link) respectively.