SB 203580

Epidermal keratinocytes were isolated from human foreskin as previously described (Halbert et al., 1992 (link)). The cells were propagated in medium 154 supplemented with human keratinocyte growth supplement, 1,000× gentamycin/amphotericin B solution (Invitrogen), and 0.07 or 0.2 mM CaCl₂.
Keratinocytes were transduced with retroviral supernatants produced from Phoenix cells (provided by G. Nolan, Stanford University, Stanford, CA) as previously described (Getsios et al., 2004 (link)). For differentiation of submerged cultures, cells were grown to confluence and switched to E-medium containing 1.8 mM Ca²⁺ for 1–6 d (Meyers and Laimins, 1994 (link)). For raft cultures, transduced cells were expanded and grown at an air–medium interface according to published protocols (Meyers and Laimins, 1994 (link)). Organotypic cultures were grown for 3–10 d, at which time they were lysed for RNA/protein analysis, embedded in optimal cutting temperature compound for frozen sections, fixed in 10% neutral-buffered formalin, and embedded in paraffin for histology or fixed in 2% paraformaldehyde/2% glutaraldehyde in cacodylate buffer for EM analysis. For some experiments, cultures were treated with 2–5 µg/ml ETA, DMSO (Thermo Fisher Scientific), 10 µM PKI166 (Novartis), 5 µM U0126 (Cell Signaling Technology), or 10 µM SB203580 (EMD).

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.

Immunofluorescence detecting Iba-1 and GFAP (microglial and astroglial activation, respectively), and BDNF/TrkB was carried out in distinct juvenile animals 7 days after burn injury (n = 3 per group). Immunofluorescent staining for p-p38 MAPK was performed after measuring the mechanical withdrawal threshold (MWT) following SB203580 administration. For spinal cord immunofluorescent staining, the rats were terminally administered anesthesia by isoflurane inhalation, followed by transcardial perfusion with heparinized saline and 4% formalin in PBS. After excision, spinal cord lumbar segments underwent postfixation overnight, storage in 20% sucrose (3 days), and sectioning using a cryostat (25 μm). After blocking (3% bovine serum albumin in 0.3% Triton X-100) for 1 h ambient, incubation were carried out overnight at 4°C with primary antibodies targeting GFAP, Iba1, BDNF, TrkB (Abcam, UK), and p-p38 MAPK (Santa Cruz, USA). This was followed by incubation with appropriate fluorescein isothiocyanate- or cyanine 3 (CY3)-conjugated secondary antibodies (Jackson, USA). Spinal cord cells that expressed p-p38 MAPK were examined by double staining with anti-NeuN (Abcam; neuronal marker), GFAP (astrocytic marker), or Iba1 (microglial marker). GFAP was stained red to distinguish it from p-p38 MAPK. Laser confocal microscopy (Zeiss LSM 510; Zeiss, Germany) was used to observe and image the dorsal horn area of the spinal cord (1 field per section). Image Pro Plus 6.0 was used for quantifying GFAP, Iba1, p-p38 MAPK, BDNF, and TrkB, based on the IOD (integrated optical density) values of immunofluorescence intensity. Measurement of signal co-localization was performed with NIH Image J program. Five spinal cord sections of each animal were randomly selected. The experimenter who performed the immunofluorescent staining was blinded to the groups of rats.

The animals were allowed a 2-day adaptation period before burn injury. The nociceptive behaviors of the rats in the Sham + Veh2, Burn + Veh2, Burn + SB203580, Sham + SB203580, and Burn + R(L) groups (n = 6 per group) were tested before the intraspinal injection, and 15, 30, 60, and 120 min upon the intraspinal injection 7 days after burn injury. The nociceptive behaviors of the other groups (n = 6 per group) were tested before burn injury and at 1, 3, 5, 7, and 14 days post-burn injury. Upon adaptation (30 min), mechanical thresholds were measured by hind paw withdrawal to a graded series of von Frey filaments (Stoelting, USA) (4–26 g) applied to the plantar area of each hind paw. The modified Dixon “up–down” technique was applied to determine the mechanical nociceptive threshold, as described previously.^{22 (link)} The experimenter was blinded to the groups of rats.