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.