Fig. 4. (A ) Representative reverse transcription–polymerase chain reaction for prodynorphin (PDYN) messenger RNAs on the ipsilateral side of spinal cords obtained from saline-or complete Freund's adjuvant (CFA)-injected mice. Spinal cord samples were prepared at 1, 3, and 6 h after saline or CFA injection (B ). The intensity of the bands was determined semiquantitatively using ImageJ (National Institutes of Health). The values for PDYN messenger RNA were normalized by the value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA. The value in CFA-injected mice is expressed as a percentage of the increase in saline-injected mice. Each column represents the mean ± SEM of three samples. Student t test: **P < 0.01, ***P < 0.001, versus saline group. (C ) Quantitative analysis of PDYN messenger RNA on the spinal cord of saline-or CFA-treated mice at 6 h after the injection. (C-i ) Amplification plots of fluorescence intensities versus PCR cycle numbers in each sample are displayed. (C-ii ) Each column represents the mean ± SEM of three samples. *P < 0.05 versus saline-treated group. (D ) Change in dynorphin A (1–17)-like immunoreactivity on the superficial laminae of the ipsilateral dorsal horn after CFA injection. Photomicrographs show immunofluorescent staining of dynorphin A (1–17) on the superficial layers of the ipsilateral spinal dorsal horn at 6 h after saline (D-i ) or CFA injection. Dynorphin A-like immunoreactivity observed on the superficial laminae of the ipsilateral dorsal horn after CFA injection (D-ii ) was significantly increased compared with that after saline injection (D-i ). Scale bars = 50 μ m.