Figure 1.
Mechanical hyperalgesia/allodynia and time course-dependent hyperactivation of astrocytes in the RVM after CCI-ION. A, Examples of S–R function curves illustrating the intensity-dependent head withdrawal responses to mechanical stimuli. There is a significant leftward shift of the curve at 1 d (green) and 14 d (red) after CCI (p < 0.001; n = 9 rats) compared with the pre-CCI baseline (blue). The leftward shift of the S–R curves indicates an increased response to suprathreshold stimuli and a decrease in EF50 value, suggesting the presence of mechanical hyperalgesia and allodynia. B, Time course of mechanical hyperalgesia and allodynia as indicated by a reduction of the EF50 values. CCI-ION results in mechanical hyperalgesia and allodynia that persists over the 28 d observation period. The rats receiving a sham operation show a moderate reduction of the EF50 values that lasted for 3 d, which is totally blocked by local tissue anesthesia with injection of 0.25% bupivacaine (twice at 1–3 d) at the intraoral incision site in sham-operated rats. CCI-ION versus sham, ***p < 0.001; sham versus naive, #p < 0.05. C, Immunohistochemistry of GFAP in the RVM. a, Low power of a tissue section at the rostral medulla level. b, Enlarged RVM region corresponding to the small rectangle area in a. Enhanced expression of GFAP is observed at 3 d (c) and 14 d (d) after CCI, compared with GFAP immunostaining in naive animal (b). D, Immunostaining of S100β in the RVM. Increased S100β expression is observed at 3 d (b), 14 d (c), and 28 d (d) after CCI, compared with naive (a). E, Western blots illustrating CCI-induced increase in GFAP in the tissues punched out from the RVM. A representative blot is shown on top, and the mean protein levels are summarized below. Compared with naive and sham-operated (−) rats, the level of GFAP is selectively increased at the 14 and 28 d time points in CCI-ION (+) rats. The increase in GFAP at 1–3 d in sham-operated and CCI-ION rats is consistent with behavioral changes at these time points (B). β-Actin was used as a loading control. The asterisks indicate significant differences from the naive rats (*p < 0.05; n = 3 per time points). F, Western blots illustrating the effect of bupivacaine, a long-lasting local anesthetic, on the incision-induced increase in GFAP expression in the RVM at 3 d after sham operation. Bupivacaine (0.25%; 0.1 ml) is injected at the intraoral incision site twice in the first 3 d (+). Saline is injected as a vehicle control (−). Compared with naive rats, the GFAP expression is increased at 3 d after sham operation (*p < 0.05; n = 3 per group), which is completely abolished by the bupivacaine (n = 3). Scale bars: Ca, 0.5 mm; Cd (for Cb–d), Dd (for Da–d), 0.025 mm. NGC, Nucleus reticularis gigantocellularis; Py, pyramidal tract. Error bars indicate SEM.
