Article Figures & Data
Figures
- Figure 1.
Double immunostaining of P2X7R and cell-specific markers in morphine-tolerated rats. A, Schematic diagram of the spinal cord. Black open square (A) marks the corresponding scope of confocal images (B, D) on the spinal cord section. White open squares (B, D) mark the corresponding scope of amplified images (C, E) on the confocal images. B, C, Double immunostaining of P2X7R and the microglial marker OX42. D, E, Double immunostaining of P2X7R and the astrocytic marker GFAP. Scale bars: B, D, 40 μm; C, E, 10 μm.
- Figure 2.
Upregulation of spinal expression of microglia and P2X7R in morphine-tolerant rats. Nine days of subcutaneous morphine (10 mg/kg, twice daily) were given to induce analgesic tolerance. In behavioral tests (A), analgesic efficacy of acute morphine (5 mg/kg, i.p.) was decreased significantly during repeated administration (#p < 0.05, ##p < 0.01, ###p < 0.001, compared with the corresponding saline group). Western bolt analysis showed that the protein levels of microglial marker Iba1 (B) and P2X7R (C) were upregulated significantly by chronic morphine treatment (B, C; #p < 0.05, ##p < 0.01, compared with saline group; n = 4). The spinal tissue samples of saline group were collected on day 10. PWT, Paw-withdrawal threshold; TFL, tail-flick latency.
- Figure 3.
Effects of P2X7R antagonist on the induction and maintenance of morphine analgesic tolerance. Chronic morphine (MOR)-induced analgesic tolerance was attenuated in von Frey test (A) and tail-flick test (B) by intrathecal BBG treatment (#p < 0.05, ###p < 0.001, compared with Saline + Saline; **p < 0.01, compared with MOR + Saline). Immunohistochemical analysis (C) showed that intrathecal BBG treatment abolished the immunoreactive upregulation of P2X7R (D) and the microglial marker OX42 (E) induced by chronic morphine (#p < 0.05, compared with Saline + Saline). Western blot analysis indicated that the protein levels of P2X7R (F), the microglial marker Iba1 (G), and phosphorylated p38 MAPK (H) with chronic morphine were also inhibited by BBG treatment (#p < 0.05, ##p < 0.01, compared with Saline + Saline; *p < 0.05, compared with MOR + Saline; n = 3). Five days of subcutaneous morphine (10 mg/kg, twice daily) produced analgesic tolerance to acute morphine challenge (5 mg/kg, i.p.) in von Frey test (I) and tail-flick test (J). After 3 d of BBG treatment (10 μm, 20 μl, i.t., 30 min before subcutaneous morphine, twice daily) did not affect the onset of morphine tolerance. Data represent means ± SEM (###p < 0.001, compared with day 0).
- Figure 4.
Intrathecal P2X7 siRNA attenuated the induction of morphine analgesic tolerance. A, Schematic showing the experimental schedule. P2X7 siRNA and control siRNA were injected intrathecally on day 0. Acute morphine analgesic tests were performed on day 8 and day 14, respectively. Chronic morphine consisted of subcutaneous morphine injection (twice daily) from day 9 to day 13. Western blot analysis (B) showed that intrathecal P2X7 siRNA significantly reduced the protein level of P2X7R in the spinal cord on day 7 and day 16 after siRNA injection (##p < 0.01, compared with Naive). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. The protein levels of Iba1 (C) and phosphorylated p38 MAPK (D) in the spinal cord were significantly lower in the P2X7 siRNA groups after chronic morphine exposure (*p < 0.05, compared with control siRNA group; n = 4). P2X7 siRNA-treated rats exhibited a significantly higher morphine analgesic potency in von Frey test (E), Hargreaves' test (F), and tail-flick test (G) after chronic morphine treatment (#p < 0.05, ###p < 0.001, compared with day 8; *p < 0.05, compared with control siRNA group). PWL, Paw-withdrawal latency.
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