FIGURE 1.

Chronic morphine treatment induces hyperalgesia and evokes ER stress in neurons of spinal cord in mice. (A) Chronic morphine treatment induced hyperalgesia in mice. Mice were subcutaneously injected with saline or morphine (5 mg/kg) twice daily for 6 consecutive days. Behavioral testing was conducted before first morphine administration every morning by tail-flick assay. Chronic administration of morphine significantly decreased tail flick latency from days 1 to 5. The saline-treated group served as control. Data was analyzed by Student’s t test (n = 8) (*P < 0.01, ***P < 0.001 vs. control). (B) Representative western blot image and analysis to quantify ER stress-related molecule levels in the spinal cord. Consecutive administration of morphine (5 mg/kg) twice daily for 6 days evokes ER stress in the spinal cord. The expression of GRP78, ATF6, IRE1α, p-eIF2, XBP1s, Caspase-12 were significantly increased in OIH mice measured by western blots (n = 4). The spinal samples were collected 1 h after the last morphine treated and determined by western blot. Significant difference was revealed following Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001 vs. control). (C) Distribution and cellular localization of GRP78 in the dorsal horn of the spinal cord before or after morphine administration. The level of GRP78 (green) was significantly increased in OIH mice compared with those in control mice. Double immunostaining of GRP78 and specific makers in OIH mice showed that GRP78 was co-localized with NeuN (red) not with astrocytic maker GFAP (red) or microglial maker Iba1 (red). Spinal samples were collected after the last administration of morphine. Scale bar: 100 μm.