Effects of intrathecal DREAM-shRNA on hyperalgesia in morphine-tolerant rats with bone cancer pain

Abstract

Objective To evaluate the effects of intrathecal DREAM-short hairpin RNA (shRNA) on hyperalgesia in morphine-tolerant rats with bone cancer pain (BCP).Methods Sixty healthy female Sprague-Dawley rats,weighing 200-230 g,in which intrathecal catheters were successfully implanted without complications,were randomly divided into 5 groups (n =12 each) using a random number table:sham operation group (group S),BCP group (group P),BCP + morphine tolerance group (group PM),BCP + morphine tolerance + vehicle group (group PMV) and BCP + morphine tolerance + RNAi group (group PMR).Bone cancer pain was induced by injection of breast cancer cells (Walker 256) 5 μl (1 × 105/tμl) into upper segment of bone marrow of the left tibia.Morphine tolerance was induced by subcutaneous injection of morphine three times a day for 5 consecutive days starting from 14th day after intra-tibia injection in PM,PMV and PMR groups.The single bolus of morphine was 10,20,30,40 and 60 mg/kg from 14th day to 18th day,respectively.Morphine 3 mg/kg was injected subcutaneously on 19th day after intra-tibia injection in PM,PMV and PMR groups.On 20th day after intra-tibia injection,normal saline 10 μl,vehicle 5 μl + normal saline 5 μl and DREAM-shRNA 5 μl + normal saline 5 μl were injected intrathecally once a day for 5 consecutive days in PM,PMB and PMR groups,respectively.The frequency of spontaneous paw withdrawal was recorded and the extent of hind limb claudication when the rats walked was scored and recorded,and thermal paw withdrawal latency (TWL) and mechanical paw withdrawal threshold (MWT) were measured 1 day before intra-tibia injection (To) and at 7,14,18,19,21,25 and 28 days after intra-tibia injection (T1-7).The animals were sacrificed after the last measurement of pain threshold at T7 and the L4 segments of the spinal cord were removed for detection of the expression of green fluorescent protein (GFP),and DREAM mRNA and protein.Results Compared with group S,the frequency of spontaneous paw withdrawal was signifioantly increased and the extent of hind limb claudication score was decreased at T2-3,5.7 in group P,while at T4-6 in PM,PMV and PMR groups,MWT was decreased at T1-3,5-7 in group P,while at T1, 3-7 in PM,PMV and PMR groups,and the expression of DREAM mRNA and protein was up-regulated in P,PM,PMV and PMR groups (P ＜ 0.01).Compared with group P,the frequency of spontaneous paw withdrawal was significantly decreased at T2-3 and increased at T4,the extent of hind limb claudication score was increased at T2-3 and decreased at T4 in PM and PMV groups,the frequency of spontaneous paw withdrawal was decreased at T2-3,6-7 and increased at T4,and the extent of hind limb claudication score was increased at T2-3,6.7 and decreased at T4 in group PMR,MWT was increased at T2-3 and decreased at T4-7 in PM and PMV groups,MWT was increased at T2-3,6-7 and decreased at T4-5 in group PMR,and the expression of DREAM mRNA and protein was up-regulated in PM and PMV groups and downregulated in group PMR (P ＜ 0.01).There was no significant difference in the indexes mentioned above at each time point between PM and PMV groups (P ＞ 0.05).Compared with PM and PMV groups,the frequency of spontaneous paw withdrawal was significantly decreased,and the extent of hind limb claudication and MWT were increased at T6-7,and the expression of DREAM mRNA and protein was down-regulated in PMR group (P ＜ 0.01).There was no significant difference in TWL at each time point among the five groups (P ＞ 0.05).In PMV and PMR groups,a large amount of green fluorescence was observed,which meant that the expression of GFP was positive.Conclusion Intrathecal DREAM-shRNA can attenuate hyperalgesia in morphine-tolerant rats with BCP. Key words: RNA interference ; Transcription factors; Injections, spinal; Bone neoplasm; Pain; Morphine; Drug tolerance; Hyperalgesia