Involvement Of Spinal Glutamate Transporters In Bone Cancer Pain On Mice

ObjectiveThe glutamate transporters are known to play important roles in central pain transmission by regulate the extracellular glutamate concentration. The glutamate transporter expression and localization altered significantly in a model of neuropathic pain, GTs were initially upregulated in early postoperative phases but downregulated in later phases. Bone cancer pain has its specific neurochemical changes, astrocyte changes were different from neuropathic pain. Whether the glutamate transporters have similar change in bone cancer pain as in the neuropathic pain has not yet been investigated.In the present study, a mice model of bone cancer pain was established and evaluated by the pain related behavior and bone destruction. Then the role of glutamate transporters in spinal cord was investigated in the nociceptive information transmitsion by using Western Blot. The last, we explored the effect of glutamate transporter activator Riluzole on the pain threshold of the bone cancer mice.Materials and MethodsPart1 Establish a Mouse Model of Bone Cancer Pain and evaluation of pain behavior and bone destruction1. Bone cancer pain modelExperiments were performed on adult, male C3H/HeJ weighting 20-25g. Osteolytic murine sarcoma cells (NCTC 2472 cells, American Type Culture Collection, ATCC) were cultured in NCTC 135 medium (Sigma) containing 10% horse sera (Sigma) and passaged weekly according to ATCC recommendations. For their administration, cells were detached by 0.25% trypsin and then centrifuged at 1000 rpm. The remaining pellet suspended in minimal essential medium (2×10~5 cells/ 20μl) and used for femur injections. For implantation, animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). The right knee of mice was incised, exposing the condyles of the distal femur. 2×10~5 NCTC 2472 cells suspended in 20μl of a minimal essential medium (α-MEM) containing 1% bovine serum albumin (BSA) were slowly injected. Control groups were injected with 20μl ofα-MEM containing 1% BSA into the medullary cavity of the distal femur. For all animals, the injection site was sealed with a dental amalgam plug to confine the cells within the intramedullary canal and followed by irrigation with sterile saline. Finally, the surgical procedure was completed with a stitch of the knee skin.2. Experimental paradigmForty mice were randomly divided into two groups, Sham group and Sarcoma group (n=20). Spontaneous lifting behavior and ambulatory score and mechanical withdraw threshold (MWT) and paw withdrawal latencies (PWL) to radiant heat stimulation were measured pre- injection and on the 1st,3rd,5th,7th,10th,14th 21st,28th day after injection. The development of the bone tumor and structural damage in the bone were assessed by X-ray scan and HE staining.3. Data analysis and statisticsData were expressed as mean±SEM. The results were analyzed by analysis of variance (ANOVA) followed either by a multiple comparison test to compare the time course of anti-thermal hyperalgesia effect and of anti-allodynia effect or by t-test in an unpaired series to compare data obtained after drug administration. Values of P<0.05 were considered as statistically significant.Part2 Expression of glutamate transporter subtype in Spinal Cord on bone cancer pain mice1. Mice bone cancer pain modelThe same as that in part one.2. Experimental paradigmThirty-five mice were randomly divided into Sham group (n=5), and Cancer pain group (n=35). The protein of three glutamate transporters (EAAT1,EAAT2,EAAT3) in the L4-5 of spinal cord were measured after the day 1,3,7,10,14 and 21. The protein content was measured by Western Blot.3. Data analysis and statisticsData were expressed as mean±SEM. The results were analyzed by analysis of variance (ANOVA).Values of P<0.05 were considered as statistically significant.Part3 Effects of glutamate transporter activator Riluzole on pain threshold in bone cancer mice1. Bone cancer pain modelThe same as that in part one.2. DrugsRiluzole was purchased from Sigma and was dissolved in 10% DMSO diluted in normal saline. The solutions were sterilized through 0.22μm filters. Drug concentrations were prepared when use.3. Experimental paradigmFifty six mice were divided into Sham group, DMSO group and CA group (Ca+DMSO) and R5 (Ca + Riluzole 5mg/kg i.p. bid) and R10 (Ca + Riluzole 10mg/kg i.p.bid ) and R20 (Ca + Riluzole 20mg/kg i.p. bid ). Flinches and move evoked pain and mechanical withdrawal threshold (MWT) using von Frey filament and paw withdrawal latency (PWL) using radiant heat applied to the plantar surface were measured at day 14,17,20 after tumor cells injected.4. Data analysis and statisticsThe same as that in Part Two.ResultsPart1 Establish a Mouse Model of Bone Cancer Pain and evaluation of pain behavior and bone destruction1. Body weightIn the group of tumor-bearing animals, the mean body weight showed a tendency to decrease during the 28-day period (P<0.05). In the Sham group of mice body weight showed an increase (P<0.05).2. Spontaneous pain lifting There were no significant flinches between Sham group and Sarcoma group before injection (P>0.05). Compared with Sham group, the mice of Sarcoma group were observed spontaneous pain after injection of tumor cells and increased significantly on days 14 till days 28(P<0.05).3. Paw withdraw latency (PWL)The PWL of the right foot of Sarcoma group decreased significantly and were lower compared with that before inoculation procedure (P<0.05). And continued to decrease until 28 day after innoculation procedures compared with Sham group.4. Mechanical withdrawl threshold (MWT)The MWT of the right foot of Sarcoma group decreased significantly on days 7 and were lower compared with that before inoculation procedure (P<0.05). And continued to decrease until 28 day after innoculation procedures compared with Sham group.5. Ambulatory scoreThe ambulatory score of the right paw decreased continuously and decreased significanty by day 14n 21 and 28 compared with the Sham group (P<0.05). The mice in Sham group returned to 4 score after procedure.6. The X-ray scanThe X-ray scan of the femur showed a significant bone destruction and pathological fracture on the 28th day.7. HistologyObservation on light microscopy found that the bone marrow cavity from the mice of the Sarcoma group was fulled with tumor cells, and the cortical bone was destroyed. The bone construction from the group of Sham did not change.Part 2 Expression of glutamate transporter subtype in Spinal Cord on bone cancer pain mice1. The protein content of EAAT1 in spinal cord on bone cancer pain miceThe protein content of EAAT1 decreased from day 10 and decreased significantly by day 14 and continuously decreased till day 21 (P<0.01). 2. The protein content of EAAT2 in spinal cord on bone cancer pain miceThe protein content of EAAT2 decreased from day 10 and decreased significantly by day 14 and continuously decreased till day 21 (P<0.01).3. The protein content of EAAT3 in spinal cord on bone cancer pain miceThe protein content of EAAT3 decreased from day 14 and continuously decreased till day 21 (P<0.01).Part 3 Effects of glutamate transporter activator Riluzole on pain threshold in bone cancer miceCompared with Ca group, the dose of 5 mg/kg and 10 mg/kg and 20 mg/kg significant increased the pain threshold (P<0.01). And the effect of 20 mg/kg can maintain till the day 20. Riluzole injected intraperitoneal has the dose-depend effect as that 20 mg/kg>10 mg/kg >5mg/kg.Conclusions1. The results of behavioral tests, radiology and histology examination suggest that mice could give rise to a distinct pattern of bone cancer-related characteristics after injected carcinoma cells.2. Downregulation of spinal glutamate transporter was involved in the forming of bone cancer pain.3. Intraperitoneal injection of Riluzole can upregulate pain threshold of bone cancer mice and can relieve the cancer pain.