| ObjectiveTo provide new basis for therapeutic methods of BCP through detecting the expression of M-CSF in the spinal cord of bone cancer pain rats and exploring the mechanism of M-CSF on pain sensitization.Methods1.Preparation of Walker 256 breast cancer cells for tibial injection Walker 256 breast cancer cells(Shanghai Suran Biotechnology Co.,Ltd.)were intraperitoneally injected into healthy,adult and male SD rats weighing about 150-180 g.The morphology of breast cancer cells implanted in the abdomen of rats were observed daily.After 5 to 7 days,formed ascites were extracted and washed with PBS solution and centrifuged(800r/min)for 3 times(5 minutes each time),then were placed on ice.2.AnimalsHealthy,adult and male SD rats weighing about 170-210 g(Jinan Pengyue Co.,Ltd.)were fed on a free diet for 12 hours each day and night at room temperature of 25(±0.5C).3.Intrathecal catheterAs reported in previous studies,PE-10 catheter was placed in subarachnoid lumbar enlargement of rats after anesthetized.4.Establishment of bone cancer pain modelThe same as previous studies,rats successfully intrathecal catheterized were injected with Walker 256 breast cancer cells(10ul 5X10~6 cells/ml)into the bone marrow cavity of the upper right tibia to establish a bone cancer pain model.5.Behavioral testingRats were placed in a transparent glass cage with wire mesh and placed in the cage for 30 minutes before the test.Von Frey fine needle was used to test the paw withdrawal threshold(PWT)of rats.Duration of the acupuncture was 5 seconds,and PWT test was repeated three times with an interval of 10 minutes.6.Western blotThe expression of M-CSF,Iba1 and purine 2X4 receptor(P2X4R)protein in the sham group and bone cancer pain group were detected by Western blot.7.Double immunofluorescenceCo-expression of Iba1(the marker of microglia activation)and P2X4R in the dorsal spinal cord of the sham group and bone cancer pain group was detected by immunofluorescence double labeling method8.Experiment groupingEstablishment of bone cancer pain model:24 rats weighing 170-210 g were randomly divided into three groups:blank control(control)group,sham group,bone cancer pain(BCP)group.PWT of rats was measured at 0,1,2,4,8 and 14 days after modeling.The expression of M-CSF,P2X4R and Iba1 in the spinal cord:16 clean and male SD rats weighing about 170-210 g were randomly divided into two groups:sham group and bone cancer pain group.The expression of M-CSF protein in the spinal cord of rats in BCP group was measured by western blot at 0,4,8 and 14 day.The co-expression of Iba1and P2X4R in the dorsal spinal cord was determined by double immunofluorescence staining.Effects of M-CSF receptor blocker(GW2580)on normal and Bone Cancer Pain:32 healthy and male SD rats were randomly divided into four groups on the 14th day of BCP establishment:sham group,sham+GW2580 group,bone cancer pain group and bone cancer pain+GW2580 group.GW2580(100uM 20ul)was injected intrathecally.The changes of PWT in rats were measured before injection and 15,30,60,90 and 120minutes after injection.The expression of Iba1 and P2X4R was detected by Western blot in the four groups after injection for 120 minutes.8.Drugs used in the experiment and their route of administrationGW2580 was purchased from MedChem Express company.Then it was injected into the subarachnoid space of rats through PE-10 tube.The volume of the injection was20 ul.Results1.There was no significant difference in PWT between control group and sham group at 0,1,2,4,8 and 14 days(P>0.05).Compared with control and sham group,there was no significant difference in PWT at 0,1 and 2 days in bone cancer pain group(P>0.05),but there was significant difference at 4,8 and 14 days(P<0.05).The lowest value of PWT was found on the 14th day,which indicated that bone cancer pain model was successfully established.2.The expression of M-CSF protein in spinal cord of the sham group and bone cancer pain group was detected by western blot.Compared with sham group,the expression of M-CSF in the spinal cord of the bone cancer pain group was increased in a time-dependent manner on 4,7 and 14 days after modeling(P<0.05).The expression of M-CSF protein was the highest on day 14,which was consistent with the change of PWT value in BCP rats.3.Compared with sham group,the PWT values of bone cancer pain group was significantly decreased before injection and 15 minutes,30 minutes,60 minutes,90minutes and 120 minutes after injection(P<0.05).Compared with bone cancer pain group,the PWT values of bone cancer pain+GW2580 group was significantly increased(P<0.05)before injection and 15,30,60,90 and 120 minutes after injection.This indicated that intrathecal injection of GW2580 could significantly relieve bone cancer pain.4.Compared with sham group and sham+GW2580 group,the expression of Iba1 and P2X4R in spinal cord of rats in bone cancer pain group was significantly increased at 120minutes after injection(P<0.05).Compared with bone cancer pain group,the expression of Iba1 and P2X4R in bone cancer pain+GW2580 group was significantly different before injection and at 120 minutes after injection(P<0.05).This indicated that GW2580plays an analgesic role through reducing the activation of microglia and the expression of P2X4R.5.Double immunofluorescence staining showed that compared with the sham group,Iba1 and P2X4R protein were both increased in the dorsal spinal horn of the BCP group rats on day 14 after modeling(P<0.05)and were co-expressed in the spinal microglia.Compared with the BCP group,Iba1 and P2X4R protein were both decreased in the dorsal spinal horn of the BCP+GW2580 group rats on day 14 after modeling(P<0.05).ConclusionIn the spinal cord of bone cancer pain rats,the expression of P2X4R and Iba1 was co-expressed in microglia and M-CSF was significantly increased.While,blocking with M-CSF receptor reduced microglial activation and P2X4R expression,which relieved bone cancer pain. |
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