Establishment Of Rat Skin Cancer Pain Model And The Role Of Syndecan2 Signaling Pathway In Cancer Pain

With the continuous development of medical level,the survival time of cancer patients has been greatly prolonged.Tumor-related pain is one of the most common symptoms in patients with advanced cancer,and the specific mechanism still needs to be explored.Pain has gradually become the most intolerable symptom of cancer.Due to the unclear mechanism leading to cancer pain and the limitations of therapeutic drugs(most of the drugs for cancer-related pain have the characteristics of tolerance and addiction),there are still about 40%of cancer patients whose pain can not be effectively controlled.At present,although the treatment of cancer pain means are very diverse,but its treatment results are still unsatisfactory.Therefore,to further study the mechanism of cancer pain and to find effective ways to relieve cancer pain has become one of the hot spots in the field of chronic pain research.Means of studying the mechanism of cancer pain mainly come from the establishment of animal models of cancer pain.Bone cancer pain model is currently the most widely used animal model,but its production is more complex,so skin cancer pain model has been widely used for its convenience.This experiment will try to establish a rat model of skin cancer pain by injecting Walker 256 breast cancer cells into the sole of the foot.At present,studying the mechanism of cancer pain and finding effective targets to alleviate cancer patients'pain have become the theme of current research on the occurrence and development of cancer.So,is there a specific target in the occurrence and development of cancer pain,which can not only relieve pain continuously and effectively,but also can not produce serious side effects?In recent years,with the deepening of research,synaptic remodeling in the spinal cord has gradually attracted our attention.The role of actin cytoskeleton in this process has become a hot topic in recent research.Syndecan2(SDC2)is specifically expressed on dendrites and plays a role in the occurrence and maturation of dendritic spines.Heparanase is an important regulator of syndecan family.Its function is to degrade the glycoprotein chain linked to the outer membrane of syndecan molecule.The degradation function of heparanase may provide necessary conditions for SDC2 monomers to be close to each other.In order to explore the role of SDC2 signaling pathway in cancer pain,we observed the changes of pain behavior by detecting heparanase,SDC2,SDC3 and intrathecal injection of heparanase inhibitor OGT2115 in rat skin cancer pain model.This study is divided into two parts.Part I:Establishment of a rat model of skin cancer painObjective:To investigate the feasibility of establishing a rat skin cancer pain animal model with cultured Walker256 rat breast cancer cells.Methods:Thirty Sprague Dawley(SD)rats weighing 180-220g were randomly divided into three groups:Naive,Sham,tumor cells implanting group(TCI group),and there were ten rats in each group.In the TCI group,200?l Walker256 cells suspension with a cell density of 4×10~7/mL was subcutaneously injected into the posterior foot of rats.Sham animals were injected with an equal volume of dead cells;the Naive group did not undergo any surgical procedure.The appearance of feet were recorded.And the volume of feet of rats were observed,as well as pain behavior.Masson staining on skin tissue bearing tumor cells was performed.And immunohistochemistry staining was used to detect activation of astrocytes and microglia in the spinal cord.Moreover,ELISA was performed to show NGF,IL-1?and TNF?concentration in skin tissue bearing tumor cells.Results:Red and swollen foot was observed in TCI rat from 2 days after TCI and became severe by time.This phenomenon existed till the end of observation.Meanwhile,foot volume kept increasing in TCI group.Slight red and swollen temporally appeared on the foot of Sham rats.A large number of tumor cell infiltration was found,and activation of astrocytes and microglia were detected in the spinal cord dorsal horn in TCI group.Significant mechanical allodynia and thermal hyperalgesia were observed from 8 to 17 days in the TCI group with statistic difference with that of Sham(all P<0.05).Expressions of NGF,IL-1?and TNF?increased in skin tissue bearing tumor cell compared with that of Sham(all P<0.05).Conclusion:A rat model of skin cancer pain was successfully established by subcutaneously injecting Walker 256 breast cancer cells.Part II:The role of SDC2 signaling pathway in cancer painObjective:To investigate the role of SDC2 signaling pathway in cancer pain.METHODS:Forty SD male rats were randomly divided into normal control group(Naive),sham inoculation group(Sham),TCI group(TCI group)and experimental group(TCI+OGT2 115),with 10 rats in each group.Rats in TCI group and experimental group were subcutaneously inoculated with Walker 256 breast cancer cell suspension of 200 ml(4 X107/ml)on the hind sole and plantar surface of rats to establish a skin cancer pain model.The sham inoculation group was injected with the same amount of inactivated cells,while the normal group was not inoculated.On the8th day after inoculation of tumor cells,rats in the experimental group were injected with a microinjector into the spinal cord sheath with heparanase sulfate inhibitor OGT215 20ul at a concentration of 6 umol.L-1.Normal control group,sham inoculation group and TCI group were not injected.The expression of heparanase,SDC2 and Syndecan3 in spinal dorsal horn was detected by immunohistochemistry and Western blotting,and pain behavior was observed.Results:Mechanical and thermal hyperalgesia appeared in TCI group 8-12 days after operation,which was significantly different from that in normal group(P<0.05),while the pain threshold in experimental group increased first and then decreased after OGT2 115 injection 8 days after operation.On the 11th day after operation,the trend of decline decreased to the level of TCI group,and then maintained the same level as that of TCI group.The expression of heparanase and SDC2 in TCI group began to increase 5 days after operation,which was significantly different from that in sham inoculation group and normal control group(P<0.05).There was no significant change in SDC3 expression in the three groups.Conclusion:SDC2 signaling pathway is involved in the occurrence of cancer pain,Intrathecal injection of OGT2115 to inhibit heparanase sulfate alleviated hyperalgesia induced by cancer pain and mechanical hyperalgesia.