Surgery Activates Peripheral Myeloid-derived Suppressor Cells To Facilitate Postoperative Cognitive Dysfunction In Mice With Tumor

Backgrounds:Postoperative cognitive dysfunction(POCD)refers to the obvious cognitive impairment after anesthesia and surgery,which may have serious consequences for quality of life and even result in irreversible cognitive deficits.The incidence of cancer has increased annually,more and more patients are having surgery due to cancer.Central inflammation is considered as a critical pathogenesis in POCD;at the same time,the relationship between tumor and inflammation is very complicated.So,does the tumor affect the occurrence of POCD in patients,and what is the mechanism?These issues are still unclear.Tumor could recruit a number of MDSCs in blood and spleen,which can transform into pro-inflammatory phenotypes under sorts of stimuli and secret amounts of pro-inflammatory cytokines.Therefore,we hypothesize that surgery can activate MDSCs recruited by tumors in peripheral immune organs,releasing a large number of pro-inflammatory factors,which enter the central center through the damaged blood-brain barrier,causing central inflammation and facilitating POCD.Purpose:To verify whether the tumor-bearing mice are more likely to develop POCD than normal mice.To explore whether its mechanism is involved in the surgical activation of myeloid-derived suppressor cells(MDSCs)recuited by tumors in peripheral blood and spleen,which can secret amounts of pro-inflammatory cytokines that induce central inflammation.This study provides a target for the prevention and treatment of POCD in tumor-bearing individuals,and offer us new ideas for the pathogenesis of POCD.Methods:In part 1,two-month-old adult male C57BL/6J(23-25g)mice were randomly divided into naive group,surgery group,tumor group and tumor+surgery group.MC38 colon cancer cells(2 x 10~5/mouse)or LLC lung cancer cells suspension(2 x 10~5/mouse)were injected subcutaneously into mice to establish a tumor-bearing mouse model.When the subcutaneous tumor had grown to 0.8-1cm in diameter,the tibial fracture internal fixation procedure was performed under the anesthesia of fentanyl and droperidol.Morris water maze(MWM)and novel object recognition test(NOR)were carried out to detect postoperative cognitive function of mice.Western blot was used to determine the relative protein expression levels of PSD-95.In part 2,in MC38 and LLC cell line tumor-bearing models,the concentration of IL-2,IL-4,IL-6,IFN-?,TNF-?,IL-17A,IL-10 and IL-1?in plasma were tested by cytometric bead array(CBA)and ELISA.Hippocampal cytokines(TNF-?,IL-6 and IL-1?)were determined with Western blot and ELISA.Real-time PCR was used to detect the mRNA levels of TNF-?,IL-6 and IL-1?in the hippocampus.Immunofluorescence was used to detect the activation of microglia.The relative protein expression levels of ZO-1 were measured with Western blot.In part 3,we select LLC cell line tumor-bearing model,weigh the weight of spleen of each group of mice.Flow cytometry was used to detect the proportion of MDSCs in peripheral blood and spleen.Intraperitoneal injection of Gr-1 neutralizing antibody abolishes MDSCs in mice,then ELISA was used to detect the expression levels of TNF-?,IL-6 and IL-1?in plasma and hippocampus of naive,surgery,tumor+surgery,tumor+surgery+Gr-1 antibody mice.NOR was used to detect the learning and memory ability of these mice after the MDSCs were neutralized.Results:In part 1,the results of MWM and NOR all suggested that adult mice without tumor implantation had no significant changes in learning and memory ability after tibial fracture surgery,but compared to the naive group these abilities of MC38 or LLC cell line tumor-bearing mice was significantly reduced after the surgery,with decreased expression of the synaptic integrity marker PSD-95 in hippocampus.In part 2,By detecting peripheral inflammatory factors in MC38 and LLC cell line tumor-bearing models at 48 h after surgery,it was found that the expression level of IL-2,IL-4,IL-6,IFN-?,TNF-?,IL-10 and IL-1?in the surgery group was not significantly different from those in the naive group,but the expression levels of TNF-?,IL-6 and IL-1?in the tumor+surgery group were significantly higher than those in the naive group,and the tendency of the three types of inflammation in the hippocampus is consistent with that in peripheral blood with the reversible decrease of tight junction protein ZO-1 in the hippocampus.Interestingly,at this time point,the relative mRNA levels of TNF-?,IL-6 and IL-1?in the hippocampus were not significantly different between the groups.Immunofluorescence assay showed that the activation of microglia did not appear in the mice from each experimental group.In part 3,flow cytometry analysis showed that the proportion of MDSCs in the blood and spleen tissues of the LLC tumor-bearing mice was significantly higher than that of the tumor-free mice.Surgery can cause acute aggregation of MDSCs at the above tissues(6h),and the proportion of MDSCs can be restored to the preoperative level at48h after surgery.At 48 h after surgery,the proportion of MDSCs in blood and spleen tissues of the tumor+surgery group was still significantly higher than that of the naive group.After ablation of peripheral MDSCs with Gr-1 antibody,the expression levels of TNF-?,IL-6 and IL-1?in peripheral blood and hippocampus of tumor+surgery+Gr-1 antibody mice were significantly lower than those of tumor+surgery mice.The results of NOR suggested that the cognitive function of tumor+surgery+Gr-1 antibody mice no longer showed a significant decrease after surgery.Conclusion:Tumor-bearing mice are more vulnerable to POCD,which is characterized by prolonged central inflammation in tumor-bearing mice.Surgery can activate MDSCs that are recruited in the blood and spleen by tumors.Activated MDSCs produce a large number of pro-inflammatory cytokines in circulation,which enter the brain through the impaired blood-brain barrier and lead to cognitive impairment.