| BackgroundArgon-helium cryoablation technology rise from the United States in recent years, At present, the technology developed rapidly, because it is simple, target specificly, broad indications, combined with timely treatment can be monitored in the target area, the small damage, exact ablation. That make this technology become one of the best means in treatment of solid tumors, greatly promoted the progress of clinical oncology therapeutics, significantly improved the condition of cancer patients, survival status and prognosis. With the recent advance out worldwide, this technique allows precise cryoablation of a wide variety of neoplasms, and has been used in the clinical treatment of cancers in liver cancer, lung cancer, pancreatic cancer, prostate cancer, kidney cancer, breast cancer. All these data suggest that argon-helium cryosurgery causes not only tumor tissue destructions but also enhancement of the host immune function through several possible mechanisms.The brain glioma patients that fail to respond to or are ineligible for chemotherapy or radiotherapy often present difficult clinical cases, for which argon-helium cryosurgery provide a valuable therapeutic option. Nevertheless, the application of this technique in the treatment of gliomas still remains preliminary, and reports of only a handful cases were available to demonstrate its good therapeutic effect in the management of gliomas without support by sufficient evidences from basic research and animal experiments. In this study, we performed argon-helium cryosurgeries in Wistar rat models bearing C6 gliomas, and observed the pathology of tumor cells,image and cellular immunity changes. we aimed to explore the mechanism of angon-helium cryosurgery in relation to the cellular immunity and provide experimental evidences for the clinical application of this technique in glioma treatment.Part I The MRI and pathology change after cryoablation on rat model bearing C6 gliomasObjective①In this study, we performed argon-helium cryosurgeries in Wistar rat models bearing C6 gliomas, and observed the postoperative tumor cell apoptosis or necrosis.②To observe the MRI change after cryoablation on wistar rat models bearing C6 gliomas.Materials and methods①we established Wistar rat models bearing subcutaneous C6 glioma and divided the rats into the normal control, sham-operated, surgical resection, and cryosurgery groups for corresponding treatments.②The postoperative changes in the findings by magnetic resonance imaging (MRI) and tumor cell morphology were observed, the cell apoptosis at the tumor foci was assessed with TUNEL assay after 6 hours,12 hours,24 hours, and analyze the tumor size by computing the volume.Results①MRI scanning displayed low signal intensity of the tumor tissues on T1-weighted images (T1WI) and high signal intensity on T2WI. After cryoablation, the tumors showed enhanced T1 signals and attenuated T2 signals, suggesting tumor necrosis following cryoablation. MRI demonstrated radical tumor resection in the surgical resection group without abnormal T1 or T2 signals in the tumor foci. In the cryosurgery group, the tumor volume tended to gradually decrease after the operation.②HE staining showed tumor cell rupture, nuclear condensation and coagulative necrosis of the C6 glioma following cryoablation. One week after the cryablation, obvious congestion and bleeding occurred with formation of granulation tissues on the margin of the ablated area. TUNEL assay reached the highest that was 53.818±5.018 under×200 visual field 12 hours after the cryoablation showed that the apoptotic cells occurred mostly around the ablated foci, presenting with typical apoptotic morphologies including yellowish brown nuclei, nuclear condensation and chromatin margination. It is significant difference in the cryosurgical group comparing with the normal control group (F=28.965,P=0.000), and significant difference in the cryosurgical group comparing with shamed-operationed group (F=28.376, P=0.000), In the normal control group and shamed-operationed group, only occasional apoptotic cells were found in the tumor foci, between which was no statistical significance (F=0.590,P=0.568)ConclusionsIn summary, Wistar rat models bearing C6 gliomas is simple,economical, accurate and inoculated under direct vision, into the tumor rate, growing 2 to 3 weeks of, tumor volume will reach 2.5-3.5cm3,Ablation on the rat back avoid the small rat cranial cavity which can easily result animal deaths caused by ablation of brain edema. Therefore, Rat bearing C6 glioma on back is an ideal animal glioma model is one. HE staining and Tunel staining show argon-helium cryoablation result to cell death by necrosis and apoptosis.PartⅡThe immunity change after cryoablation on rat model bearing C6 gliomasObjectiveTo evaluate the cellular immunity change after cryoablation on wistar rat models bearing C6 gliomas and provide experimental evidences for the clinical application of this technique in glioma treatment.Materials and methods①we established Wistar rat models bearing subcutaneous C6 glioma and divided the rats into the normal control, sham-operated, surgical resection, and cryosurgery groups for corresponding treatments.②The CD4+,CD8+,F480 positive cells were detected by immunohistochemistry after 14 days.③Flow cytometry was performed for analysis of the T lymphocyte subset,NK lymphocyte subset changes.④the ratio of Th1/Th2 were assessed with flow cytometry at 7 days,14 days,21 days following the cryosurgery.⑤Elisa was performed to analyse the serum IL-4,IL-10,IL-12,IFN-γat 7 days,14 days,21 days following the cryosurgery.Results①The CD4+,CD8+,F480 positive cells were were found to obviously increase on days 7,14 and 21 after the operation by immunohistochemistry after 14 days, showing significant differences from those in the other 2 groups (P<0.05). but, no significant differences between normal control and shamed operation group (P>0.05)②Compared with the preoperative levels, the percentages of CD3+,CD4+,CD14+,CD16+56 cells in the surgical resection group decreased significantly 7 days after the operation followed by gradual increase, but recovered the preoperative level till 14 days after the operation (P＜0.05). In the cryosurgery group, the CD3+,CD4+,CD14+,CD16+56 cells were found to obviously increase on days 7,14 and 21 after the operation, showing significant differences from those in the other 3 groups (P＜0.05). The CD3+,CD4+,CD14+,CD16+56 cells showed no significant differences between the normal control and sham-operated groups at 7,14 and 21 days after the operation. In all the 4 groups, CD8+cells increased with time after the operation, but no significant differences were found between the 4 groups at each of the time points for measurement (P＜0.001).③Compared with the preoperative levels, the percentages of IFN-y positive spleen cells in the surgical resection group decreased significantly 7 days after the operation followed by gradual increase, but recovered the preoperative level till 21 days after the operation (P<0.05). In the cryosurgery group, the IFN-y positive spleen cells by flow cytometer were found to obviously increase on days 7,14 and 21 after the operation, showing significant differences from those in the other 3 groups (P<0.05). The IFN-y positive cells showed no significant differences between the normal control and sham-operated groups at 7,14 and 21 days after the operation. n all the 4 groups, IL-4 positive cells increased with time after the operation, but no significant differences were found between the 4 groups at each of the time points for measurement.④In normal control group and shamed-operationed group, the serum IL-4 increased with time measured by enzyme-linked immunosorbent assay, however, the serum IL-4 decreased with time 7 days after the surgical resection, that come to a stable state, then no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05). the serum IL-4 decreased with time 7 days after the cryosurgical treatment, that come to a stable state, then no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05). and no significant differences were found at each of the time points days 14 and days 21 for measurement in the 4 groups (P>0.05). In normal control group and shamed-operationed group, the serum IL-10 increased with time measured by enzyme-linked immunosorbent assay, however, no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05).the serum IL-10 decreased with time 7 days after the surgical resection, that come to a stable state, then no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05). the serum IL-10 decreased with time 7 days after the cryosurgical treatment, that come to a stable state, then no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05). and no significant differences were found at each of the time points days 14 and days 21 for measurement in the 4 groups (P>0.05).In normal control group and shamed-operationed group, the serum IL-12 increased with time measured by enzyme-linked immunosorbent assay, however, it is significant difference at each of the time points days 14 and days 21 for measurement (P>0.05).the serum IL-12 decreased with time 7 days after the surgical resection, however, no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05). the serum IL-12 decreased with time 7 days after the cryosurgical treatment, that come to a stable state, then no significant differences were found at each of the time points days 14 and days 21 for measurement in the cryosurgical group(P>0.05). Multiple comparison showed no significant differences were found at each of the time points days 7 in the 4 groups(F=3.238, P=0.033), it is significant differences at the time points days 14 (F=18.539, P= 0.001) and days 21 for measurement in the 4 groups (F=19.235, P=0.000), the lowest is 6.318±1.073 in the cryosurgical group.In normal control group and shamed-operationed group, the serum IFN-y decreased with time measured by enzyme-linked immunosorbent assay, the serum IFN-y increased with time after the surgical resection, that come to a stable state after 14 days, the serum IFN-y increased with time after the cryosurgical treatment, the highest is at days 14 after cryosurgery. Multiple comparison showed no significant differences were found before operation, Cryosurgical group>Surgical resection group>shamed operation group, Cryosurgical group>Surgical resection group>Normal control group, it is significant differences at each time points days in the 4 groups. but, no significant differences were found in the normal control group and shamed-operationed group(F=0.443, P= 0.582). ConclusionsUsing argon-helium cryoablation Wistar rats bearing C6 glioma model, using immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay, and other means can effectively induce, enhance the tumor immunity against tumor cells, But the anti-tumor immunity after 3 weeks have decreased, so that such anti-tumor immunity may only act on the body for some time, how to maintain and promote the immune response to argon-helium refrigeration and frozen the specific immune mechanism is our furthe issue. But this experiment provide important reference data in treatment of glioma patients and even the central nervous system tumors for argon-helium cryosurgery in clinical management of gliomas.
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