| Objectives:The purpose of the first part of this study was to model and validate the effectiveness,safety and bioeffect mechanism of irreversible electroporation(IRE)for bone metastases.The second part of the trial aimed to design a surgical protocol for bone metastases and compare the relationship of ablation effect caused by different needle distances and different pulse modes.Methods:Part Ⅰ:The study of the effectiveness,safety,local immunity and biological mechanism of IRE for the treatment of paraveroneal bone metastases.Rabbits bone metastases model were established by tissue embedding method,and then 25 tumor-bearing rabbits were selected and divided into observation group and anatomical group.In the observation group,14 negatively tumor-bearing rabbits were selected and divided into 2 groups,one for the control group and one for the IRE group,with 7 rabbits in each group.The IRE group ablated the tumor in the left inguinal parafemoral near the neurovascular area,while the control group was untreated.The safety of IRE ablation was assessed by the changes of preoperative and postoperative neuromotor function.The tumor and blood vessels were monitored by ultrasound every 3 days after surgery.The tumor growth curves were drawn,and the tumor tissues in the operated area of the observation group of tumor-bearing rabbits were taken with a 20G biopsy needle under ultrasound guidance on days 1,4,7 and 14 after surgery,and the tumor tissues were examined by histopathological examination,including deoxynucleotidyl transferase nick end labeling(TUNEL)staining,CD45+staining,CD4+staining,CD 8+ staining,CD4+CD25+ staining,to evaluate the effectiveness,immune effect and biological effect mechanism of IRE.Thirteen tumor-bearing rabbits were selected for the anatomical group.One rabbit was dissected before surgery as a blank control,and the remaining rabbits were ablated at the left inguinal parafemoral near the neurovascular.Two rabbits were dissected on postoperative days 1,7,14,21 and 28,respectively,and the vessels,nerves and muscles were separated,fixed and embedded,and stained with hematoxylin-eosin(H&E),Masson trichrome(Masson),Luxol Fast Blue(LFB)and S-100 protein immunohistochemistry to compare the pathology of the two groups to further evaluate the safety of IRE ablation.Part Ⅱ:Study of the ablation effect induced by IRE at different needle distances and different pulse emission modes.Thirteen blank rabbits were selected and divided into 2 groups.The first group including 8 rabbits was again divided according to the distance of the two needle tips from the neurovascular:group D1(needle tip connection perpendicular to the nerve,nerve located at the midpoint of the two needle tips),group D2(needle tip located on the side of the nerve,two needle connections parallel to the nerve,0 mm from the nerve),group D3(needle tip located on the side of the nerve,two needle connections parallel to the nerve,10mm from the nerve),group Group D4(needle tip on the side of the nerve,two needle lines parallel to the nerve,5 mm from the nerve).Four groups were stimulated with the same parameters,and the nerve,blood vessels and muscles were dissected 7 days after surgery,and HE staining,Masson staining,Luxol Fast Blue(LFB)staining,and S-100 protein immunohistochemical staining were performed to evaluate the functional effects on the nerve,blood vessels and muscles.In the second group of 5 rabbits,the left hind limb was pulsed in group 1 and the right hind limb was pulsed in group 2.The left hind limb was treated with positive ablation needle firing 200 pulses,and the right hind limb was treated with positive and negative ablation needle firing 100 pulses respectively,and the rest of the parameters were the same.The tissues near number 1(+)and number 2(-)ablation needles were biopsied in the left hind limb one week after surgery,and the tissues near the negative ablation needles were biopsied in the right hind limb,fixed,embedded and stained with HE,Masson,LFB and S-100 protein immunohistochemistry to compare the pathology of the two groups to evaluate the safety of IRE ablation.Results:Part Ⅰ:The success rate of tissue embedding method was 100%,and the survival rate of tumor-bearing rabbits was 97.5%.In the study of IRE ablation effectiveness and local immunity,the tumor was edematous 1 day after ablation,gradually necrotic 7 days after surgery,and the ablated necrotic area was fixed in volume and no longer increased in size 10-14 days after surgery.The cells showed a continuous apoptotic state 1-4 days after ablation.In the control group,the tumor volume increased exponentially after surgery.The rabbits started to die gradually after 10 days postoperatively.In the IRE group,the volume was also measured to increase gradually after surgery due to the gradual necrosis of the ablated area,and there were still statistically significant differences between the two groups at 4,7,10 and 13 days postoperatively without considering the tumor activity.In the local immune study,it was found that CD45+infiltrating cells were significantly elevated at 1 day postoperatively and decreased after 1 week postoperatively,CD8+T cells were significantly elevated at 1 week postoperatively compared to the control group,with statistical differences at 4 and 7 days postoperatively,CD4+T cells were also significantly elevated at 4 days postoperatively,with statistical differences compared to the control group,and decreased at 7 days postoperatively,and the percentage of Treg cells gradually increased at 1 week postoperatively.Contrary to the trend of these indicators,it decreased at 2 weeks postoperatively and was statistically different compared to the control group.In the IRE ablation safety study,there were no serious complications during or after ablation,and there was no statistical difference in postoperative weight loss between the two groups in the observation group.The motor function of the hormonal rabbits in the IRE group decreased on postoperative day 1 and gradually recovered afterwards,with statistically significant differences between the two groups on postoperative days 1,4,7,and 10,and no statistical differences between the two groups on postoperative day 13.The motor function of the hormonal rabbits in the IRE group continued to improve on postoperative day 16.Pathological examination of the nerves revealed no abnormalities in axons and myelin sheaths,no significant fibrous tissue hyperplasia,and vacuolation of some Schwann cells at 1 day postoperatively.Half(1/2)of the axons were apparently swollen 1 week after surgery,and occasional axonal fragmentation was seen,and ellipsoid bodies containing swollen axons and fragmented myelin sheaths were visible.Interfibrillar fibrous tissue hyperplasia with fragmented myelin sheaths and occasional Schwann cells are visible.The number of swollen axons gradually decreased after 2-4 weeks postoperatively,and the axons gradually returned to their original shape,and the ellipsoid decreased.The fibrous tissue within the nerve bundle further increased at 2-3 weeks postoperatively,but no increase in fibrous tissue was seen at 4 weeks postoperatively,and most of the myelin sheaths gradually returned to their preoperative shape at 4 weeks postoperatively,and no obvious Schwann cell hyperplasia was seen.In the vascular pathology 1 day after surgery,the 3-layer structure of the vessels was still visible,the endothelium existed,the nuclei of the endothelial cells mostly lost their original shape and were no longer distributed along the endothelial alignment,the nuclei of the cells in the midmembrane were half solidified,the staining deepened,and the overall shape of the vessels did not change and was round-like;1 week after surgery,the vessels obviously had a decreased endothelial/epithelial ratio,and the wave shape of part of the endothelium was seen to partially disappear,and the endothelial layer could not be identified,and the endothelium was medial in some directions Fibrous tissue proliferation could be seen,filled with inflammatory cells,the morphological demarcation of intima-media myocytes was unclear,smooth muscle cells were less in some directions,fibrous connective tissue increased,and the thickness of the vessel wall was uneven in all directions;2 weeks after surgery,the wave shape of the intima-media was blurred,the intima/epima ratio increased,fibrous connective tissue further increased,the thickness of the vessel wall was more uniform in all directions,and the fibrous tissue of the epima-media increased;3 weeks after surgery,the intima-media The nucleus still did not return to normal shape,and the mid-membrane was further thickened;there were no significant changes in the intima,mid-membrane and outer membrane of the vessels at 4 weeks after surgery compared with those at 3 weeks after surgery.In the ultrasound examination,small vessel shadows were still visible in the tumor on the ultrasound 4 days after surgery,and no thrombus was seen in the superficial femoral artery.Postoperatively,there was significant destruction of muscle fibers,widening of muscle fiber bundle spacing,followed by cellular infiltration,increase in fibrous connective tissue and thickening of muscle fibers,and gradual recovery.Part Ⅱ:In the study of the nanosecond pulse surgery protocol it can be found that the motor function of the distance group D2 was statistically different from the remaining three groups at 4 days postoperatively;at 7 days postoperatively it can be seen that the neuromotor function of all groups of rabbits showed recovery,while the D1 group and the D2 and D3 groups were still statistically different from each other.Neuropathology showed that the degree of nerve damage in the D2 group was significantly higher than that in the D1,D3 and D4 groups,with the lowest nerve damage in the D4 group and the most severe nerve damage in the D2 group.Vascular pathology showed the lowest percentage of postoperative mesenteric smooth muscle cell area in the D2 group,which was statistically different between the D2 and D3 and D4 groups(p<0.05),and also statistically different between the D3 and D4 groups in comparison(p<0.05).In exploring the ablation effects caused by different pulse firing modalities,it could be found that there was no significant difference in postoperative motor function between the pulse 1 and pulse 2 groups.The damage was slightly greater in the pulse 1 group at a than at b and in the pulse 2 group,but there were more new Schwann cells.There was no statistically significant difference in the percentage of mesangial cells between pulse 1 group at a,pulse 1 group at b and pulse 2 group.There was no statistically significant difference in the percentage of muscle area between pulse 1 group a and b in muscle pathology.Conclusion:An animal model suitable for IRE was successfully established,and based on this,IRE was performed to verify the safety and efficacy of operating next to the vascular nerve.Determination of treatment parameters and electrodes arrangement.By investigating electrophysiological phenomena of muscle twitching,immune activation and temperature change,the following patterns were found in this project,which provide experimental and theoretical basis for the clinical application of IRE ablation of bone metastases.1、VX2 tumor tissue encapsulation method has high tumorigenic rate and rapid growth,which can be used for the study of local treatment of metastases.2、IRE can effectively control tumor growth and promote continuous apoptosis after ablation,with good effectiveness.3、The anti-tumor immune response is enhanced within 1 week after IRE.4、The therapeutic effect on bone metastases depends on the lethal effect of the output energy on the cells of different tissues in the treatment area,and the electric field strength depends on the output voltage and electrode spacing,which are proportional to the output voltage and inversely proportional to the electrode spacing.Evaluation of the therapeutic effect requires a combination of the above factors and separate tests of cell lethality in the corresponding target tissues under different treatment parameters to confirm the limits of effectiveness and safety.There is no lethal damage to blood vessels and nerves after IRE,the basic structure still exists,and gradually recovers after 1 week postoperatively,the function exists and gradually returns to normal,with high safety.5、Different ways of distributing needles lead to differences in the effect of acting tissues.The closer the distance to the ablation needle,the greater the damage.The closer to the ablation needle,the more damage will be done.When the same number of pulses are emitted,the bi-directional emission is smaller and more uniform than the uni-directional emission. |
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