Experimental Study On Goat Livers With Ultrasound Imaging Guided Percutaneous Irreversible Electroporation

Electroporation occurs when the application of an electric field with electric field intensity of kV/cm and pulse duration ofμs to ms grade across a cell alters the transmembrane potential. Thus, the lipid bilayer structure is disrupted and small nanopores are created in the cell membrane, and the nanopores then allow micromolecules and macromolecules to be transported into and out of the cells. Electroporation could be used as reversible electroporation (RE) or irreversible electroporation (IRE) due to the nanopores recover or not. This process when used in RE has been used in medicine and research for drug or macromolecule delivery into cells, such as electrochemotherapy (ECT). With sufficiently high voltage, these pores become permanent and contribute to cell death by interfering with cell homeostasis.This is the mechanism of IRE ablation method. IRE tumor treatment process doesn't depend on chemotherapeutics which might take some disadvantages of toxicity and drug resistance. In the past findings, researchers had fully certified the extensive application prospect of IRE though tumor cells and solid tumor ablation treatment experiments. But in the past, IRE only could be used on body surface or superficial tumor ablation, because lack of some equipment to help for the location of the electrodes placement accurately. With non-thermal cell death and a markedly decreased treatment time, IRE provides a potential new ablation method that can be operated in a well-controlled and focused manner under image monitoring (such as ultrasound). In this study, we used an in vivo goat liver model to evaluate the clinical feasibility of IRE imagine-guided by US, the mechanism of cell death by using histochemical analysis, the effectiveness of tissue destruction near bile ducts and vessels, and the correlation of ablation areas as measured by using US versus gross section examination.Part 1 Investigation of the feasibility and safety with ultrasound imaging guided irreversible electroporation on goat liversChapter 1 Parameter selection of goats'liver ablation with IREObjective:To select the most suitable IRE ablation parameters under fixed electric field intensity, permanent duration and frequency.Methods:IRE induced by 30 to 150 pieces of pulses(Pulse electric fields with a 2-cm probe distance, permanent duration 100μs, frequency 1Hz and voltage 2000V) were performed on goats'livers directly after abdomen discission. 30 pieces of pulses were increased for each group. Temperature of the target area was measured immediately after IRE ablation. All the animals were scarified 24 hours post IRE. Specimens were collected respectively for TTC stain. The largest ablation diameters of specimens were measured by electronic sliding caliper.Results:It was shown that there was dose-dependence result between the dosage of the electric field and diameters of the ablation zone. Necrosis area enlarged gradually with the increasing of the pulse pieces of electric field. When the hepatic tissue was exposed to pulsed electric fields with 120 pieces of pulses, permanent duration 100μs, frequency 1Hz and voltage 2000V, the diameters of ablation area appeared to be obvious. Necrospy-based measurement demonstrated highly consistency with FEM-anticipated ablation zones. The dose in group 120 pieces of pulses may be considered as the optimal dosage for the hepatic tissue ablation in goat's livers. There was no significant difference between 120 pulses and 150 pulses group in diameter comparison of the ablation zones (P>0.05). And 120 pulses group had significant difference when compared with 30 pulses, 60 pulses and 90 pulses group separately (P<0.05). The temperature of the injuring tissues was measured at the same time, and there was no significant difference before and after IRE ablation procedure (P>0.05).The temperature of the ablation area was under the regular injure level instantly post IRE.Conclusion:120 pieces of pulses was the most optimal IRE ablation parameters under fixed pulse electric fields with 2-cm probe distance, permanent duration 100μs, frequency 1Hz and voltage 2000V. Electric field created by IRE was devoid of any joule heating and therefore, non-thermal ablation was achieved. Since it does not depend on heat, IRE can create a focal tumor ablation area which was independent of any heat sink effect.Chapter 2 Feasibility investigations and characteristic of US imaging changes with ultrasound imaging guided IRE on goat liversObjective: To investigate the proper method of percutaneous irreversible electroporation (IRE) on goat livers under ultrasonic guidance, and observe the characteristic of ultrasound imaging changes.Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) applied on the electrodes and placement of the electrodes had been localized into goats'liver under ultrasound guidance though the animal skin to the target area. The treated area was observed by real-time ultrasound scanning and gross appearance observation at the time of 0, 24hours, 3d, 7d and 14d after IRE ablation. Results: Ultrasound imaging guidance was accurate to focus on the target area. Imagines captured by the ultrasound after IRE procedure were quite different compared with the normal liver imaging. Real-time US images obtained during IRE show hypoechoic areas of ablation with hyperechoic region in close proximity to the probes. Sharp demarcation of IRE-ablated zone is well visualized immediately after the procedure. This ablated area was well correlated with pathologic measurements. US images obtained after 24 hours showed that area of hypoechogenicity becomes hyperechogenic, and seems to have shrunk when compared with US images obtained istantly. US images obtained after 3 days post IRE shows that gray scale of ablation area became more hyperechogenic, and boundary of the ablation area was quite clear. US images obtained after 7 days shows the most hyperechogenic gray scale of ablation area. Meanwhile, the circumsciption injured zone became irregular for hyperplasia of the fibrous connective tissue. 14 days post IRE, US images obtained showed insignificant hyperechogenic images with high point-like echo in the center. The ablation area shrunk obviously. US images correlated well with gross appearance observation at the time of 0, 24hours, 3d, 7d and 14d after IRE ablation.Conclusion: With real-time monitoring by ultrasonography and well-controlled ablation of the target tissue, percutaneous IRE can provide a novel and unique ablative method for IRE tumor ablation. There was regular changement of US images after IRE ablation. Image-guided provides the fundamental experimental work for future studies on IRE clinical application and follow-up investigation.Chapter3 Security investigation with ultrasound imaging guided irreversible electroporation on goat liversObjective: To investigate the influence of IRE ablating to the liver of goat on general condition, and the security of IRE ablation method.Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) applied on the electrodes and placement of the electrodes had been localized into goats'liver under ultrasound guidance though the animal skin to the target area. Heart rate, respiration frequency, and electrophysiology (ECG) changes between pre and post IRE ablation were compared. Postoperative complications and venous blood analyzing including liver function, kidney function, complete blood count and myocardial enzyme (CK - MB) were compared 0 hour, 24 hours, 3 days, 7 days and 14 days after IRE ablation.Results: The respiratory frequency and heart rate of goat increased in the process of IRE ablation compared with in rest preoperation, and resumed to the condition of preoperation after IRE ablation. No significant statistic difference was showed between preoperation and postoperation (P>0.05). The ALT and AST progressively increased in postoperation 24h to 3rd day, and returned to normal level in postoperation 7th day. No significant statistic difference was showed between pre and post ablation in liver function, kidney function, complete blood count and myocardial enzyme (CK-MB)level (P>0.05). Instantly after IRE ablation, the ECG showed normal and no arrhythmia of electrophysiology changes. No complications including intraperitoneal hemorrhage, infection, and neighbouring organs damages (gallbladder, greater omentum, intestinal tract, etc) have been found.Conclusion: IRE ablation was security and validity, and no obvious complications was observed.Part 2 Intraprocedural US accuracy for IRE ablation area monitoringObjective: To evaluate the difference between intraprocedural US and gross section measurements and to analysis the accuracy and controllability of monitoring the ablation zones by US.Methods: Pulsed electric fields (PEFs) with a 2-cm probe distance, permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) applied on the electrodes and placement of the electrodes had been localized into goats'liver under ultrasound guidance though the animal skin to the target area.The ablation zone was monitored and measured in real time with US, which was repeated both immediately after the IRE procedure (D1) and again 24 hours after the IRE ablation procedure (D2), and the gross section (D3) was measured by electric vernier caliper after the animal was sacrificed by taken an overdose of anesthetic agent. Correlations between D1 and D2, D2 and D3, D1 and D3, D3 and D4(24 hours post IRE ablation when 120 pieces of pulses were performed on goat livers directly after abdomen discission ) were statistically analyzed separately.Results: There was no significant statistic difference showed between D3 and D4[(35.96±2.52)mm](24 hours post IRE ablation when 120 pieces of pulses were performed on goat livers directly after abdomen discission)(P>0.05). Among the three sets of data (D1, D2, and D3), the maximum diameter measured by intraprocedural US immediately after IRE ablation procedure [D1,(39.58±2.13)mm] was the largest diameter, and followed was the data measured by US 24 hours after the procedure [D2(,37.07±3.51)mm]. The two sets were statically different(P<0.05). The gross section measurement [D3,(36.44±2.04)mm] which was measured 24 hours after IRE ablation was the smallest, but it's not significant statistic difference between D2 and D3(P>0.05). D1 showed a well linear correlation with D3(r=0.949).Conclusion:Ultrasound-guided percutaneous IRE on goats'liver ablation had the same effect with IRE ablation areas when pulses were performed on goat livers directly after abdomen discission. In the percutaneous IRE ablation procedure, data measured by intraprocedural US correlated well with gross section measurement. Data measured by intraprocedural US immediately after IRE ablation procedure was larger than the same set of data measured by US and though gross section measurement 24 hours post IRE ablation, but correlated well with the latter one. US provided an accurate and available method for IRE target area monitoring.Part 3 Research on histological features of goat livers post IRE ablationChapter 1 Histopathology and ultra-structure changes of goat livers post IRE ablationObjective: To investigate the histopathology and ultra-structure changes of goat livers with percutaneous irreversible electroporation (IRE) treatment.Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) were performed on the electrodes which were placed under ultrasound guidance through the animal skin to the target area. The treated area was observed by hematoxylin and eosin (HE) stain, Glucose-6-phosphatse (G-6-P)stain, Succinodehydrogenase (SDH) stain, Cholangioles Pyridine silver stain and reticular fibers silver stain at the time of 0hour, 24hours, 3days, 7days, 14days after IRE ablation. The function of G-6-P and SDH enzymes and histological feature of liver cells, cholangioles and reticular fibers were all observed by light microscope. Ultra-structure changes of liver cells were observed by transmission electron microscope at the time of 0hour, 24hours, and 3days after IRE ablation.Results: Complete hepatic cell death was observed after the IRE treatment and there was a sharp demarcation between the ablated zone and the non-ablated zone. By 14 days the ablated lesions was almost completely replaced by fibrous scartissue. Ultra-structure changes of liver cells observed by TEM showed that there were rupture of cell membranes, swelling and necrosis of mitochondrion, extension of rough endoplasmic reticulum and destruction of capillaries continuity in the ablated zone. Stain of G-6-P, SDH and cholangioles were all negative 24 hours post IRE. Endothelial cells of the cholangioles and capillaries and reticular cells were ablated at the same time.Conclusion: Percutaneous IRE can provide a novel and unique method in ablating living cells with lipid bilayer membranes, such as liver cells, endothelial cells of the capillaries, cholangioles or reticular cells. The activity function of G-6-P and SDH in hepatic cells was lost at the same time. Chapter 2 Effect of IRE ablation on the structure of blood vesselsObjective: To investigate the effect of IRE ablation on the structure of blood vessels.Methods: Pulsed electric fields (PEFs) with permanent duration (100μs), frequency (1Hz), voltage (2000V) and pulses (120 pieces) were performed on the electrodes which were placed under ultrasound guidance through the animal skin to the target area. The treated area was observed by real-time ultrasound scanning and color doppler flow imaging at the time of 0hour, 24hours, 3d, 7d and 14d after IRE ablation. At gross section examination, blood vessel sections in the ablation area were fixed in 4% paraformaldehyde and embedded with paraffin. Histological changes of the treated area were observed by HE stain, VG stain and Masson stain.Results: Gross section examination showed that structural integrity of vascular within IRE-ablated zone (including the intrahepatic large vessels with diameters of more than 10mm and small intrahepatic vessels with diameters of less than 2mm). The imaging of two-dimensional ultrasound showed that blood vessels were intact and there was color flow angiography in blood vessels. Endothelial cells of were blood vessels not ablated by IRE. The blood vessel wall structures which mainly formed by vascular elastic and collagenous structures, peri-cellular matrix proteins and vascular smooth muscle structures were not damaged by IRE ablation. Conclusion: IRE-ablation destroyed capillaries and cholangioles, but retained important vital structure such as hepatic arteries, hepatic veins and bile duct and perivascular elastic fiber structure and collagen fiber structure in the ablation zone. It suggested that IRE would not be affected by the heat sink effect of blood flow, which ensured effective transmission and accumulation of electric field energy. Thus, IRE could produce a more complete ablation effect within the lesion area, even with a large vessel traversing the ablation zone.Chapter 3 Apoptosis of goat livers induced by irreversible electroporationObjective:To investigate the impact of IRE ablation on apoptosis and necrosis of goat liver cells, and the relationship between these biological changes and distribution of electric fields.Methods:IRE induced by 120 pieces of pulses(Pulsed electric fields (PEFs) with permanent duration 100μs, frequency 1Hz and voltage 2000V) were performed on goats livers. The electrodes were placed under ultrasound guidance via the animal skin to the target area. All the animals were scarified 24 hours after IRE ablation. Specimens were collected respectively from the center of ablated zone and the margin of ablated zone. Normal hepatic tissue was also collected as control. The specimens were observed by hematoxylin and eosins (HE) stain. Apoptosis was detected by DNA ladder and TUNEL. Hepatic cells were analyzed with flow cytometry (FCM) for cell cycle changes.Results:Complete hepatic cell death were observed after IRE treatment. In the center of ablated zone (electrode point), coagulative necrosis was obvious. Apoptotic cells stained by TUNEL were found mainly at the margin of the ablated zone. Nucleus and cytoplasm of apoptotic cells were positively stained as pitch black and light buffy color respectively. DNA ladder analysis detected ladder strap with a length of 400 to 1000bp at the margin of ablated zone. Hypodiploid peak was found with FCM analysis in the hepatic cells from this area.Conclusion:IRE induced liver cell death was by the means of coagulative necrosis in the center of ablation area (electrode point), while programmed cell death by the means of apoptosis was gradually involved as the decay of electric field strength at the margin of ablated zone. IRE could ablate tumor beyond the area of macroscopic observation. It is benefit to reducing recurrence and metastasis in surrounding area after IRE tumor ablation.