Analysis of safety and efficacy of irreversible electroporation hepatic ablation with high-frequency bipolar pulse in swine / 中华消化外科杂志

Objective@#To investigate the safety and efficacy of irreversible electroporation (IRE) hepatic ablation with high-frequency bipolar pulse in swine.@*Methods@#The experimental study was conducted. A total of 18 swines of either gender, aged (6.8+ 0.8)months with a range of 5.5-8.0 months, were collected from Animal Laboratory Center of Army Medical University. were randomly divided into 15 in experimental group and 3 in control group. The swines in experimental group underwent IRE hepatic ablation with high-frequency bipolar pulse, and 3 swines were chose randomly and underwent enhanced CT examination immediately after ablation, and at 3, 7, 14, and 28 days after ablation. The liver tissues were taken for histopathological examination. The swines in the control group underwent IRE hepatic ablation with high-frequency monopolar burst, and was performed enhanced CT examination at 3 days after ablation. Liver tissues were taken for histopathological examination. Observation indicators: (1) comparison of muscle contraction of siwnes between two groups; (2) imaging performance on enhanced CT after IRE ablation in the experimental group; (3) hepatic histopathological findings after IRE ablation in the experimental group; (4) comparison of apoptotic index in the ablation zone between two groups. The measurement data with normal distribution were expressed as Mean±SD, and comparison between groups was performed by the independent sample t test.@*Results@#(1) Comparison of muscle contraction between two groups: swines in both groups underwent ablation successfully. The degree of muscle contraction was (9.8±0.4)m/s2 and (48.6±0.5)m/s2 in the experimental group and in the control group, respectively, showing statistically significant difference between the two groups (t=-163.50, P<0.05). (2) Imaging performance on enhanced CT after IRE ablation in the experimental group: the enhanced CT examination of swines immediately after IRE ablation showed a low-density shadow and clear boundary in the ablation zone. There was no obvious abnormality in the ablation zone and its adjacent large vessels. No serious complications occurred after the ablation. The boundary between the ablation zone and the normal liver tissue of the experimental group gradually became blurred over time, and the ablation zone was gradually replaced by normal liver tissue. The ablation zone at the 28 days after ablation was significantly reduced or even disappeared on imaging of enhanced CT examination.The maximum diameter of the ablation zone was (1.81±0.17)cm immediately after ablation, (1.75±0.19)cm at the 3 days after ablation, (1.32±0.22)cm at the 7 days after ablation, (0.65±0.14)cm at the 14 days after ablation, (0.28±0.10)cm at the 28 days after ablation, respectively. (3) Hepatic histopathological findings after IRE ablation in the experimental group: the HE staining of ablated tissue immediately after ablation showed that the cells in the ablation zone were swollen, arranged disorderly, and bleeding was observed around some of the needles.The bile ducts and blood vessels were intact in the ablation zone, and a large number of deeply stained nuclei were seen at 3 days after ablation, some of the nucleus and apoptotic bodies were partially dissolved or cleaved. A large number of inflammatory cell were infiltrated around the ablation zone. Intact vascular and biliary endothelial cells were observed by von Willebrand factor staining, a larger number of apoptotic cells with deeply stained nuclei in the ablation zone were observed by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining, and partial deposited dark brown calcium salt was seen by Von Kossa staining. More newborn hepatocytes were observed growing from the periphery of the ablation zone to the center at the 7, 14, 28 days after ablation. Smooth muscle cell proliferation was observed at 14 and 28 days after ablation. The ablation zone was replaced by new cells on 28 days after ablation. (4) Comparison of apoptotic index in the ablation zone between two groups: the apoptotic index of the ablation zone was significantly higher in the experimental group than in the control group on the 3 days after operation (76.67%±0.04% vs. 64.03%±0.05%, t=4.79, P<0.05).@*Conclusion@#IRE hepatic ablation of swine using high-frequency bipolar pulse is safe and reliable, and it has more apoptotic cells than IRE ablation with high-frequency monopolar burst.

Analysis of safety and efficacy of irreversible electroporation hepatic ablation with high-frequency bipolar pulse in swine / 中华消化外科杂志

Objective To investigate the safety and efficacy of irreversible electroporation (IRE) hepatic ablation with high-frequency bipolar pulse in swine.Methods The experimental study was conducted.A total of 18 swines of either gender,aged (6.8+0.8)months with a range of 5.5-8.0 months,were collected from Animal Laboratory Center of Army Medical University.were randomly divided into 15 in experimental group and 3 in control group.The swines in experimental group underwent IRE hepatic ablation with high-frequency bipolar pulse,and 3 swines were chose randomly and underwent enhanced CT examination immediately after ablation,and at 3,7,14,and 28 days after ablation.The liver tissues were taken for histopathological examination.The swines in the control group underwent IRE hepatic ablation with high-frequency monopolar burst,and was performed enhanced CT examination at 3 days after ablation.Liver tissues were taken for histopathological examination.Observation indicators:(1) comparison of muscle contraction of siwnes between two groups;(2) imaging performance on enhanced CT after IRE ablation in the experimental group;(3) hepatic histopathological findings after IRE ablation in the experimental group;(4) comparison of apoptotic index in the ablation zone between two groups.The measurement data with normal distribution were expressed as Mean±SD,and comparison between groups was performed by the independent sample t test.Results (1) Comparison of muscle contraction between two groups:swines in both groups underwent ablation successfully.The degree of muscle contraction was (9.8±0.4)m/s2 and (48.6±0.5) m/s2 in the experimental group and in the control group,respectively,showing statistically significant difference between the two groups (t =-163.50,P＜0.05).(2) Imaging performance on enhanced CT after IRE ablation in the experimental group:the enhanced CT examination of swines immediately after IRE ablation showed a low-density shadow and clear boundary in the ablation zone.There was no obvious abnormality in the ablation zone and its adjacent large vessels.No serious complications occurred after the ablation.The boundary between the ablation zone and the normal liver tissue of the experimental group gradually became blurred over time,and the ablation zone was gradually replaced by normal liver tissue.The ablation zone at the 28 days after ablation was significantly reduced or even disappeared on imaging of enhanced CT examination.The maximum diameter of the ablation zone was (1.81±0.17) cm immediately after ablation,(1.75±0.19) cm at the 3 days after ablation,(1.32±0.22)cm at the 7 days after ablation,(0.65±0.14)cm at the 14 days after ablation,(0.28±0.10)cm at the 28 days after ablation,respectively.(3) Hepatic histopathological findings after IRE ablation in the experimental group:the HE staining of ablated tissue immediately after ablation showed that the cells in the ablation zone were swollen,arranged disorderly,and bleeding was observed around some of the needles.The bile ducts and blood vessels were intact in the ablation zone,and a large number of deeply stained nuclei were seen at 3 days after ablation,some of the nucleus and apoptotic bodies were partially dissolved or cleaved.A large number of inflammatory cell were infiltrated around the ablation zone.Intact vascular and biliary endothelial cells were observed by yon Willebrand factor staining,a larger number of apoptotic cells with deeply stained nuclei in the ablation zone were observed by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining,and partial deposited dark brown calcium salt was seen by Von Kossa staining.More newborn hepatocytes were observed growing from the periphery of the ablation zone to the center at the 7,14,28 days after ablation.Smooth muscle cell proliferation was observed at 14 and 28 days after ablation.The ablation zone was replaced by new cells on 28 days after ablation.(4) Comparison of apoptotic index in the ablation zone between two groups:the apoptotic index of the ablation zone was significantly higher in the experimental group than in the control group on the 3 days after operation (76.67％±0.04％ vs.64.03％±0.05％,t=4.79,P＜0.05).Conclusion IRE hepatic ablation of swine using high-frequency bipolar pulse is safe and reliable,and it has more apoptotic cells than IRE ablation with high-frequency monopolar burst.

The anti-tumor efficacy of nanosecond pulsed electric fields on the mouse with melanoma xenograft in vivo / 生物医学工程学杂志

This study was conducted to investigate the anti-tumor efficacy of nanosecond pulsed electric fields (nsPEFs) on the mouse with A375-GFP melanoma xenograft in vivo. In vivo fluorescence image analysis system was used in this study to evaluate the effects of nsPEFs on human melanoma A375 cell xenograft. On the Day 90 af ter pulse delivery, the skin that had contained A375 cell xenograft was surgically excised and pathologically evalua ted. The changes of scar were recorded by digital camera. The experiment revealed that significant changes in fluorescence value trend and amplitude were found in the treated group from those in the control group. The fluorescence of tumor in the treated group decreased mostly 48 h after the treatment and completely disappeared 10 d after the treatment, while that in control group was increased gradually. Surgical excision of the area confirmed a complete pathologic response. Within a few days after the nsPEFs treatment, a hard scab formed at the treatment region. The scab fell off by the end of the second week. As time went on, the scar gradually became faded and all xenograft tumors were disappeared without recurrence. From the experiment, we learn that nsPEFs can bring good therapeutic effect. It may provide a new approach for the clinical treatment of superficial tumors.

Induction of apoptosis of ovarian cancer cells and influence on Fas-mediated apoptosis pathway by nanosecond pulsed electric fields / 生物医学工程学杂志

This paper is to investigate the apoptosis effect of ovarian cancer SKOV3 cells induced by nanosecond plused electric fileds (nsPEFs) and to study its influence on Fas-mediated apoptosis. SKOV3 cell were exposed to the 45kV/cm of field intensity, 30 pulses, and 50ns, 100ns, and 200ns of pulse width, respectively. Flow cytometry were used to assay apoptosis. Agarose gel electrophoresis was used to detect DNA ladder. Real time PCR (RT-PCR) and Western blot analysis were used to measure the expression level of Fas, FasL, caspase-8 and Bid. Flow cytometry results revealed that the late apoptosis rates and (or) necrosis were significantly higher than those in control group (3.03% +/- 0.57%) (P < 0.05), with apoptosis rates and (or) necrosis being (18.31 +/- 0.65%), (45.55% +/- 3.71%), (47.47% +/- 7.01%) in the groups of 50ns, 100ns, 200ns, respectively. A typical DNA ladder pattern of internucleosomal fragmentation was observed in the groups of 50ns and 100ns, but not clear in the 200ns group. RT-PCR results revealed that the mRNA expression of Fas, FasL, caspase8 and Bid were significantly increased in groups of 50ns, 100ns, but significantly decreased in group of 200ns (P < 0.05). Meanwhile, Western blot analysis demonstrated that the Fas, FasL, Caspase-8 and Bid expression were significantly higher in groups of 50ns, 100ns, but significantly lower in group of 200ns (P < 0.05). It indicated that 45kV/cm, 50ns, 100ns nsPEFs could induce apoptosis in ovarian cancer SKOV3 cells and activate Fas-mediated apoptosis pathway.

Effects of electric pulses on liver cancer cells: apoptosis induction and decrease of mitochondrial transmembrane potential / 生物医学工程学杂志

In order to investigate the effects of electric pulses on cancer cells, we carried out the experiments with exposing HepG2 and L02 to electric pulses (1 kV/cm, l00 micros, 1 Hz) for different lengths of time (8 s, 15 s, 30 s, 60 s). Annexin V-FITC Kit and Flow cytometry were used to study the apoptosis of treated cells. The results showed that the electric pulses of 1 kV/cm, l00 micros, 1 Hz for 8 s could not induce tumor cells apoptosis. Apoptosis was observed when tumor cells were stimulated for 15 s and longer, and the apoptosis percentage increased with the increase of stimulation time. Furthermore, tumor cells were more sensitive than normal cells in response to electrical pulses. Rhodamine 123 and Laser Scanning Confocal Microscope (LSCM) were used to make a real-time study of mitochondrial transmembrane potential (Deltapsim) when the tumor cells were exposed to electric pulses for 60 s. No significant change of Deltapsim was observed within 30 s stimulation. After that, the Deltapsim increased sharply and declined later, suggesting that the mitochondrial pathway may be one of the apoptosis mechanism induced by electric pulses.

Analysis of frequency-domain and window effect for cellular inner and outer membranes subjected to pulsatile electric field / 生物医学工程学杂志

Based on multi-layer dielectric model of spherical biological cell, a simulating method of frequency characteristics of inner and outer membranes is presented in this paper. Frequency-domain analysis showed that inner and outer membranes subjected to pulsed electric field exhibit band-pass and low-pass filter characteristics, respectively. A calculating method of the transmembrane potential of inner and outer membranes induced by time-varying electric field was introduced, and the window effect between electric field and transmembrane potential was also analyzed. When the duration is reduced from microsecond to sub-microsecond, and to nanosecond, the target induced was from the outer membrane to inner membrane gradually. At the same time, the field intensity should be increased to induce corresponding bioelectric effects. Window effect provides theoretical guidance to choosing reasonable parameters for application of pulsatile electric field in tumor treatment.

Focusing properties of picosecond electric pulses in non-invasive cancer treatment / 生物医学工程学杂志

In the light of optical theory, we advanc an ultra-wideband impulse radiating antenna (IRA) which is composed of an ellipsoidal reflector and a cone radiator. The high-intensity ultra-short electric pulses radiated by IRA can be transferred into the deep target in tissue non-invasively and be focused effectively. With the focused picosecond electric pulses, the organelles (mitochondria) transmembrane potential shall change to collapse under which the tumor cells will be targetly induced to apoptosis, so the method of non-invasive treatment of tumors would be achieved. Based on the time-domain electromagnetic field theory, the propagation characteristics of picosecond electric pulses were analyzed with and without the context of biological tissue, respectively. The results show that the impulse characteristics of input pulse were maintained and the picosecond electric pulses can keep high resolution in target areas. Meanwhile, because of the dispersive nature of medium, the pulse amplitude of the pulses will attenuate and the pulse width will be broadened.

Experimental study on effects of energy controllable steep pulses on cytoskeleton of human ovarian cancer cells SKOV3 / 生物医学工程学杂志

The aim of our study was to determine the effects of energy controllable steep pulse (ECSP) on the cytoskeleton of human ovarian cancer cells SKOV3. SKOV3 cells were divided into five groups under ECSP treatment with different parameters (frequency, pulse duration, peak value of voltage). The positive control group included SKOV3 cells treated with volchicine; the negative control group included SKOV3 cells subjected to sham-lightning stroke. Rhodamine-phalloidine was used to label microfilament directly. After using immunofluorescence to label microbules, we observed them by means of Confocal Laser Scanning Microscope. Making specimen and using electronmicroscope, we observed the ultramicrostructure of cystoskeleton. The results showed that ECSP-treated-SKOV3 cells lost their normal cystoskeleton network structure. There were obvious microfilament disaggregation, diffused skeleton protein, and disappearance of cystoskeleton network structure. Also noticeable were microbule disaggregation, reduction of pseudopod, obvious microfilament disaggregation, permutation disorder and structure disappearance. Moreover, this effect bears a direct relation with dosage.

Recent advances on physical ablation for tumor / 生物医学工程学杂志

Physical ablation is a new kind of tumor treatment which directly acts on local solid tumors to eradicate or destroy tumor tissues by use of various advanced physical techniques. Physical ablation can be classified by physical characteristics as thermal ablate therapy (TAT), cryoablation and electrical ablation. Recent studies and technical trend of these three physical ablation treatments are reviewed in this paper.

The study of apoptosis and mechanism of cells exposed to steep pulse / 生物医学工程学杂志

This experiment was designed to study the apoptosis and related mechanism of adherent liver tumor cells (SMMC-7721) and adherent normal liver cells (HL-7702) when they were exposed to the steep pulse generated by the steep pulse apparatus for tumor treatment. The results showed that the steep pulse of 200 V could induce tumor cells apoptosis. The tumor cells presented with their apoptosis when they were exposed to the steep pulse from 200 V to 250 V. Laser scanning confocal microscopy was used to make a real time study of calcium burst when the adherent tumor cells were exposed to the steep pulse. The results showed:On the condition of no extracellular Ca2+, the concentration of Ca2+ in tumor cells exposed to the steep pulse of 150 V did not change; the concentration of Ca2+ in tumor cells exposed to the steep pulse of 200 V decreased; the concentration of Ca2+ in tumor cells exposed to the steep pulse of 250 V decreased more evidently. On the condition of existing extracellular Ca2+, the concentration of Ca2+ in tumor cells exposed to the steep pulse of 150 V did not change; the concentration of Ca2+ in tumor cells exposed to the steep pulse of 200 V decreased little; the concentration of Ca2+ in tumor cells exposed to the steep pulse of 250 V reduced little, too. Maybe the change of calcium burst in the tumor cells is the mechanism of apoptosis when cells are exposed to the steep pulse.

Research progress of nanosecond pulsed electric field applied to intracellular electromanipulation / 生物医学工程学杂志

In recent years, many experts have done some researches on experiment and mechanism of intracellular electromanipulation (IEM) under nanosecond pulsed electric field (nsPEF). The experiment results have shown that nsPEF could not induce electroporation of cell membrane, but could induce intracellular effects such as apoptosis, calcium release, enhancement of gene expression, and fragmentation of DNA and chromosome. In order to account for the phenomenon, researchers believe that when the pulse width of the pulsed electric field is larger than the charging time of plasma membrane, the pulsed electric field mainly targets on the outer membrane of cell; and that the effect of the pulsed electric field on nucleus and nuclear membrane increases with the decrease of the pulse width. It is also believed that the effect of electroporation changes from the outer membrane to intracellular electromanipulation when the pulse width decreases to a value being smaller than the charging time of plasma membrane.

Steep pulse changes the expression of tissue factor in ovarian tumor / 生物医学工程学杂志

As a micro-wound and target-aimed technology without special limitation, Electric Pulses have been widely researched in tumor treatment and the effects have been demonstrated by a series of experiments, yet the mechanism has not been explained clearly. In this experiment, energy controllable steep pulse (ECSP) was used to treat nude mice bearing human ovarian tumor, and the result was compared with that of the control group. The expression of an important coagulant factor-tissue factor (TF) was analyzed, as TF was also a tumor indicator of invasion and metastasis, the result may indicate the relationship among ECSP, thrombosis and tumor invasion. In this study, to shed light on the mechanism of tumor treatment in electrical fields, nude mice bearing ovarian tumors were randomly divided into the treated group and the untreated group. We treated the former group and took out the tumor instantly. The thrombosis and necrosis of ovarian tumor were observed under microscope. The expression of TF was analyzed by SP immunohistochemistry and RT-PCR. Lower level of TF expression was noticed in the tumor tissue treated by ECSP, and more apparent thrombosis was also seen in this group. The results make it clear that ECSP can accelerate thrombosis and consume coagulant factors such as TF, and that low expression of TF in tumor tissue can cut out the signal paths of tumor invasion. So it is suggested that ECSP may restrain tumor invasion and metastasis by modulating thrombosis.

Effect of steep pulsed electric fields on the immune response of tumor-bearing Wistar mice / 生物医学工程学杂志

This study sought to evaluate the effect of steep pulsed electric fields (SPEFs) on the immune response of Wistar mice inoculated with Walker256 sarcoma. Thirty mice were randomly divided into three groups: control group (group A, inoculated with Walker256 sarcoma, not treated), treatment group (group B, inoculated with Walker256 sarcoma, treated by SPEFs), and normal control group (group C, inoculated with normal saline, not treated). Tumor size was measured before and every 3 days after treatment by vernier caliper. MTT methods were used to assess the lymphocytes proliferation and the natural killer (NK) cells activity. TNF-a activity was measured by ELISA. Statistical analysis was performed utilizing the SPSS10.0 software package. The experiment results revealed that tumor growth was significantly inhibited in group B as compared with group A (P < 0.01), and that lymphocytes proliferation, NK cells activity and TNF-a activity in group B were not significantly different from those in group C (P = 0.953, P = 0.130, P = 0.080, respectively) but markedly higher than those in group A (P < 0.05). The results also showed that SPEFs could not only kill tumor cells but also induce antitumor immune response and improve the immune function of the host efficiently.

Experimental study of the lethal effects of steep pulsed electric field on cancer cells in BALB/c mice / 生物医学工程学杂志

This study was conducted to observe the lethal effects of steep pulsed electric field(SPEF) on the cancerous squamous cells in BALB/c mice. Female mice were inoculated with the solution of cervical cancer cells line. The tumor-bearing mice of the experiment group were exposed to SPEF. Then, the histomorphological changes were examined and compared between experiment group and control group. The histological and antitumor assay showed that SPEF could selectively injure tumor cells and inhibit cell proliferation. After treatment, pyknosis, karyoclasis and karyolysis of cancer cells were observed under light and electron mircroscope. More serious changes appeared some days later. These results indicate that SPEF have lethal effects on cervical cancer. It may prolong the survival period of tumor-bearing mice.

A qualitative study of in vivo pulsed electric field distribution model in rabbit liver tissues / 生物医学工程学杂志

Pulsed electric fields (PEFs) with fixed frequency, width and gradually increased peak value of voltage was applied to 30 healthy rabbit liver tissues. The specific aims were to explore the feasibility of establishing a model of in vivo PEFs distribution in healthy rabbit liver tissues and to provide important references for clinical electrochemotherapy and for electrotransfer. Repeated experiment and self-comparison statistics design were implemented. The rabbit underwent the experiment under intravenous anesthesia and their liver tissues, after exposure to PEFs, were sent for HE staining. Necrotic borderline was visible 3 days after PEFs application, the necrotic shape of concentric circle was evident around the electrodes under optical microscope at lower voltage, as voltage increasing, two necroses in the shape of concentric circle gradually enlarged; nuclei with chromatin condensation, fragmentation and lysis alterations were seen in the middle region between the needles; concentric circles changed into ellipse fusiform and finally overlaped each other forming irregular necrosis contours. Cell cavitation and tissues ischemia were also observed within electric field. The shape of tissue necrosis from the experiment was noted to correlate with theoretic simulation of electric field distribution. Therefore, rabbit liver tissues can be a good carrier for in vivo modeling of electric field distribution when the lethal effects of PEFs in tissues are investigated. PEFs also show safety for the surrounding normal tissue while causing damage or injury to the target area therapeutically.

Effects of steep pulsed electric fields on cancer cell proliferation and cell cycle / 生物医学工程学杂志

To assess study the cytocidal and inhibitory effects of steep pulsed electric fields (SPEFs) on ovarian cancer cell line SKOV3, the cancer cell suspension was treated by SPEFs with different parameters (frequency, pulse duration, peak value of voltage). Viability rate and growth curves of two test groups (high dosage and low dosage of SPEFs) and one control group were also measured. The DNA contents and cell cycle were analyzed by flow cytometry (FCM). Different dosing levels of SPEFs exerted obviously different effects on cancer cell viability. With the enhancement of each pulse parameter, the viability rate was promoted and the inhibitory effect on the proliferation of treated cells was more evident. The cells exposed to SPEFs grew slower than the control. The ratio of S+G2/M phase cells was decreased, which restrained the DNA synthesis and division, but the ratio of G0/G1 phase cells was increased in the treated groups. It was also indicated that the SPEFs blocked the cell transition from G0/G1 phase to S+G2/M phase. There was a significant difference in cell cycle between treated group and control group (P<0.01). Lethal effects of SPEFs were represented by inhibiting the cancer cell proliferation at the cell level and by influencing the cell cycle at the DNA level.

Effect of steep pulsed electric fields on survival of tumour-bearing mice / 生物医学工程学杂志

To investigate the lethal effect of steep pulsed electric fields (SPEFs) on cancer cells and the life-prolonging effect of SPEFs on the survival of tumour-bearing mice, this study was carried out with the use of SPEFs to treat 40 BALB/C mice inoculated by cervical cancer. The lethal effect on cancer cells and the life-prolonging effect on tumour-bearing mice were examined and compared between the experiment group and control group. The survival periods of the experiment group and control group were 52.05 days and 33.03 days, respectively. There was a significant difference in survival curve between the two groups. The results confirmed the inhibitiory effect and lethal effect of SPEFs on cancer cells. SPEFs can prolong the survival period of tumour-bearing mice.

The research progress of using electroporation therapy in treatment of tumor / 生物医学工程学杂志

In these years, the electrical technology is widely applied in the study of biomedical engineering. Using electroporation therapy (EPT) to treat tumor is associated with biomedical engineering, electrical new technology, computer technology and microelectronic technology, which is a new marginal subject. Many experts have studied the mechanism and clinical treatment of the cell membrane electroporation phenomenon under electrical fields. These researches have shown that the membrane electroporation can stimulate the transport and intake of various drugs, which improves the tumoricidal effect of these drugs. The researchers have also been exploring the phenomenon that irreversible electrical breakdown (IREB) of cell membrane under high electrical fields and steep pulses leads to the death of tumor.