Cryosurgery with pulsed electric fields

This study explores the hypothesis that combining the minimally invasive surgical techniques of cryosurgery and pulsed electric fields will eliminate some of the major disadvantages of these techniques while retaining their advantages. Cryosurgery, tissue ablation by freezing, is a well-established minimally invasive surgical technique. One disadvantage of cryosurgery concerns the mechanism of cell death; cells at high subzero temperature on the outer rim of the frozen lesion can survive. Pulsed electric fields (PEF) are another minimally invasive surgical technique in which high strength and very rapid electric pulses are delivered across cells to permeabilize the cell membrane for applications such as gene delivery, electrochemotherapy and irreversible electroporation. The very short time scale of the electric pulses is disadvantageous because it does not facilitate real time control over the procedure. We hypothesize that applying the electric pulses during the cryosurgical procedure in such a way that the electric field vector is parallel to the heat flux vector will have the effect of confining the electric fields to the frozen/cold region of tissue, thereby ablating the cells that survive freezing while facilitating controlled use of the PEF in the cold confined region. A finite element analysis of the electric field and heat conduction equations during simultaneous tissue treatment with cryosurgery and PEF (cryosurgery/PEF) was used to study the effect of tissue freezing on electric fields. The study yielded motivating results. Because of decreased electrical conductivity in the frozen/cooled tissue, it experienced temperature induced magnified electric fields in comparison to PEF delivered to the unfrozen tissue control. This suggests that freezing/cooling confines and magnifies the electric fields to those regions; a targeting capability unattainable in traditional PEF. This analysis shows how temperature induced magnified and focused PEFs could be used to ablate cells in the high subzero freezing region of a cryosurgical lesion.     

Selective freezing of target biological tissues after injection of solutions with specific thermal properties

Cryosurgery is a minimally invasive surgical technique that employs the destructive effect of freezing to eradicate undesirable tissues. This paper proposes a flexible method to control the size and shape of the iceball by injecting solutions with specific thermal properties into the target tissues, to enhance freezing damage to the diseased tissues while preserving the normal tissues from injury. The cryosurgical procedure was performed using a minimally invasive cryoprobe cooled by liquid nitrogen (LN2) to obtain deep regional freezing. Several needle thermocouples were applied simultaneously to record the transient temperature to detect the freezing effect on the tissues. Simulation experiments on biological tissue (fresh pork) were performed in vitro and four different liquids were injected into the test materials; these were distilled water, an aqueous suspension of aluminum nanoparticles in water, ethanol, and a 10% solution of the cryoprotective agent dimethyl sulfoxide (Me2SO). The experimental results demonstrate that the localized injection of an appropriate solution could enhance the tumor-killing effect without altering the freezing conditions. The study also suggests the potential value of combining cryosurgery with other therapeutic methods, such as electrical, chemical, and thermal treatments, to develop new clinical modalities in the near future.     

Tumor ablation with irreversible electroporation

We report the first successful use of irreversible electroporation for the minimally invasive treatment of aggressive cutaneous tumors implanted in mice. Irreversible electroporation is a newly developed non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the cell membrane. Mathematical models of the electrical and thermal fields that develop during the application of the pulses were used to design an efficient treatment protocol with minimal heating of the tissue. Tumor regression was confirmed by histological studies which also revealed that it occurred as a direct result of irreversible cell membrane permeabilization. Parametric studies show that the successful outcome of the procedure is related to the applied electric field strength, the total pulse duration as well as the temporal mode of delivery of the pulses. Our best results were obtained using plate electrodes to deliver across the tumor 80 pulses of 100 micros at 0.3 Hz with an electrical field magnitude of 2500 V/cm. These conditions induced complete regression in 12 out of 13 treated tumors, (92%), in the absence of tissue heating. Irreversible electroporation is thus a new effective modality for non-thermal tumor ablation.     

Enhanced electroporation in plant tissues via low frequency pulsed electric fields: influence of cytoplasmic streaming

Pulsed electric fields (PEF) are known to be effective at permeabilizing plant tissues. Prior research has demonstrated that lower pulse frequencies induce higher rates of permeabilization, but the underlying reason for this response is unclear. Intriguingly, recent microscopic observations with onion tissues have also revealed a correlation between PEF frequency and the subsequent speed of intracellular convective motion, i.e., cytoplasmic streaming. In this paper, we investigate the effect of cytoplasmic streaming on the efficacy of plant tissue permeabilization via PEF. Onion tissue samples were treated with Cytochalasin B, a known inhibitor of cytoplasmic streaming, and changes in cellular integrity and viability were measured over a wide range of frequencies and field strengths. We find that at low frequencies (f < 1 Hz), the absence of cytoplasmic streaming results in a 19% decrease in the conductivity disintegration index compared with control samples. Qualitatively, similar results were observed using a microscopic cell viability assay. The results suggest that at low frequencies convection plays a statistically significant role in distributing more conductive fluid throughout the tissue, making subsequent pulses more efficacious. The key practical implication is that PEF pretreatment at low frequency can increase the rate of tissue permeabilization in dehydration or extraction processes, and that the treatment will be most effective when cytoplasmic streaming is most active, i.e., with freshly prepared plant tissues.     

What forces can determine the formation of highly specific protein-protein complexes?

A software package was designed and used in a detailed study of the contact regions (interfaces) of a large number of protein-protein complexes using the PDB data. It appeared that for about 75% of the complexes the amino acid composition of the subunit surface in the contact region is not essential. Thus one may suggest that, along with the amino acid residues at the interface, the residues in the interior of the globules substantially contribute to protein-protein recognition. Such interactions between quite remote residues are most probably of electrical nature, and are involved in recognition by contributing to the overall electric field created by the protein molecule; the configuration of this field is perhaps the definitive factor of recognition. The overall field of the protein molecule is additively built of the fields created by each constituent residue, and it can be calculated as a sum of the fields created by the protein multipole (aggregate of 'partial' electric charges assigned to every atom of the protein molecule). Preliminary calculations of the remote electrostatic interaction have been performed for ribonuclease subunits in vacuum. The results are indicative of a real possibility that the electric field created by the protein multipole can strongly influence the mutual orientation of molecules before Brownian collisions.     

Effects of Postmortem Delay and Temperature on Neurotransmitter Receptor Binding in a Rat Model of the Human Autopsy Process

Abstract: Studies of neurotransmitter and drug receptor alterations in neurodegenerative disorders have contributed to our understanding of the pathophysiology of these conditions. The effect of postmortem delay in freezing tissue after death and prolonged storage of tissue prior to analysis on receptor binding assays are potential artifacts that may limit interpretation of the effects of disease on receptor populations. We used a rat model of the human autopsy process to study the effects of increasing postmortem delay and storage time on N -methylscopolamine (NMS), p -aminoclonidine (PAC), flunitrazepam (FLU), and spiperone binding in a variety of rat brain regions. The rat brains were cooled using a temperature-controlled environment and thermistor probe to follow cooling curves obtained in human brain. Brains were cooled to either room temperature (22°C) or refrigerator temperature (4°C). For three of the four receptors, receptor binding decreased as postmortem delay before freezing increased, particularly in tissue cooled to room temperature. Unlike binding at other receptor sites, FLU binding increased with increasing postmortem delay to freezing. Different effects on K _D and B _max were noted for each ligand studied. No effects of the freezing process itself or storage at -80°C were detectable.     

A statistical model for multidimensional irreversible electroporation cell death in tissue

Background  

Irreversible electroporation (IRE) is a minimally invasive tissue ablation technique which utilizes electric pulses delivered by electrodes to a targeted area of tissue to produce high amplitude electric fields, thus inducing irreversible damage to the cell membrane lipid bilayer. An important application of this technique is for cancer tissue ablation. Mathematical modelling is considered important in IRE treatment planning. In the past, IRE mathematical modelling used a deterministic single value for the amplitude of the electric field required for causing cell death. However, tissue, particularly cancerous tissue, is comprised of a population of different cells of different sizes and orientations, which in conventional IRE are exposed to complex electric fields; therefore, using a deterministic single value is overly simplistic.     

Surface transmission probe for noninvasive measurements of dielectric properties of organ tissues at frequencies between 1 MHz and 300 MHz

A probe is described for determining the dielectric permittivity and dielectric loss of organ tissues at very high frequencies (VHF) in a non-violating manner. Instead of the usual reflection coefficient measurement, with this probe the electromagnetic wave transmission coefficient is measured between two coplanar electrodes on the organ surface which are capacitively coupled by the electric fringing fields extending into the organ. An appropriately constructed transmission probe can sense the dielectric behaviour of tissue into deeper organ regions than a reflection probe of corresponding size. Results are reported of transmission probe measurements on various liquids with known dielectric properties for the purpose of probe testing and on a pig liver during ischemia.     

Microsecond and nanosecond electric pulses in cancer treatments

New local treatments based on electromagnetic fields have been developed as non‐surgical and minimally invasive treatments of tumors. In particular, short electric pulses can induce important non‐thermal changes in cell physiology, especially the permeabilization of the cell membrane. The aim of this review is to summarize the present data on the electroporation‐based techniques: electrochemotherapy (ECT), nanosecond pulsed electric fields (nsPEFs), and irreversible electroporation (IRE). ECT is a safe, easy, and efficient technique for the treatment of solid tumors that uses cell‐permeabilizing electrical pulses to enhance the activity of a non‐permeant (bleomycin) or low permeant (cisplatin) anticancer drug with a very high intrinsic cytotoxicity. The most interesting feature of ECT is its unique ability to selectively kill tumor cells without harming normal surrounding tissue. ECT is already used widely in the clinics in Europe. nsPEFs could represent a drug free, purely electrical cancer therapy. They allow the inhibition of tumor growth, and interestingly, nsPEF can target intracellular organelles. However, many questions remain on the mechanism of action of these pulses. Finally, IRE is a new ablation procedure using pulses that provoke the permanent permeabilization of the cells resulting in their death. This technique does not result in any thermal effect, which is its main advantage in current physical ablation technologies. For both the nsPEF and the IRE, the preservation of the normal tissue, which is characteristic of ECT, has not yet been shown and their safety and efficacy still have to be investigated thoroughly in vivo and in the clinics. Bioelectromagnetics 33:106–123, 2012. © 2011 Wiley Periodicals, Inc.     

Electrical injury mechanisms: dynamics of the thermal response

The thermal response of the human upper extremity to large electric currents was examined using an axisymmetric unidimensional model containing bone, skeletal muscle, fat, and skin in coaxial cylindrical geometry. Appropriate thermal and electrical properties were assigned to each tissue, and the tissue response to joule heating was determined by a finite-element numerical technique. We found that when the tissues are electrically in parallel, skeletal muscle sustained the largest temperature rise and then heated adjacent tissues. Thus, when bone is not in series with other tissues, joule heating of bone is unlikely to be responsible for thermal damage to adjacent tissue. In addition, the effect of tissue perfusion on the thermal response was found to be essential for rapid cooling of the centrally located tissues.     

Electrical impedance tomography of cell viability in tissue with application to cryosurgery

Tissue damage that is associated with the loss of cell membrane integrity should alter the bulk electrical properties of the tissue. This study shows that electrical impedance tomography (EIT) should be able to detect and image necrotic tissue inside the body due to the permeabilization of the membrane to ions. Cryosurgery, a minimally invasive surgical procedure that uses freezing to destroy undesirable tissue, was used to investigate the hypothesis. Experimental results with liver tissue demonstrate that cell damage during freezing results in substantial changes in tissue electrical properties. Two-dimensional EIT simulations of liver cryosurgery, which employ the experimental data, demonstrate the feasibility of this application.     

Galvanic apparent internal impedance: An intrinsic tissue property

Using basic galvanic cell principles, the ability of tissues to generate electrical current through electrolysis was characterized. Studying Zn/Cu electrolysis in animal organs revealed a fundamental and measurable tissue-specific property - the galvanic apparent internal impedance (GAII), that is most likely related to the salt bridge function of tissues delineated by electrodes. Further to the fundamental knowledge acquired, GAII enables a new diagnostic method to distinguish between tissue types and to determine their health status without a need for expensive calibration, as often required when external power source is used. We demonstrated the GAII sensitivity in detecting tissue ablation with microwave heating or irreversible electroporation. The results open the way for a novel, inexpensive self-powered tissue diagnostic system for a wide range of applications such as minimally invasive tissue health status, ischemia, hydration, real time intra-operative control of minimally invasive surgery, medical imaging, virtual biopsy and many others.     

The use of electric fields in tissue engineering: A review

The use of electric fields for measuring cell and tissue properties has a long history. However, the exploration of the use of electric fields in tissue engineering is only very recent. A review is given of the various methods by which electric fields may be used in tissue engineering, concentrating on the assembly of artificial tissues from its component cells using electrokinetics. A comparison is made of electrokinetic techniques with other physical cell manipulation techniques which can be used in the construction of artificial tissues.Key words: tissue engineering, electric field, microenvironment, electrokinetics, dielectrophoresis, polarity     

时域相干刺激研究进展

脑深部电刺激已成为许多神经和精神疾病的有效治疗方法。然而,侵入性的电极植入会带来手术并发症的风险,并且刺激靶区在植入后很难改变。经颅磁刺激和经颅电刺激等非侵入性刺激方法为调节大脑功能提供了新的途径。但是,尚未证明这些非侵入性脑刺激方法可以直接调节脑深部神经元活动而不影响皮层神经元。因此,这些方法主要用于调节大脑表层脑区的神经活动。时域相干（temporal interference,TI）刺激是通过两个高频电场相互作用,产生低频包络调节神经活动的一种非侵入式脑深部电刺激的新方法,该方法有望解决无创脑深部刺激的需求。本文首先介绍TI刺激的概念以及安全性,然后阐述TI刺激现有研究中的电场分析方法,并讨论电场分析相关的生理模型建模方法和仿真平台以及TI刺激诱发场分布的研究进展与在动物和人体中的应用进展。最后,本文展望了TI刺激技术未来发展方向,以期为无创脑深部刺激研究提供新的研究思路。     

Prospects on clinical applications of electrical stimulation for nerve regeneration

Regenerative capability is limited in higher vertebrates but present in organ systems such as skin, liver, bone, and to some extent, the nervous system. Peripheral nerves in particular have a relatively high potential for regeneration following injury. However, delay in regrowth or growth, blockage, or misdirection at the injury site, and growth to inappropriate end organs may compromise successful regeneration, leading to poor clinical results. Recent studies indicate that low-intensity electrical stimulation is equivalent to various growth factors, offering avenues to improve these outcomes. We present a review of studies using electric and electromagnetic fields that provide evidence for the enhancement of regeneration following nerve injury. Electric and electromagnetic fields (EMFs) have been used to heal fracture non-unions. This technology emerged as a consequence of basic studies [Yasuda, 1953; Fukada and Yasuda, 1957] demonstrating the piezoelectric properties of (dry) bone. The principle for using electrical stimulation for bone healing originated from the work of Bassett and Becker [1962], who described asymmetric voltage waveforms from mechanically deformed live bone. These changes were presumed to occur in bone during normal physical activity as a result of mechanical forces, and it was postulated that these forces were linked to modifications in bone structure. Endogenous currents present in normal tissue and those that occur after injury were proposed to modify bone structure [Bassett, 1989]. These investigators proposed that tissue integrity and function could be restored by applying electrical and/or mechanical energy to the area of injury. They successfully applied electrical currents to nonhealing fractures (using surgically implanted electrodes or pulsed currents using surface electrodes) to aid endogenous currents in the healing process. A considerable technological improvement was made with the noninvasive application of EMFs [Bassett et al., 1974] to accelerate fracture repair. This newer technique allowed the treatment of hard tissues without the complications of invasive electrode insertion. In addition, soft tissue injuries were now accessible for treatment by electromagnetic fields. In this article, we will first define the basic problems encountered in nerve injury and regeneration, and then review both in vitro and in vivo studies on the use of electric and electromagnetic fields to stimulate the healing process.     

What Forces Can Determine the Formation of Highly Specific Protein–Protein Complexes?

A software package was designed and used in a detailed study of the contact regions (interfaces) of a large number of protein–protein complexes using the PDB data. It appeared that for about 75% of the complexes the amino acid composition of the subunit surface in the contact region is not essential. Thus one may suggest that, along with the amino acid residues at the interface, the residues in the interior of the globules substantially contribute to protein–protein recognition. Such interactions between quite remote residues are most probably of electrical nature, and are involved in recognition by contributing to the overall electric field created by the protein molecule; the configuration of this field is perhaps the definitive factor of recognition. The overall field of the protein molecule is additively built of the fields created by each constituent residue, and it can be calculated as a sum of the fields created by the protein multipole (aggregate of partial electric charges assigned to every atom of the protein molecule). Preliminary assessment of the remote electrostatic interaction has been performed for ribonuclease subunits in vacuum. The results are indicative of a real possibility that the electric field created by the protein multipole can strongly influence the mutual orientation of molecules before Brownian collision.     

Intracranial Nonthermal Irreversible Electroporation: In Vivo Analysis

Nonthermal irreversible electroporation (NTIRE) is a new minimally invasive technique to treat cancer. It is unique because of its nonthermal mechanism of tumor ablation. Intracranial NTIRE procedures involve placing electrodes into the targeted area of the brain and delivering a series of short but intense electric pulses. The electric pulses induce irreversible structural changes in cell membranes, leading to cell death. We correlated NTIRE lesion volumes in normal brain tissue with electric field distributions from comprehensive numerical models. The electrical conductivity of brain tissue was extrapolated from the measured in vivo data and the numerical models. Using this, we present results on the electric field threshold necessary to induce NTIRE lesions (495–510 V/cm) in canine brain tissue using 90 50-μs pulses at 4 Hz. Furthermore, this preliminary study provides some of the necessary numerical tools for using NTIRE as a brain cancer treatment. We also computed the electrical conductivity of brain tissue from the in vivo data (0.12–0.30 S/m) and provide guidelines for treatment planning and execution. Knowledge of the dynamic electrical conductivity of the tissue and electric field that correlates to lesion volume is crucial to ensure predictable complete NTIRE treatment while minimizing damage to surrounding healthy tissue.     

A Numerical Investigation of the Electric and Thermal Cell Kill Distributions in Electroporation-Based Therapies in Tissue

Electroporation-based therapies are powerful biotechnological tools for enhancing the delivery of exogeneous agents or killing tissue with pulsed electric fields (PEFs). Electrochemotherapy (ECT) and gene therapy based on gene electrotransfer (EGT) both use reversible electroporation to deliver chemotherapeutics or plasmid DNA into cells, respectively. In both ECT and EGT, the goal is to permeabilize the cell membrane while maintaining high cell viability in order to facilitate drug or gene transport into the cell cytoplasm and induce a therapeutic response. Irreversible electroporation (IRE) results in cell kill due to exposure to PEFs without drugs and is under clinical evaluation for treating otherwise unresectable tumors. These PEF therapies rely mainly on the electric field distributions and do not require changes in tissue temperature for their effectiveness. However, in immediate vicinity of the electrodes the treatment may results in cell kill due to thermal damage because of the inhomogeneous electric field distribution and high current density during the electroporation-based therapies. Therefore, the main objective of this numerical study is to evaluate the influence of pulse number and electrical conductivity in the predicted cell kill zone due to irreversible electroporation and thermal damage. Specifically, we simulated a typical IRE protocol that employs ninety 100-µs PEFs. Our results confirm that it is possible to achieve predominant cell kill due to electroporation if the PEF parameters are chosen carefully. However, if either the pulse number and/or the tissue conductivity are too high, there is also potential to achieve cell kill due to thermal damage in the immediate vicinity of the electrodes. Therefore, it is critical for physicians to be mindful of placement of electrodes with respect to critical tissue structures and treatment parameters in order to maintain the non-thermal benefits of electroporation and prevent unnecessary damage to surrounding healthy tissue, critical vascular structures, and/or adjacent organs.     

Suprazero cooling conditions significantly influence subzero permeability parameters of mammalian ovarian tissue

To model the cryobiological responses of cells and tissues, permeability characteristics are often measured at suprazero temperatures and the measured values are used to predict the responses at subzero temperatures. The purpose of the present study was to determine whether the rate of cooling from +25 to +4 degrees C influenced the measured water transport response of ovarian tissue at subzero temperatures in the presence or absence of cryoprotective agents (CPAs). Sections of freshly collected equine ovarian tissue were first cooled either at 40 degrees C/min or at 0.5 degrees C/min from 25 to 4 degrees C, and then cooled to subzero temperatures. A shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine ovarian tissue sections. After ice was induced to form in the extracellular fluid within the specimen, the sample was frozen from the phase change temperature to -50 degrees C at 5 degrees C/min. Replicate samples were frozen in isotonic medium alone or in medium containing 0.85 M glycerol or 0.85 M dimethylsulfoxide. The water transport response of ovarian tissue samples cooled at 40 degrees C/min from 25 to 4 degrees C was significantly different (confidence level >95%) from that of tissue samples cooled at 0.5 degrees C/min, whether in the presence or absence of CPAs. We fitted a model of water transport to the experimentally-derived volumetric shrinkage data and determined the best-fit membrane permeability parameters (L(pg) and E(Lp)) of equine ovarian tissue during freezing. Subzero water transport parameters of ovarian tissue samples cooled at 0.5 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.06 to 0.73 microm/min.atm and E(Lp) = 6.1 to 20.5 kcal/mol. The corresponding parameters of samples cooled at 40 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.04 to 0.61 microm/min.atm and E(Lp) = 8.2 to 54.2 kcal/mol. Calculations made of the theoretical response of tissue at subzero temperatures suggest that the optimal cooling rates to cryopreserve ovarian tissue are significantly dependent upon suprazero cooling conditions.     

Modulation of hippocampal rhythms by subthreshold electric fields and network topology

Theta (4–12 Hz) and gamma (30–80 Hz) rhythms are considered important for cortical and hippocampal function. Although several neuron types are implicated in rhythmogenesis, the exact cellular mechanisms remain unknown. Subthreshold electric fields provide a flexible, area-specific tool to modulate neural activity and directly test functional hypotheses. Here we present experimental and computational evidence of the interplay among hippocampal synaptic circuitry, neuronal morphology, external electric fields, and network activity. Electrophysiological data are used to constrain and validate an anatomically and biophysically realistic model of area CA1 containing pyramidal cells and two interneuron types: dendritic- and perisomatic-targeting. We report two lines of results: addressing the network structure capable of generating theta-modulated gamma rhythms, and demonstrating electric field effects on those rhythms. First, theta-modulated gamma rhythms require specific inhibitory connectivity. In one configuration, GABAergic axo-dendritic feedback on pyramidal cells is only effective in proximal but not distal layers. An alternative configuration requires two distinct perisomatic interneuron classes, one exclusively receiving excitatory contacts, the other additionally targeted by inhibition. These observations suggest novel roles for particular classes of oriens and basket cells. The second major finding is that subthreshold electric fields robustly alter the balance between different rhythms. Independent of network configuration, positive electric fields decrease, while negative fields increase the theta/gamma ratio. Moreover, electric fields differentially affect average theta frequency depending on specific synaptic connectivity. These results support the testable prediction that subthreshold electric fields can alter hippocampal rhythms, suggesting new approaches to explore their cognitive functions and underlying circuitry.