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.     

Development and estimation of a novel cryoprobe utilizing the Peltier effect for precise and safe cryosurgery

We have developed a novel cryoprobe for skin cryosurgery utilizing the Peltier effect. The four most important parameters for necrotizing tissue efficiently are the cooling rate, end temperature, hold time and thawing rate. In cryosurgery for small skin diseases such as flecks or early carcinoma, it is also important to control the thickness of the frozen region precisely to prevent necrotizing healthy tissue. To satisfy these exacting conditions, we have developed a novel cryoprobe to which a Peltier module was attached. The cryoprobe makes it possible to control heat transfer to skin surface precisely using a proportional-integral-derivative (PID) controller, and because it uses the Peltier effect, the cryoprobe does not need to move during the operation. We also developed a numerical simulation method that allows us to predict the frozen region and the temperature profile during cryosurgery.We tested the performance of our Peltier cryoprobe by cooling agar, and the results show that the cryoprobe has sufficient cooling performance for cryosurgery, because it can apply a cooling rate of more than 250 °C/min until the temperature reaches −40 °C. We also used a numerical simulation to reconstruct the supercooling phenomenon and examine the immediate progress of the frozen region with ice nucleation. The calculated frozen region was compared with the experimentally measured frozen region observed by an interferometer, and the calculation results showed good agreement. The results of numerical simulation confirmed that the frozen region could be predicted accurately with a margin of error as small as 150 μm during use of the cryoprobe in cryosurgery. The numerical simulation also showed that the cryoprobe can control freezing to a depth as shallow as 300 μm.     

An analytical study of cryosurgery in the lung.

The process of freezing in healthy lung tissue and in tumors in the lung during cryosurgery was modeled using one-dimensional close form techniques and finite difference techniques to determine the temperature profiles and the propagation of the freezing interface in the tissue. A thermal phenomenon was observed during freezing of lung tumors embedded in healthy tissue, (a) the freezing interface suddenly accelerates at the transition between the tumor and the healthy lung, (b) the frozen tumor temperature drops to low values once the freezing interface moves into the healthy lung, and (c) the outer boundary temperature has a point of sharp inflection corresponding to the time at which the tumor is completely frozen.     

Measurement of water transport during freezing in mammalian liver tissue: Part II--The use of differential scanning calorimetry.

There is currently a need for experimental techniques to assay the biophysical response (water transport or intracellular ice formation, IIF) during freezing in the cells of whole tissue slices. These data are important in understanding and optimizing biomedical applications of freezing, particularly in cryosurgery. This study presents a new technique using a Differential Scanning Calorimeter (DSC) to obtain dynamic and quantitative water transport data in whole tissue slices during freezing. Sprague-Dawley rat liver tissue was chosen as our model system. The DSC was used to monitor quantitatively the heat released by water transported from the unfrozen cell cytoplasm to the partially frozen vascular/extracellular space at 5 degrees C/min. This technique was previously described for use in a single cell suspension system (Devireddy, et al. 1998). A model of water transport was fit to the DSC data using a nonlinear regression curve-fitting technique, which assumes that the rat liver tissue behaves as a two-compartment Krogh cylinder model. The biophysical parameters of water transport for rat liver tissue at 5 degrees C/min were obtained as Lpg = 3.16 x 10(-13) m3/Ns (1.9 microns/min-atm), ELp = 265 kJ/mole (63.4 kcal/mole), respectively. These results compare favorably to water transport parameters in whole liver tissue reported in the first part of this study obtained using a freeze substitution (FS) microscopy technique (Pazhayannur and Bischof, 1997). The DSC technique is shown to be a fast, quantitative, and reproducible technique to measure dynamic water transport in tissue systems. However, there are several limitations to the DSC technique: (a) a priori knowledge that the biophysical response is in fact water transport, (b) the technique cannot be used due to machine limitations at cooling rates greater than 40 degrees C/min, and (c) the tissue geometric dimensions (the Krogh model dimensions) and the osmotically inactive cell volumes Vb, must be determined by low-temperature microscopy techniques.     

Effect of varying freezing and thawing rates in experimental cryosurgery

Six different freezing/thawing programs, which varied freezing rate, duration of freezing, and thawing rates, were used to investigate the effect of these factors on cell destruction in dog skin. The range of tissue temperatures produced was from -15 to -50 degrees C. The extent of destruction was evaluated by skin biopsies 3 days after cold injury. In single, short freezing/thawing cycles, the temperature reached in the tissue was the prime factor in cell death. Longer freezing time and slow thawing were also important lethal factors which increased destruction of cells. Cooling rate, whether slow or fast, made little difference in the outcome. The experiments suggested that present-day, commonly employed cryosurgical techniques, which feature fast cooling, slow thawing, and repetition of the freeze/thaw cycle, should be modified by the use of maintenance of the tissue in the frozen state for several minutes and slow thawing. Thawing should be complete before freezing is repeated. These modifications in technique will maximize tissue destruction, an important consideration in cancer cryosurgery.     

An analytical study on the thermal effects of cryosurgery on selective cell destruction

The aim of cryosurgery is to kill cells within a closely defined region maintained at a predetermined low temperature. To effectively kill cells, it is important to be able to predict and control the cooling rate over some critical range of temperatures and freezing states in order to regulate the spatial extent of injury during any freeze-thaw protocol. The objective of manipulating the freezing parameters is to maximize the destruction of cancer cells within a defined spatial domain while minimizing cryoinjury to the surrounding healthy tissue. An analytical model has been developed to study the rate of cell destruction within a liver tumor undergoing a freeze-thaw cryosurgical process. Temperature transients in the tumor undergoing cryosurgery have been quantitatively investigated. The simulation is based on solving the transient bioheat equation using the finite volume scheme for a single or multiple-probe geometry. Simulated results show good agreement with experimental data obtained from in vivo clinical study. The calibrated model has been employed to study the effects of different freezing rates, freeze-thaw cycle(s), and multi-probe freezing on cell damage in a liver tumor. The effectiveness of each treatment protocol is estimated by generating the cell survival-volume signature and comparing the percentage of cell damaged within the ice-ball. Results from the model show that employing freeze-thaw cycles has the potential to enhance cell destruction within the cancerous tissue. Results from this study provide the basis for designing an optimized cryosurgical protocol which incorporates thermal effects and the extent of cell destruction within tumors.     

Real time ultrasonic monitoring of hepatic cryosurgery

Cryosurgery has a number of advantages that make it particularly appealing in the treatment of liver cancer. However, a major problem in the clinical application of hepatic cryosurgery is the lack of a precise means of monitoring the freezing process in situ. Preliminary investigations on simulated tissue have shown that standard ultrasonography is capable of accurately determining the amount of frozen material during a cryosurgical procedure. To extend these results to living tissue, cryosurgery was performed, in vivo, on the livers of four mongrel dogs. An ultrasound imaging device using a new intraoperative ultrasound transducer monitored the entire process in real time. The results indicate that the entire freezing and thawing cycle can be monitored easily using real time ultrasound. During freezing, the solidification interface can be seen to move through the tissue allowing clear imaging of the cryolesion. After complete thawing, the cryolesion became less echogenic than before freezing and was therefore distinguishable under ultrasound. Postsurgical pathologic examination showed excellent correlation between the lesion size and its ultrasonic image.     

Do physical forces contribute to cryodamage?

To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the “two factor theory.” The present study deals with a third, largely ignored component—mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70–110, 250–500, and 1,000–1,250 µm, as a means of altering the surface area with which the cells come in contact. Post‐thaw evaluation included motility at 0 h and after 3 h at 37°C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra‐container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area‐to‐volume ratio, the intra‐container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing. Biotechnol. Bioeng. 2009; 104: 719–728 © 2009 Wiley Periodicals, Inc.     

Evaluation of the impedance technique for cryosurgery in a theoretical model of the head

Bioimpedance is a noninvasive technique that produces information on the electrical characteristics of tissue inside the body from currents injected and electrical potentials measured on the surface of the body. Because freezing causes a large increase in tissue electrical impedance we thought that it may also cause significant changes in the surface electrical potential making the bioimpedance technique suitable for noninvasive monitoring and imaging of cryosurgery. To evaluate the feasibility of the bioimpedance technique in cryosurgery we examined, as a case study, a theoretical model for the electrical potentials during brain cryosurgery. A three-dimensional spherical model was used to calculate the change in the electrical potential distribution in the head as a function of the current source location and the size of the frozen lesion in the brain. The numerical calculations were executed using the finite volume method and the iterative successive over relaxation method. The results demonstrate that, indeed, freezing inside the head produces measurable changes in the electrical potential on the outer surface-the scalp.     

An in vivo study of antifreeze protein adjuvant cryosurgery

Cryosurgery employs freezing to destroy undesirable tissue. However, under certain thermal conditions, frozen tissues survive. The survival of frozen undesirable tissue may lead to complications, such as recurrence of cancer. In a study of nude mice with subcutaneous metastatic prostate tumors, we showed that the preoperative injection of a phosphate-buffered saline solution with 10 mg/ml antifreeze protein of type I into the tumor prior to freezing enhances destruction under thermal conditions which normally yield cell survival. This suggests that the adjunctive use of antifreeze proteins in cryosurgery may reduce the complications from undesirable tissues that survive freezing.     

A mathematical model for the freezing process in biological tissue

A mathematical model has been developed to study the process of freezing in biological organs. The model consists of a repetitive unit structure comprising a cylinder of tissue with an axial blood vessel (Krogh cylinder) and it is analysed by the methods of irreversible thermodynamics. The mathematical simulation of the freezing process in liver tissue compares remarkably well with experimental data on the structure of tissue frozen under controlled thermal conditions and the response of liver cells to changes in cooling rate. The study also supports the proposal that the damage mechanism responsible for the lack of success in attempts to preserve tissue in a frozen state, under conditions in which cells in suspension survive freezing, is direct mechanical damage caused by the formation of ice in the vascular system.     

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.     

Effects of egg yolk, glycerol and the freezing rate on the viability and acrosomal structures of frozen ram spermatozoa.

The influence of egg yolk, glycerol and the freezing rate on the survival of ram spermatozoa and on the structure of their acrosomes after freezing was investigated. Egg yolk was shown to be beneficial not only during chilling but also during freezing; of the levels examined, 1-5% gave the greatest protection. Although the presence of glycerol in the diluent improved the survival of spermatozoa, increasing concentrations produced significant deterioration of the acrosomes. With closely controlled linear cooling rates, no overall difference was detected in the survival of spermatozoa frozen at rates between 6 and 24 degrees C per min. However, a significant interaction between freezing rate and the inclusion of glycerol in the diluent showed that glycerol was less important at the highest freezing rate. A sudden cooling phase near to the freezing point following the release of the latent heat of fusion was not detrimental to spermatozoa.     

Effect of cooling and rewarming rates on glycerolated human erythrocytes.

Human erythrocytes suspended in a sodium-free buffered salt solution containing glycerol in 1 m concentration (1 part of packed cells to 4 parts buffered salt solution) were frozen by slow, moderately rapid, or very rapid cooling to various subzero C temperatures. The frozen specimens, after a 5-min storage period at a given temperature, were thawed at low, moderately high, or very high rates. The hemolysis in the frozen and thawed samples was measured by a colorimetric determination of the hemoglobin released from the damaged cells. At ?10 °C, the highest freezing temperature employed, nearly 100% recovery of intact erythrocytes was obtained irrespective of the cooling and rewarming conditions. The extent of the hemolysis after exposure to lower freezing temperatures depended upon the cooling and rewarming conditions. Moderately rapid and very rapid freezing to, and thawing from temperatures below ?40 °C permitted significantly higher recoveries of intact cells than the other freezing/ thawing combinations. In the temperature range ?15 to ?30 °C the combination slow cooling and slow rewarming afforded maximum protection. Very rapid freezing/ slow thawing was the most damaging combination throughout the entire freezing range. The results were interpreted in part by a conventional two-factor analysis, lower cooling rates allowing concentrated salts to determine hemolysis, higher cooling rates destroying the cells by intracellular freezing. Apparent anomalies were explained in terms of a generalized “thermal/osmotic” shock according to which the erythrocytes were subject to greater hemolysis the higher the rates of cooling and/or warming.     

The effect of cooling rate and of dimethyl sulfoxide concentration on the ultrastructure of neonatal rat heart cells after freezing and thawing

The ultrastructure of neonatal rat heart cells in suspension and in tissue culture after freezing at optimal, suboptimal, and supraoptimal cooling rates with 2.5, 5, 7.5, and 10% DMSO was investigated. The effect of DMSO treatment only on the structure of the cells was also studied. A comparison was made with the survival in culture.Without freezing, increasing DMSO concentrations caused an increase of morphological damage, correlating with a decrease of the survival in culture. With 2.5% DMSO there was no difference with untreated cells. At higher DMSO concentrations, the ultrastructural damage increased from spaces between cell membrane and cytoplasm at 5% DMSO to interrupted cell membranes, swollen or destroyed mitochondria, and nuclei with clumped chromatin at 10% DMSO.After freezing at optimal or nonoptimal cooling rates with 5 or 7.5% DMSO, the ultrastructure correlated well with the survival. After freezing with 2.5 or 10% DMSO at optimal or nonoptimal cooling rates, differences in survival were found, which were not reflected in the ultrastructure of the cell. After 8 days of culturing, cells which were frozen at all the different cooling rates and DMSO concentrations appeared to have a normal structure.     

Development of reformative surgery method using partial freezing for the liver

To minimize surgical stresses including blood loss and operation time to the patients during hepatic resection, we studied the feasibility of a combination of a partial liver freezing technique and shape-memory alloy, which also enables a free-designed resection curve. In this surgical procedure, the region surrounding a tumor in the liver is frozen to excise and prevent hemorrhage. The liver was frozen by a Peltier module. The effects of cooling rate and freezing temperature on the excision force that arise between a scalpel and the liver are carried out experimentally as a basic research for partial freezing surgical procedures. A porcine liver was used as a liver sample. The physical properties were estimated by using the finite element method based on the heat transfer characteristics of the liver. Isolation of the liver was conducted using a scalpel attached to the end-effector of a 3 degrees of freedom robot. In the experiments, the minimum excision force was obtained at a temperature between 272 K and 275 K; therefore, it is preferable that the liver be excised within this temperature range. Lowering of the cooling rate decreases the excision force even if the temperature of the liver remains unchanged. The lower the temperature of the liver is, the larger the increment rate of excision force is with regard to the cooling rate.     

Basic investigations on the freezing of human lymphocytes

Human lymphocytes were frozen at constant cooling rates in the range 2.4 to 1000 degrees K/min without cryoadditive on the cold stage of a thermally defined cryomicroscope. The volume loss due to water efflux was quantified optically for the cooling rates 2.4, 12, 48, and 120 degrees K/min. The likelihood of the formation of intracellular ice was determined as function of the cooling rate. Intracellular crystallization temperatures were obtained for ice formation during both cooling and rewarming. A theoretical analysis of the cell volume loss during freezing was compared to the experimental data and used for an indirect determination of the water permeability of the cells. A relative optimum of the cooling rate is predicted theoretically under the assumption of a critical level of intracellular salt concentration near the eutectic temperature. The dependence of survival and cooling rate was determined cryomicroscopically by simultaneously applying the FDA/EB fluorescence viability test. The optimal cooling rate of about 35 degrees K/min was also found for 2-ml samples frozen within the range of cooling rates of interest. The results show that for freezing in physiological saline solution (1) the optimum of the cooling rate is theoretically predictable, (2) cryomicroscopical data are significant for freezing of samples of larger volume, and (3) the lethal type of intracellular crystallization is cooling rate dependent and distinguishable from innocuous types.     

Capture of spatially homogeneous chemical reactions in tissue by freezing.

A useful technique in studying the saturation of hemoglobin in erythrocytes or myoglobin in tissue is cryophotometry, in which tissue is frozen for later spectrophotometric analysis. A general question associated with this technique is whether the freezing process alters the chemical state. This paper presents a theoretical analysis of the simplest model relevant to that question. We study the effect of rapid cooling on a spatially homogeneous chemical reaction. The analysis shows that changes during freezing are negligible near the boundary to which the heat sink is applied, but can be significant deeper in the sample. The distance from the boundary at which the changes during freezing become appreciable can be expressed simply in terms of the chemical reaction rates and the thermal diffusivity of the tissue. Detailed results are given for the case of oxygen and myoglobin in skeletal muscle.     

An investigation of the effects from a urethral warming system on temperature distributions during cryoablation treatment of the prostate: A phantom study

Introduction of urethral warmers to aid cryosurgery in the prostate has significantly reduced the incidence of urethral sloughing; however, the incidence rate still remains as high as 15%. Furthermore, urethral warmers have been associated with an increase of cancer recurrence rates. Here, we report results from our phantom-based investigation to determine the impact of a urethral warmer on temperature distributions around cryoneedles during cryosurgery. Cryoablation treatments were simulated in a tissue mimicking phantom containing a urethral warming catheter. Four different configurations of cryoneedles relative to urethral warming catheter were investigated. For each configuration, the freeze–thaw cycles were repeated with and without the urethral warming system activated. Temperature histories were recorded at various pre-arranged positions relative to the cryoneedles and urethral warming catheter. In all configurations, the urethral warming system was effective at maintaining sub-lethal temperatures at the simulated surface of the urethra. The warmer action, however, was additionally demonstrated to potentially negatively impact treatment lethality in the target zone by elevating minimal temperatures to sub-lethal levels. In all needle configurations, rates of freezing and thawing were not significantly affected by the use of the urethral warmer. The results indicate that the urethral warming system can protect urethral tissue during cryoablation therapy with cryoneedles placed as close as 5 mm to the surface of the urethra. Using a urethral warming system and placing multiple cryoneedles within 1 cm of each other delivers lethal cooling at least 5 mm from the urethral surface while sparing urethral tissue.     

Effects of cooling rate and glycerol concentration on the structure of the frozen kidney: assessment by cryo-scanning electron microscopy

An experimental technique, employing a directional solidification stage for controlled freezing of tissue samples and low-temperature scanning electron microscopy for observation of the structure of the frozen-hydrated samples, was used to study freezing processes in the kidney. Parametric studies in which the cooling rate during freezing and the concentration of glycerol in the tissue were varied confirmed the results of earlier freeze-substitution studies. The results suggest a mechanism for ice propagation in the kidney similar to that already proposed for the liver, in which ice originates in, and is subsequently propagated through, the peritubular vasculature. The ice front dehydrates the cells and tubular structures encountered in its path, thus preventing intraluminal freezing. At higher rates of cooling and increased concentrations of glycerol there is less dehydration of cortical structure and intraluminal freezing occurs.