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.     

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.     

Fast inverse prediction of the freezing front in cryosurgery

Cryosurgery has become a well-established technique for the ablation of undesirable tissues such as tumors and cancers. The motivation for this study is to improve the efficacy and safety of this technique. This study presents an inverse heat transfer method for monitoring the motion of the freezing front from a cryoprobe. With the help of a thermocouple inserted into the layer of diseased tissue, the inverse heat transfer method estimates simultaneously the blood perfusion rate and the thermal conductivities of both frozen and unfrozen tissues. This information is then fed to the Pennes bioheat equation that: (1) calculates the time-varying temperature distribution inside the layer of tissue and (2) predicts the motion of the freezing front. The effect of the most influential parameters on the inverse predictions is investigated. These parameters are (1) the initial guesses for the unknown Levenberg-Marquardt polynomial parameters of the thermo-physical properties; (2) the temperature of the cryoprobe; (3) the heat transfer coefficient of the impinging jet of liquid nitrogen; and (4) the noise on the temperature data recorded by the thermocouple probe. Results show that the proposed inverse method is a promising alternative to ultrasound and Magnetic Resonance Imaging (MRI) for monitoring the motion of the freezing front during cryosurgery. For all the cryogenic scenarios simulated, the predictions of the inverse model remain accurate and stable.     

Probing field-induced tissue polarization using transillumination fluorescent imaging

Despite major successes of biophysical theories in predicting the effects of electrical shocks within the heart, recent optical mapping studies have revealed two major discrepancies between theory and experiment: 1), the presence of negative bulk polarization recorded during strong shocks; and 2), the unexpectedly small surface polarization under shock electrodes. There is little consensus as to whether these differences result from deficiencies of experimental techniques, artifacts of tissue damage, or deficiencies of existing theories. Here, we take advantage of recently developed near-infrared voltage-sensitive dyes and transillumination optical imaging to perform, for the first time that we know of, noninvasive probing of field effects deep inside the intact ventricular wall. This technique removes some of the limitations encountered in previous experimental studies. We explicitly demonstrate that deep inside intact myocardial tissue preparations, strong electrical shocks do produce considerable negative bulk polarization previously inferred from surface recordings. We also demonstrate that near-threshold diastolic field stimulation produces activation of deep myocardial layers 2-6 mm away from the cathodal surface, contrary to theory. Using bidomain simulations we explore factors that may improve the agreement between theory and experiment. We show that the inclusion of negative asymmetric current can qualitatively explain negative bulk polarization in a discontinuous bidomain model.     

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.     

An Improved Method for In Situ Freezing of Cat Brain for Metabolic Studies

This study introduces a new method for rapid freezing of the cat brain. The method employed a Styrofoam box which was fitted around the head of the animal. Liquid nitrogen was poured into the box until the head was submerged. Temperature changes in three brain sites (ventral hypothalamus, the fourth ventricle, and the corpus callosum) and levels of labile carbohydrate metabolites (glycogen, glucose, ATP, P-creatine, and lactate) in five brain regions (cortex, thalamus, midbrain, cerebellum, and pons) frozen by the box method were compared with those frozen by a conventional cup method in which liquid nitrogen was poured into a hollow Styrofoam cup placed on top of the skull. The box method shortened the time of arrival of the freezing front and improved the freezing rate. The time required to bring the tissue to -20 degrees C was shortened, from 20 min at the ventral hypothalamus and 10-12 min at the fourth ventricle with the cup method, to less than 5 min at both sites with the box technique. Continued perfusion of brainstem prior to freezing was demonstrated. Levels of metabolites frozen by either method were similar. Lactate levels in any of the five brain regions studied by either method were not elevated, indicating no ischemic change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Roles of Head-Activation and Head-Inhibition Potentials in Pattern Formation of Hydra: Analysis of a Multi-Headed Mutant Strain

SYNOPSIS. Lateral grafting of tissue was used to compare therelative head-activation and head-inhibition potentials of differentHydra strains. A small piece of tissue taken from one polyp,when grafted to another polyp, induces formation of a head structurewhen the relative head-activation potential of the donor tissueis sufficiently (i.e., more than some threshold value) higherthan the relative head-inhibition potential of the recipienttissue. It was found that a multi-headed mutant strain (mh-1),which produces many extra heads along its body column, has significantlyhigher head-activation and significantly lower head-inhibitionpotentials than the standard wild-type strain. This suggeststhat these potentials play important roles in hydra morphogenesis,and that an imbalance between the two potentials is responsiblefor the developmental abnormality of mh-1. The significanceof this finding is discussed in light of the "positional information"model proposed by Wolpert and his associates and the "lateralinhibition" model proposed by Gierer and Meinhardt.     

The process of freezing and the mechanism of damage during hepatic cryosurgery

Experiments were performed to correlate the structures of liver tissue frozen during cryosurgery, liver frozen at various constant cooling rates, and unfrozen, dried normal liver. The results show that during freezing of tissue ice forms and propagates along the vascular system, expanding during freezing at low cooling rates. This expansion occurs over most of the region frozen during cryosurgery and may be one of the mechanisms of damage to tissue during cryosurgery.     

Feasibility test of a sapphire cryoprobe with optical monitoring of tissue freezing

This article describes a sapphire cryoprobe as a promising solution to the significant problem of modern cryosurgery that is the monitoring of tissue freezing. This probe consists of a sapphire rod manufactured by the edge-defined film-fed growth technique from Al₂O₃ melt and optical fibers accommodated inside the rod and connected to the source and the detector. The probe's design enables detection of spatially resolved diffuse reflected intensities of tissue optical response, which are used for the estimation of tissue freezing depth. The current type of the 12.5-mm diameter sapphire probe cooled down by the liquid nitrogen assumes a superficial cryoablation. The experimental test made by using a gelatin-intralipid tissue phantom shows the feasibility of such concept, revealing the capabilities of monitoring the freezing depth up to 10 mm by the particular instrumentation realization of the probe. This justifies a potential of sapphire-based instruments aided by optical diagnosis in modern 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.     

An electrodiffusive neuron-extracellular-glia model for exploring the genesis of slow potentials in the brain

Within the computational neuroscience community, there has been a focus on simulating the electrical activity of neurons, while other components of brain tissue, such as glia cells and the extracellular space, are often neglected. Standard models of extracellular potentials are based on a combination of multicompartmental models describing neural electrodynamics and volume conductor theory. Such models cannot be used to simulate the slow components of extracellular potentials, which depend on ion concentration dynamics, and the effect that this has on extracellular diffusion potentials and glial buffering currents. We here present the electrodiffusive neuron-extracellular-glia (edNEG) model, which we believe is the first model to combine compartmental neuron modeling with an electrodiffusive framework for intra- and extracellular ion concentration dynamics in a local piece of neuro-glial brain tissue. The edNEG model (i) keeps track of all intraneuronal, intraglial, and extracellular ion concentrations and electrical potentials, (ii) accounts for action potentials and dendritic calcium spikes in neurons, (iii) contains a neuronal and glial homeostatic machinery that gives physiologically realistic ion concentration dynamics, (iv) accounts for electrodiffusive transmembrane, intracellular, and extracellular ionic movements, and (v) accounts for glial and neuronal swelling caused by osmotic transmembrane pressure gradients. The edNEG model accounts for the concentration-dependent effects on ECS potentials that the standard models neglect. Using the edNEG model, we analyze these effects by splitting the extracellular potential into three components: one due to neural sink/source configurations, one due to glial sink/source configurations, and one due to extracellular diffusive currents. Through a series of simulations, we analyze the roles played by the various components and how they interact in generating the total slow potential. We conclude that the three components are of comparable magnitude and that the stimulus conditions determine which of the components that dominate.     

A solution method for the determination of cardiac potential distributions with an alternating current source

A recently presented solution method for the bidomain model (Johnston et al. 2006), which involves the application of direct current for studying electrical potential in a slab of cardiac tissue, is extended here to allow the use of an applied alternating current. The advantage of using AC current, in a four-electrode method for determining cardiac conductivities, is that instead of using 'close' and 'wide' electrode spacings to make potential measurements, increasing the frequency of the AC current redirects a fraction of the current from the extracellular space into the intracellular space. The model is based on the work of Le Guyader et al. (2001), but is able to include the effects of the fibre rotation between the epicardium and the endocardium on the potentials. Also, rather than using a full numerical technique, the solution method uses Fourier series and a simple one dimensional finite difference scheme, which has the advantage of allowing the potentials to be calculated only at points, such as the measuring electrodes, where they are required. The new alternating current model, which includes intracellular capacitance, is used with a particular four-electrode configuration, to show that the potential measured is affected by changes in fibre rotation. This is significant because it indicates that it is necessary to include fibre rotation in models, which are to be used in conjunction with measuring arrays that are more complex than those involving simply surface probes or a single vertical probe.     

Synthesis of voltage-sensitive fluorescence signals from three-dimensional myocardial activation patterns

Voltage-sensitive fluorescent dyes are commonly used to measure cardiac electrical activity. Recent studies indicate, however, that optical action potentials (OAPs) recorded from the myocardial surface originate from a widely distributed volume beneath the surface and may contain useful information regarding intramural activation. The first step toward obtaining this information is to predict OAPs from known patterns of three-dimensional (3-D) electrical activity. To achieve this goal, we developed a two-stage model in which the output of a 3-D ionic model of electrical excitation serves as the input to an optical model of light scattering and absorption inside heart tissue. The two-stage model permits unique optical signatures to be obtained for given 3-D patterns of electrical activity for direct comparison with experimental data, thus yielding information about intramural electrical activity. To illustrate applications of the model, we simulated surface fluorescence signals produced by 3-D electrical activity during epicardial and endocardial pacing. We discovered that OAP upstroke morphology was highly sensitive to the transmural component of wave front velocity and could be used to predict wave front orientation with respect to the surface. These findings demonstrate the potential of the model for obtaining useful 3-D information about intramural propagation.     

Current playground surface test standards underestimate brain injury risk for children

Playgrounds surface test standards have been introduced to reduce the number of fatal and severe injuries. However, these test standards have several simplifications to make it practical, robust and cost-effective, such as the head is represented with a hemisphere, only the linear kinematics is evaluated and the body is excluded. Little is known about how these simplifications may influence the test results. The objective of this study was to evaluate the effect of these simplifications on global head kinematics and head injury prediction for different age groups. The finite element human body model PIPER was used and scaled to seven different age groups from 1.5 up to 18 years old, and each model was impacted at three different playground surface stiffness and three head impact locations. All simulations were performed in pairs, including and excluding the body. Linear kinematics and skull bone stress showed small influence if excluding the body while head angular kinematics and brain tissue strain were underestimated by the same simplification. The predicted performance of the three different playground surface materials, in terms of head angular kinematics and brain tissue strain, was also altered when including the body. A body and biofidelic neck need to be included, together with suitable head angular kinematics based injury thresholds, in future physical or virtual playground surface test standards to better prevent brain injuries.     

Cryosurgery of normal and tumor tissue in the dorsal skin flap chamber: Part I--thermal response

Current research in cryosurgery is concerned with finding a thermal history that will definitively destroy tissue. In this study, we measured and predicted the thermal history obtained during freezing and thawing in a cryosurgical model. This thermal history was then compared to the injury observed in the tissue of the same cryosurgical model (reported in companion paper (Hoffmann and Bischof, 2001)). The dorsal skin flap chamber, implanted in the Copenhagen rat, was chosen as the cryosurgical model. Cryosurgery was performed in the chamber on either normal skin or tumor tissue propagatedfrom an AT-1 Dunning rat prostate tumor. The freezing was performed by placing a approximately 1 mm diameter liquid-nitrogen-cooled cryoprobe in the center of the chamber and activating it for approximately 1 minute, followed by a passive thaw. This created a 4.2 mm radius iceball. Thermocouples were placed in the tissue around the probe at three locations (r = 2, 3, and 3.8 mm from the center of the window) in order to monitor the thermal history produced in the tissue. The conduction error introduced by the presence of the thermocouples was investigated using an in vitro simulation of the in vivo case and found to be <10 degrees C for all cases. The corrected temperature measurements were used to investigate the validity of two models of freezing behavior within the iceball. The first model used to approximate the freezing and thawing behavior within the DSFC was a two-dimensional transient axisymmetric numerical solution using an enthalpy method and incorporating heating due to blood flow. The second model was a one-dimensional radial steady state analytical solution without blood flow. The models used constant thermal properties for the unfrozen region, and temperature-dependent thermal properties for the frozen region. The two-dimensional transient model presented here is one of the first attempts to model both the freezing and thawing of cryosurgery. The ability of the model to calculate freezing appeared to be superior to the ability to calculate thawing. After demonstrating that the two-dimensional model sufficiently captured the freezing and thawing parameters recorded by the thermocouples, it was used to estimate the thermal history throughout the iceball. This model was used as a basis to compare thermal history to injury assessment (reported in companion paper (Hoffmann and Bischof, 2001)).     

An Analysis and Comparison of Convergence and Uniqueness of Time-Independent Bone Adaptation Models

A recently presented solution method for the bidomain model (Johnston et al. 2006), which involves the application of direct current for studying electrical potential in a slab of cardiac tissue, is extended here to allow the use of an applied alternating current. The advantage of using AC current, in a four-electrode method for determining cardiac conductivities, is that instead of using ‘close’ and ‘wide’ electrode spacings to make potential measurements, increasing the frequency of the AC current redirects a fraction of the current from the extracellular space into the intracellular space.

The model is based on the work of Le Guyader et al. (2001), but is able to include the effects of the fibre rotation between the epicardium and the endocardium on the potentials. Also, rather than using a full numerical technique, the solution method uses Fourier series and a simple one dimensional finite difference scheme, which has the advantage of allowing the potentials to be calculated only at points, such as the measuring electrodes, where they are required.

The new alternating current model, which includes intracellular capacitance, is used with a particular four-electrode configuration, to show that the potential measured is affected by changes in fibre rotation. This is significant because it indicates that it is necessary to include fibre rotation in models, which are to be used in conjunction with measuring arrays that are more complex than those involving simply surface probes or a single vertical probe.     

Towards a computational method for imaging the extracellular potassium concentration during regional ischemia

We investigate the possibility of using body surface potential maps to image the extracellular potassium concentration during regional ischemia. The problem is formulated as an inverse problem based on a linear approximation of the bidomain model, where we minimize the difference between the results of the model and observations of body surface potentials. The minimization problem is solved by a one-shot technique, where the original PDE system, an adjoint problem, and the relation describing the minimum, are solved simultaneously. This formulation of the problem requires the solution of a 5 × 5 system of linear partial differential equations. The performance of the model is investigated by performing tests based on synthetic data. We find that the model will in many cases detect the correct position and approximate size of the ischemic regions, while some cases are more difficult to locate. It is observed that a simple post-processing of the results produces images that are qualitatively very similar to the true solution.     

Experimental cryogenic injury of the palate: Observations pertinent to cryosurgical destruction of tumors

In experiments using cryosurgical apparatus to freeze the canine palate in situ, observations were made on techniques of producing tissue destruction. Several time-temperature schedules of freezing were studied. The results showed the great tolerance of palatal tissues to extremely low temperatures for short time periods. Melanocytes were extraordinarily sensitive to cold injury. Tissue necrosis increased with duration of freezing, but repeated freezing was lethal and obviously critical for successful cryosurgical destruction. Thermocouples must be used in clinical cryosurgery to insure that lethal tissue temperatures (colder than ?50 °C) are attained. The incidence of sequestration in the canine palate showed the need for use of proper technique and suitable precautions in the cryosurgical treatment of human palatal tumors.     

Genetic analysis of developmental mechanisms in hydra. X. Morphogenetic potentials of a regeneration-deficient strain (reg-16)

Morphogenetic potentials of hydra tissue involved in head or foot formation were examined in a standard wild-type strain (105) and a mutant strain (reg-16) which has a very low head regenerative but a nearly normal foot regenerative capacity (T. Sugiyama and T. Fujisawa, 1977, J. Embryol. Exp. Morphol. 42, 65-77). Hydra tissue has two types of morphogenetic potentials to control head formation: the potential to form head structure (head-activation potential) and the potential to inhibit head formation (head-inhibition potential). It also has two types of morphogenetic potentials to control foot formation: foot-activation and foot-inhibition potentials. A lateral tissue grafting procedure (G. Webster and L. Wolpert, 1966, J. Embryol. Exp. Morphol. 16, 91-104), was used to examine and compare the relative levels of these potentials in the normal and the mutant strains. The potential levels were examined along the body axis of the intact animals and also in the regenerating animals after head removal. The results obtained show that the potentials involved in head formation are highly abnormal, whereas the potentials involved in foot formation are apparently normal in the mutant strain (reg-16). This suggests that the abnormal potentials are related in some way to, and may be responsible for, the reduced head regenerative capacity in the mutant strain reg-16.