The pattern of cell survival in the pig liver following one freeze-thaw cryosurgery cycle.

BACKGROUND: It is well established that in cryosurgery some cells can survive one freeze thaw cycle and that surviving cells are found at the margin of the frozen lesion. Numerous techniques are being developed to ensure the survival of frozen cells to the margin of the frozen region. OBJECTIVE: We thought that it would be of fundamental interest to observe the pattern of cell survival in a liver treated with one freeze-thaw cycle. MATERIALS AND METHODS: We performed six ultrasound-guided single freeze-thaw cryosurgery procedures on the liver of four Landrace pigs, using two cryosurgery probes separated by 25 mm inserted in parallel. Treated organs were removed 24 hours after the cryosurgery and processed for histology with hematoxylin and eosin. The tissues were analyzed with a digital slice scanner. RESULTS: We found an unexpected pattern of cell survival; sheets of live cells, about 200 µm in width, that follow the network of interlobular connective tissue septae to a distance of several millimeter from the outer edge of the one freeze-thaw cycle cryosurgery treated lesion. The sheets of live cells surround lobule cores that have undergone complete coagulative necrosis. In addition, larger blood vessels, as far as 5 mm from the outer rim of the treated lesion, have a major and complex effect on cell survival with large areas of completely necrotic and completely alive cells intermixed. CONCLUSION: This study may have value as a baseline for developing new cryosurgery protocols designed to ablate cells to the margin of the frozen lesion.

Prostate cancer treatment with Irreversible Electroporation (IRE): Safety, efficacy and clinical experience in 471 treatments.

BACKGROUND: Irreversible Electroporation (IRE) is a novel image-guided tissue ablation technology that induces cell death via very short but strong pulsed electric fields. IRE has been shown to have preserving properties towards vessels and nerves and the extracellular matrix. This makes IRE an ideal candidate to treat prostate cancer (PCa) where other treatment modalities frequently unselectively destroy surrounding structures inducing severe side effects like incontinence or impotence. We report the retrospective assessment of 471 IRE treatments in 429 patients of all grades and stages of PCa with 6-year maximum follow-up time. MATERIAL AND FINDINGS: The patient cohort consisted of low (25), intermediate (88) and high-risk cancers (312). All had multi-parametric magnetic resonance imaging, and 199 men had additional 3D-mapping biopsy for diagnostic work-up prior to IRE. Patients were treated either focally (123), sub-whole-gland (154), whole-gland (134) or for recurrent disease (63) after previous radical prostatectomy, radiation therapy, etc. Adverse effects were mild (19.7%), moderate (3.7%) and severe (1.4%), never life-threatening. Urinary continence was preserved in all cases. IRE-induced erectile dysfunction persisted in 3% of the evaluated cases 12 months post treatment. Mean transient IIEF-5-Score reduction was 33% within 12-month post IRE follow-up and 15% after 12 months. Recurrences within the follow-up period occurred in 10% of the treated men, 23 in or adjacent to the treatment field and 18 outside the treatment field (residuals). Including residuals for worst case analysis, Kaplan Maier estimation on recurrence rate at 5 years resulted in 5.6% (CI95: 1.8-16.93) for Gleason 6, 14.6% (CI95: 8.8-23.7) for Gleason 7 and 39.5% (CI95: 23.5-61.4) for Gleason 8-10. CONCLUSION: The results indicate comparable efficacy of IRE to standard radical prostatectomy in terms of 5-year recurrence rates and better preservation of urogenital function, proving the safety and suitability of IRE for PCa treatment. The data also shows that IRE, besides focal therapy of early PCa, can also be used for whole-gland ablations, in patients with recurrent PCa, and as a problem-solver for local tumor control in T4-cancers not amenable to surgery and radiation therapy anymore.

Time-dependent Effects of Pressure during Preservation of Rat Hearts in an Isochoric System at Subfreezing Temperatures.

BACKGROUND: Isochoric freezing systems enable ice-free preservation of biological matter at subfreezing temperatures under the increased hydrostatic pressure. OBJECTIVE: To examine the effects of pressure and exposure period on rat hearts preserved in an isochoric chamber. MATERIALS AND METHODS: Rat hearts were preserved in the UW solution in isochoric chambers at temperatures from -2°C to -8°C and pressure from the atmospheric level to 78 MPa for up to eight hours, with and without the addition of glycerol. Hearts were evaluated via Langendorff perfusion and HE histology. RESULTS: Hearts were compromised quickly as pressure increased, suggesting an acute time-pressure sensitivity. With the addition of 1 M glycerol, which reduces the pressure experienced at a given temperature, the survival time at -4°C was doubled. CONCLUSION: The enhanced hydrostatic pressure encountered during isochoric preservation yields time-dependent negative effects on the heart, which can potentially be alleviated by the addition of a cryoprotectant.

The effect of isochoric freezing on mammalian cells in an extracellular phosphate buffered solution.

Isochoric (constant volume) freezing has been recently suggested as a new method for cell and organ preservation. As a first step in studying the effect of isochoric freezing on mammalian cells, Madin-Darby canine kidney epithelial cells (MDCK), were frozen in an isochoric system, in a simple extracellular phosphate buffered solution to -10 °C (96.5 MPa), - 15 °C (162 MPa) and -20 °C (205 MPa) for 60 and 120 min. Cell membrane integrity and cell metabolism were studied with a Live/Dead cell vitality assay and flow cytometry. We found that cell survival decreases with an increase in pressure (lower temperatures) and time of exposure. For example, 60% of cells survived 60 min at - 10 °C and only 18% survived 120 min at this temperature. Negligible survival was measured at - 20 °C. This study may serve as the baseline towards further research on techniques to optimize the effects of isochoric freezing on living biological matter.

Cryoelectrolysis for Treatment of Atrial Fibrillation: A First Order Feasibility Study.

　　BACKGROUND: Catheter based treatment of atrial fibrillation (AFib) involves ablation of groups of cells near the pulmonary vein. OBJECTIVE: The goal of this study was to evaluate the technological feasability of a new technology for catheter-based ablation for treatment of AFib that combines freezing with electrolysis. MATERIALS AND METHODS: The study was performed on a pH dye stained gel phantom of the pulmonary vein. Freezing was induced with a cryosurgical probe inserted in the vein and electrolysis was delivered through the probe with a DC power supply. RESULTS: Visual recording of colorimetric changes in pH demonstrate that electrolytic products can propagate through the frozen tissue phantom. For example, a voltage of 9 V and a current of 40 mA delivered through a -15 degree C cryosurgical probe produced an electrolysis impacted rim of over 7 mm width in 2 min. CONCLUSION: This early stage experimental work suggests that cryoelectrolysis may have potential for treatment of AFib.

Non-ionizing radiofrequency electromagnetic waves traversing the head can be used to detect cerebrovascular autoregulation responses.

Monitoring changes in non-ionizing radiofrequency electromagnetic waves as they traverse the brain can detect the effects of stimuli employed in cerebrovascular autoregulation (CVA) tests on the brain, without contact and in real time. CVA is a physiological phenomenon of importance to health, used for diagnosis of a number of diseases of the brain with a vascular component. The technology described here is being developed for use in diagnosis of injuries and diseases of the brain in rural and economically underdeveloped parts of the world. A group of nine subjects participated in this pilot clinical evaluation of the technology. Substantial research remains to be done on correlating the measurements with physiology and anatomy.

Germicide wound pad with active, in situ, electrolytically produced hypochlorous acid.

We describe a new wound dressing technology that can actively generate an inorganic germicide agent, in situ, within the wound pad. The technology provides real time control over the quantitative, spatial and temporal delivery of the germicide. The identity of the germicide is hypochlorous acid (HClO). The HClO is produced in a flexible wound pad, made of a composite of thin (micrometer scale) layers of various materials, with different electrochemical properties that enhance HClO production. Active control over the production of HClO is achieved by control of the pH and of the electric potential across the layers. The effectiveness of the Active HClO Pad (AHClOP) concept is demonstrated in a study on sterilization of E. coli in a deep wound contamination simulating gel. The performance of the AHClOP is compared with that of four commercial wound dressings. Results show that the AHClOP can sterilize throughout the gel, while the commercial dressings cannot.

Irreversible electroporation on the small intestine.

BACKGROUND: Non-thermal irreversible electroporation (NTIRE) has recently been conceived as a new minimally invasive ablation method, using microsecond electric fields to produce nanoscale defects in the cell membrane bilayer and induce cell death while keeping all other molecules, including the extracellular matrix, intact. Here, we present the first in vivo study that examines the effects of NTIRE on the small intestine, an organ whose collateral damage is of particular concern in the anticipated use of NTIRE for treatment of abdominal cancers. METHODS: A typical NTIRE electrical protocol was applied directly to the rat small intestine and histological analysis was used to examine the effect of NTIRE over time. RESULTS: The application of NTIRE led to complete cell ablation in the targeted tissue, but the animal did not show any physiological effects of the procedure and the intestine showed signs of recovery, developing an epithelial layer 3 days post treatment and regenerating its distinct layers within a week. CONCLUSION: Our results indicate that this novel procedure can be used for abdominal cancer treatment while minimising collateral damage to adjacent tissues because of the unique ability of the NTIRE ablation method to target the cell membrane.

In vivo non-thermal irreversible electroporation impact on rat liver galvanic apparent internal resistance.

Non-thermal irreversible electroporation (NTIRE) is a biophysical phenomenon which involves application of electric field pulses to cells or tissues, causing certain rearrangements in the membrane structure leading to cell death. The treated tissue ac impedance changes induced by electroporation were shown to be the indicators for NTIRE efficiency. In a previous study we characterized in vitro tissue galvanic apparent internal resistance (GAIR) changes due to NTIRE. Here we describe an in vivo study in which we monitored the GAIR changes of a rat liver treated by NTIRE. Electrical pulses were delivered through the same Zn/Cu electrodes by which GAIR was measured. GAIR was measured before and for 3 h after the treatment at 15 min intervals. The results were compared to the established ac bioimpedance measurement method. A decrease of 33% was measured immediately after the NTIRE treatment and a 40% decrease was measured after 3 h in GAIR values; in the same time 40% and 47% decrease respectively were measured by ac bioimpedance analyses. The temperature increase due to the NTIRE was only 0.5 °C. The results open the way for an inexpensive, self-powered in vivo real-time NTIRE effectiveness measurement.

The detection of brain ischaemia in rats by inductive phase shift spectroscopy.

Ischaemia in the brain is an important clinical problem that is often monitored and studied with expensive devices such as MRI and PET, which are not readily available in low economical resource parts of the world. We have developed a new less expensive tool for non-invasive monitoring of ischaemia in the brain. This is a first feasibility study describing the concept. The system is based on the hypothesis that electromagnetic properties of the tissue change during ischaemia and that measuring the electromagnetic properties of the bulk of the brain with non-contact means can detect these changes. The apparatus we have built and whose design we describe here consists of two electromagnetic coils placed around the head. The system measures the bulk change in time of the phase difference between the electromagnetic signal on the two coils in a range of frequencies. A mathematical model simulating the device and the measurement is also introduced. Ischaemia was induced in the brain of rats by occlusion of the right cerebral and carotid arteries. Experimental subjects were monitored for 24 h. Inductive phase shift measurements were made at five frequencies in the range of 0.1-50 MHz eight times during the observation period. An ex vivo estimation of the percentage of necrosis in the ischemic subjects at t = 24 h was done. The mathematical model was also applied to the experimental tested situation. The results of both experiments and theory show significant phase shifts increase as a function of frequency and ischaemia time. The theoretical and experimental results suggest that the tested technique has the potential to detect the processes and level of ischaemia in the brain by non-invasive, continuous, bulk volumetric monitoring with a simple and inexpensive apparatus.

The effect of brain hematoma location on volumetric inductive phase shift spectroscopy of the brain with circular and magnetron sensor coils: a numerical simulation study.

This numerical simulation study addressed the effects of the location of a discrete brain hematoma on the volumetric inductive phase shift of the brain measured with an induction circular sensor coil and an induction magnetron sensor coil. The theoretical study simulates the brain cavity as a circular sphere transversely centered with respect to the circular and magnetron sensor coils. As a case study for the effects of hematoma location, we employed similar size simulated spherical hematomas placed at three different positions from the center of the brain outward. A three-dimensional finite element analysis of the field equations in the frequency range from 100 kHz to 100 MHz revealed a substantial effect of hematoma location on the ability of both the circular and magnetron sensors to detect the hematomas. In particular it was found that there are frequencies, which may be related to resonance, at which the occurrence of the hematomas has no effect on the volumetric inductive phase shift of the brain. Furthermore it was found that the relative sensitivity of circular and magnetron sensor coils with respect to the occurrence of hematoma varies with the location of the hematoma.

Tissue ablation with irreversible electroporation.

This study introduces a new method for minimally invasive treatment of cancer-the ablation of undesirable tissue through the use of irreversible electroporation. Electroporation is the permeabilization of the cell membrane due to an applied electric field. As a function of the field amplitude and duration, the permeabilization can be reversible or irreversible. Over the last decade, reversible electroporation has been intensively pursued as a very promising technique for the treatment of cancer. It is used in combination with cytotoxic drugs, such as bleomycin, in a technique known as electrochemotherapy. However, irreversible electroporation was completely ignored in cancer therapy. We show through mathematical analysis that irreversible electroporation can ablate substantial volumes of tissue, comparable to those achieved with other ablation techniques, without causing any detrimental thermal effects and without the need of adjuvant drugs. This study suggests that irreversible electroporation may become an important and innovative tool in the armamentarium of surgeons treating cancer.

Treatment of cancer with cryochemotherapy.

Cryosurgery employs freezing to destroy solid tumours. However, frozen cells can survive and cause cancer recurrence. Bleomycin, an anticancer drug with a huge intrinsic cytotoxicity is normally not very effective because it is nonpermeant. We report that freezing facilitates bleomycin penetration into cells making it toxic to cryosurgery surviving cells at concentrations that are non-toxic systemically.

Cryosurgical monitoring using bioimpedance measurements--a feasibility study for electrical impedance tomography.

The effectiveness of cryosurgery in treating tumors is highly dependent on knowledge of freezing extent, and therefore relies heavily on real-time imaging techniques for monitoring. Electrical impedance tomography (EIT), which utilizes tissue impedance variation to construct an image, is very well suited to cryosurgery since frozen tissue impedance is much higher than that of unfrozen tissue. In this study, we explore cryosurgical monitoring as a previously uninvestigated application for EIT. The feasibility of bio-impedance measurements to detect ice front propagation is demonstrated by freezing planar tissue samples one-dimensionally while measuring impedance along a linear array. The experimental results compare favorably to a simple finite element model designed to provide an electrical field visualization tool.

Transplantation of mammalian livers following freezing: vascular damage and functional recovery.

We transplanted rat livers cryopreserved at high subzero temperatures with a protocol that mimics freezing in freeze-tolerant animals. The results of nine transplants show that: (a) every single transplanted liver produced bile, which suggests that the cryopreserved livers retained some physiological function; (b) eight of the animals survived between 2 and 4 h with loss of microvascular integrity which suggests that transplantation failure is related to the circulation and tests of bile production are not indicative of transplantation success; and (c) one animal survived for 5 days with an intact circulation which might be due to an unidentified technical variation or could indicate that when the circulation recovers animals with transplanted livers survive.

Cryosurgery.

Cryosurgery is a surgical technique that employs freezing to destroy undesirable tissue. Developed first in the middle of the nineteenth century it has recently incorporated new imaging technologies and is a fast growing minimally invasive surgical technique. A historical review of the field of cryosurgery is presented, showing how technological advances have affected the development of the field. This is followed by a more in-depth survey of two important topics in cryosurgery: (a) the biochemical and biophysical mechanisms of tissue destruction during cryosurgery and (b) monitoring and imaging techniques for cryosurgery.

Viability of deformed cells.

Most of the researchers in the field of cryobiology believe that the mechanism of damage during freezing with low cooling rates is chemical and related to the hypertonicity of the extracellular solution. However, there is some evidence to indicate that cells may be destroyed during freezing also by compression between ice crystals. We have developed an experimental procedure to study the effect of cell compression on viability. Using human prostate primary adenoma cancer cells we show that cell viability decreases steeply when cells are compressed to 30% of their original diameter. If uniform expansion of cell membrane is assumed, this corresponds to a 50% increase in the cell membrane surface area. A simple mathematical model shows that the temperature at which the compression effect may cause cell damage is related to the spacing between ice crystals. When the ice crystals are spaced at distances comparable to the cell diameter the model combined with our experimental data predicts compression damage at about -1.8 degrees C. This is consistent with experimental observation on frozen cell destruction in the presence of antifreeze proteins.

A histological analysis of liver injury in freezing storage.

As part of a more extensive study on the use of high subzero freezing for cryopreservation of mammalian livers we have tried to single out the effects of freezing and thawing on tissue damage. We compared the morphology of livers after freezing and thawing with what we considered an optimal high subzero cryopreservation protocol with the morphology of livers preserved under the same thermal conditions and in the same solution in a supercooled state, without freezing. The results show that while hepatocytes survive high subzero cryopreservation, detachment of endothelial cells occurs in every freezing experiment. On the other hand, the endothelial cells in livers that are not frozen are intact. This suggests that endothelial cell damage is caused by freezing and may be an important factor in high subzero freezing cryopreservation of the liver.

Effect of thermal variables on human breast cancer in cryosurgery.

There is a growing interest in the use of cryosurgery to treat breast cancer, following recent breakthroughs in noninvasive imaging and in cryotechnology, as well as the recent success of cryosurgery in treating various types of cancer. However, since haphazard freezing does not guarantee tissue destruction, in order to apply this technique effectively it is essential to determine the thermal parameters that produce complete destruction of malignant tissue. This study seeks to quantitatively identify the relationship between thermal variables and the degree of freezing damage to human breast cancer cells. In order to do this, human breast cancer and normal cells were frozen with controlled thermal parameters using a directional solidification apparatus. Cell viability was determined after thawing using trypan blue, and correlated to the thermal variables used during freezing. Cellular damage is observed to increase with increasing cooling rates, due to the higher probability of intracellular ice formation. A double freeze thaw cycle significantly increases the extent of cell damage, and is sufficient to ensure complete cell destruction at final freezing temperatures of -40 degrees C for a 25 degrees C/min cooling rate, and -20 degrees C for a 50 degrees C/min cooling rate. The correlations between cell death and thermal parameters are qualitatively identical for all the cell types in this study, although there is some variation from one cell type to another in the overall susceptibility to freezing damage. The correlations established in this study can be used to design systematic and optimal breast cryosurgery protocols.