Biomedical subjects
Publications and source records attributed to B Rubinsky.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The thermodynamics of intracellular ice nucleation are important in low-temperature biology for understanding and controlling the process of cell destruction by freezing. We have developed a new apparatus and technique for studying the physics of intracellular ice nucleation. Employing the principle of directional solidification in conjunction with light microscopy, we can generate information on the temperature at which ice nucleates intracellularly as a function of the thermal history the cells experience. The methodology is introduced, and results with primary prostatic cancer cells are described.
Antifreeze proteins (AFPs) depress the freezing temperature of a solution in a non-colligative manner, by arresting the growth of ice crystals. The kinetics of this effect, studied here for the first time using a new technique called temperature gradient thermometry, are consistent with an adsorption-mediated inhibitory mechanism. The results obtained by this approach provide a new experimental basis for understanding AFP interaction with ice.
BACKGROUND AND OBJECTIVES: Imaging monitored cryosurgery is emerging as an important minimally invasive surgical technique for treatment of cancer. Although imaging allows excellent control over the process of freezing itself, recent studies show that at high subzero temperatures cells survive freezing. Antifreeze proteins (AFP) are chemical compounds that modify ice crystals to needle-like shapes that can destroy cells in cellular suspensions. The goal of this study was to determine whether these antifreeze proteins can also destroy cells in frozen tissue and serve as chemical adjuvants to cryosurgery. METHODS: Livers from six rats were excised, perfused with solutions of either phosphate-buffered saline (PBS) or PBS with 10 mg/ml AFP-I, and frozen with a special cryosurgery apparatus. Lobes were frozen with one or two freeze-thaw cycles and the cell viability was examined with a two stain fluorescent dye test and histological assessment. RESULTS: A significant percentage of hepatocytes survive freezing on the margin of a frozen cryolesion. AFP significantly increase cellular destruction in that region apparently through formation of intracellular ice. CONCLUSIONS: This preliminary study demonstrates that antifreeze proteins may be effective chemical adjuvants to cryosurgery.
OBJECTIVES: Recent studies show that prostate adenocarcinoma cells can survive cryosurgery and that cell destruction depends on the specific thermal parameters used during freezing. The goal of this preliminary study is to determine whether certain chemical compounds, known as antifreeze proteins, can induce complete human primary prostatic adenocarcinoma cell destruction by freezing, regardless of the thermal parameters used. The study also examines the mechanism by which antifreeze proteins bring about cell destruction. METHODS: Antifreeze proteins were added to solutions containing human primary prostatic adenocarcinoma cells. The cells were frozen with controlled thermal parameters using a directional solidification apparatus attached to a light microscope. Cell viability was determined after thawing as a function of antifreeze protein concentration and cooling rate during freezing. RESULTS: The dose response study shows that for all the cooling rates tested, 10-mg/mL solutions of antifreeze protein cause the complete destruction of human primary prostatic adenocarcinoma cells frozen to a temperature at which, without these proteins, the cells survive freezing. Light microscopy shows that the lethal effect of the antifreeze proteins is related to the formation of intracellular ice in the frozen cells. CONCLUSIONS; This preliminary study has demonstrated that antifreeze proteins have the ability to generate complete destruction of prostatic adenocarcinoma cells frozen to high subzero temperatures irrespective of the cooling rates used during freezing. This suggests that introducing antifreeze proteins into undesirable tissues prior to freezing may increase the efficacy and the control over tissue destruction by cryosurgery.
Cryosurgery currently is being used clinically to treat tumors in internal organs such as the liver and prostate. Although performed at present under ultrasound monitoring, magnetic resonance imaging (MRI)-guidance of these procedures not only permits monitoring of the frozen region during cryosurgery but also makes it possible to determine the temperature distribution in the frozen region, which is not possible using ultrasound monitoring. A good estimate of the region of destruction in the tissue can be obtained from correlating the temperature distribution and the time course of the freezing with the image of the frozen region. Unfortunately, MR pulse sequence-based temperature determination techniques such as diffusion, relaxation time, and chemical shift cannot be used for measuring the temperature in the frozen region because the T2 of the frozen regions is so short that there is effectively no RF signal from the frozen region. This paper describes a numerical technique for determining the two dimensional temperature distribution in the frozen region during MR image-guided cryosurgery of normal liver in rabbits. The technique involves solving the energy equation numerically in the frozen region to determine the temperature distribution there. The boundary conditions needed to solve the equation are determined from MR images of the frozen tissue during cryosurgery and from the measured temperature of the cryoprobe. The calculated temperature in the frozen region is then correlated with the damaged region (cryolesion) determined from post mortem histologic evaluation.
OBJECTIVES: Recent advances in imaging technology and cryotechnology have rekindled interest in prostate cryosurgery. Cryosurgery, however, cannot be applied precisely without knowing how the thermal variables used during the procedure affect tissue destruction. The goal of this article is to provide quantitative values for the relationship between thermal variables during freezing and the destruction of human primary prostatic adenocarcinoma cells. METHODS: Human primary prostatic adenocarcinoma cells were frozen with controlled thermal parameters, using a directional solidification apparatus. Cell viability was determined after thawing, using trypan blue and a two-dye fluorescent test and correlated to the thermal variables used during freezing. RESULTS: Human primary prostatic adenocarcinoma cells are damaged by intracellular chemical damage when frozen with cooling rates lower than 5 degrees C/min and by intracellular ice formation when frozen with cooling rates higher than 25 degrees C/min. A double freeze/thaw cycle is required to ensure complete cell destruction at high subzero temperatures, which must be lower than -40 degrees C for the low cooling rates and lower than -19 degrees C for the higher cooling rate. CONCLUSIONS: Haphazard freezing does not necessarily destroy tissue during cryosurgery; however, quantitative data on the relation between thermal variables and frozen cell destruction can provide the means for performing cryosurgery more precisely and with greater control over the outcome of the procedure.
Cryosurgery of the skin is a common treatment for both benign and malignant skin cancers. Monitoring the depth of the frozen lesion during cryosurgery, either by estimation based on the lateral spread of freeze at the skin surface or via thermocouples, may be inaccurate because of the heterogeneous nature of tissue. We describe an integrated cryosurgical probe and magnitude resonance imaging probe which we use to obtain high resolution MR images of skin, subcutaneous muscle and the frozen lesion during cryosurgery.
We have tested a protocol that uses a new cryoprotective solution for preserving mammalian livers in a frozen state. This protocol is based upon our studies on the mechanisms that freeze tolerant animals use to survive freezing in nature. The cryoprotective solution contains glycerol and antifreeze proteins, both of which are found in freeze tolerant animals. Whole rat livers were frozen to -3 degrees C, maintained at that temperature for 6 hours and then warmed to 37 degrees C. Post thawing bile production and microscopic analysis of tissue slices were used to verify liver function and tissue morphology. We conclude that antifreeze proteins used in the concentrations chosen here have a protective effect on the whole liver during freezing.
Normal and malignant human breast tissues were obtained from resection surgery and frozen on a directional solidification stage with controlled and uniform cooling rates. The frozen samples were freeze substituted and examined with a light microscope. It was observed that in both normal and malignant tissue, ice forms first in the connective tissue. The ice propagates along the connective tissue, which surrounds fat cells in normal adipose tissue and clumps of tightly packed malignant cells in the diseased tissue. This mode of freezing affects the osmotic response of malignant cells, causing intracellular ice crystal formation at unusually low cooling rates.