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.     

Cancer cryotherapy: evolution and biology

Since the inception of cryosurgery in the 1850s, landmark advances in chemistry, physics, materials science, and biology have culminated in the sophisticated cryosurgical devices currently in use. Effective cryosurgical tissue injury depends on four criteria: 1) excellent monitoring of the process; 2) fast cooling to a lethal temperature; 3) slow thawing; and 4) repetition of the freeze-thaw cycle. Meeting these criteria depends on understanding the imaging technology used to visualize the iceball, the type of cryogen used, the size of the probe, and probe arrangement. Third-generation cryosurgical equipment offers advantages over previous designs. These machines rely on argon for freezing but also use helium to warm probes and accelerate the treatment process, and they offer additional safety by being able to rapidly arrest iceball formation. Metallurgic advances have led to the development of thinner probes, which have been easily adapted to perineal templates similar to those used for prostate brachytherapy.     

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.     

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.     

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.     

Advances in the design of special cryosurgical apparatus in China

Advances in the design of special cryobiomedical apparatus and a review of the trend of developments in the field of cryosurgery in China are discussed. The typical structure of two special cryoprobes for treatment deep in the body and the technology of designing these probes are presented in detail. Some cases which are treated successfully with the above cryoprobes will also be discussed. The experimental aspects of heat transfer in frozen tissue and of the temperature profiles both of a human brain during surgery and of the cryoprobe are described. Other improvements in the field of cryosurgical devices, e.g., four main ways of attaching freezing tips to cryoprobes during surgery and an LN2 transfer tube with high dexterity are also presented. Finally, the development of commercial cryosurgical apparatus in China is also discussed.     

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.     

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.     

Pre-conditioning cryosurgery: cellular and molecular mechanisms and dynamics of TNF-α enhanced cryotherapy in an in vivo prostate cancer model system

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4h and diminished over several days. Early events (4-24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.     

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)).     

Critical temperature for skin necrosis in experimental cryosurgery

To investigate the minimal lethal freezing temperature required to produce skin necrosis in dogs, multiple skin sites were frozen with cryosurgical equipment. Tissue temperatures were recorded from thermocouple sites placed at diverse distances, usually 5 mm from the edge of the freezing probe. In single freezing cycles of about 3 min duration, tissue temperatures in the range of 0 to ?60 °C were produced. Punch biopsies of the skin at the thermocouple sites 3 days after freezing injury provided tissues for estimation of viability by histologic examination.The histologic findings permitted classification of the biopsy tissue into three groups, that is, viable, borderline, or necrotic. When classified as borderline, the division between the necrotic and viable tissue was evident on the histologic slide. The viable specimens were scattered through the 0 to ?35 °C range. All specimens frozen to ?10 °C or warmer were viable. In biopsies classified as borderline, the range of viability extended from ?11 ° to ?50 °C. The necrotic biopsies covered a range of ?14 ° to ?50 °C. Cell death was certain at temperatures colder than ?50 °C. The data showed cryosurgical freezing conditions produced a range of temperatures in which viability or death of tissue may occur and that the ranges of viability and necrosis overlapped to a great extent.The wide range of temperatures at which cells were viable shows the need to achieve tissue temperatures in the range of ?50 °C in the cryosurgical treatment of cancer.     

Pre-conditioning cryosurgery: Cellular and molecular mechanisms and dynamics of TNF-α enhanced cryotherapy in an in vivo prostate cancer model system

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4 h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4 h and diminished over several days. Early events (4–24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.     

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.     

Long-term follow-up post-cryosurgery in a sheep breast model.

This study constitutes the advanced stage of an ongoing project for the development of cryosurgical devices and techniques for breast cryosurgery. The current study focuses on the long-term follow-up post-cryosurgery in a sheep breast model. Results of this study indicate that the cryotreatment site in a sheep breast model cannot be identified up to 5 months post-cryosurgery by means of ultrasound, mammography, or MRI. Histology findings of this study further indicate that there is no gross or microscopic difference between lesions that have been subject to one versus three freeze/thaw cycles. Under either cryosurgical protocol, there is a main cryoinjured region that has uniform destruction of epithelium and healing scar formation and a transition zone of damaged lobules without acini, surrounded by healthy tissues. The cryoinjured region at 5 months post-cryosurgery was found to be about half the diameter of the ultrasound-imaged frozen region during the cryoprocedure. This study shows that, in terms of recovery and regeneration, surgical excision appears to have an advantage over cryosurgery, which results in a more rapid healing process. Based on observations that the cryoinjured region is no smaller than the ultrasound-imaged ice-ball and that the typical thickness of the transition zone is up to 5 mm, a conservative use of the cryosurgical device developed for the current study in an ultrasound-monitored cryoprocedure requires at least 5 mm safety margins of the frozen region radius around the target region.     

Cryoimmunotherapy: Continuing studies toward determining a rational approach for assessing the candidacy of the prostatic cancer patient for cryoimmunotherapy and postoperative responsiveness. An interim report

Cryosurgery (in situ freezing) is a recognized method for the controlled cryogenic destruction of benign and malignant tissues and has been efficaciously employed for the treatment of prostatic cancer. In situ freezing constitutes an antigenic stimulus capable of generating a specific immunologic response against autologous antigens of the tissue frozen, ergo, cryostimulation. The phenomenon of cryostimulation has, in light of reports of metastatic tumor destruction, suggested that cryosurgery may be applicable not only for ablation of a primary tumor, but also as a means of inducing or augmenting host resistance to the tumor, ergo, cryoimmunotherapy. For cryosurgery to be effectual as a means of immunotherapy several factors, categorically referred to as cryosensitivity, must be fulfilled. Cryosensitivity is presently suggested to be reflective of the (i) immunogenicity of the prostate and (ii) immunocompetence of the host. The majority of prostatic cancer patients possess varying degrees of immunocompetence. Immunotherapy under such conditions may, rather than augmenting or inducing host resistance to a tumor, act in an antagonistic manner resulting in enhancement of the tumor. This knowledge has directed initial efforts of this study group toward establishment of critical criteria to evaluate the immunocompetency of the prospective cryosurgical patient. Once the degree of immunocompetency has been established, this may serve as a means to stage this patient immunologically, i.e., immunostage. Similar to traditional staging of the anatomic spread and histological grade of tumor to aid in selecting therapy and predicting outcome, immunostaging may provide adjunctive criteria toward determining a patient's suitability for cryoimmunotherapy. Directed toward improving criteria on which to immunostage patients, our initial recommendations for assessment of tumor-host immunologic responsiveness have been redefined. The principal objective being elimination, for the most part, of nonspecific correlates and maintenance and addition of specific correlates of humoral- and cell-mediated tumor-directed immunity. Preliminary studies suggest a possible relationship between a patient's immunostage and the intensity of immunologic responsiveness following cryosurgery. But, more important, the direction of the immune response, i.e., tumoricidal vs tumor enhancing. Together with observations of the association of humoral immunity with enhancement of tumor metastases and cellular immunity with regression of tumor, reports of the adjunctive utilization of modulators of the immunologic effects of cryostimulation hold promise for the more effective clinical utilization of cryoimmunotherapy.     

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.     

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.     

Numerical simulation for heat transfer in prostate cancer cryosurgery

A comprehensive computational framework to simulate heat transfer during the freezing process in prostate cancer cryosurgery is presented. Tissues are treated as nonideal materials wherein phase transition occurs over a temperature range, thermophysical properties are temperature dependent and heating due to blood flow and metabolism are included. Boundary conditions were determined at the surfaces of the commercially available cryoprobes and urethral warmer by experimental study of temperature combined with a mathematical optimization process. For simulations, a suitable computational geometry was designed based on MRI imaging data of a real prostate. An enthalpy formulation-based numerical solution was performed for a prescribed surgical protocol to mimic a clinical freezing process. This computational framework allows for the individual planning of cryosurgical procedures and objective assessment of the effectiveness of prostate cryosurgery.     

Cryosurgery of the monkey (macaque) prostate: I. Humoral immunologic responsiveness following cryostimulation

The effect of in situ freezing of the monkey (macaque) prostate on the development of antibodies reactive with allogeneic and autologous extract preparations of the cranial and caudal lobe of prostatic tissue and their tissue specificity were evaluated by the method of tanned cell hemagglutination. A representative percentage of the animals receiving cryosurgery developed antibodies to prostatic tissue components; however the intensity of this response was considerably modest when contrasted to that obtained following similar treatment of the rabbit prostate (coagulating gland) and generally did not appear, as in the latter, to increase to any significant degree following multiple freezing. The possible relationship of this modest humoral response to the “cryosensitivity” of the target organ and of the animals evaluated in the present study, i.e., the concentration of glandular secretions (autoantigens), physiologic state (elaboration of androgen) and immunocompetence, are considered.     

Cryoelectrolysis; an acute case study in the pig liver

We report results from an acute, single case study in the pig liver on the effects of a tissue ablation protocol (we named cryoelectrolysis) in which 10 min of cryosurgery, with a commercial cryosurgical probe, are delivered after 10 min of electrolysis generated by a current of about 60 mA. The histological appearance of tissue treated with cryoelectrolysis is compared with the appearance of tissue treated with 10 min of cryosurgery alone and with 10 min of electrolysis alone. Histology done after 3 h survival shows that the mixed rim of live and dead cells found around the ablated lesion in both cryosurgery and electrolytic ablation is replaced by a sharp margin between life and dead cells in cryoelectrolysis. The appearance of the dead cells in each, cryoelectrolysis, cryosurgery and electrolytic ablation is different. Obviously, this is an acute study and the results are only relevant to the conditions of this study. There is no doubt that additional acute and chronic studies are needed to strengthen and expand the findings of this study.