Imaging of interstitial cryotherapy--an in vitro comparison of ultrasound, computed tomography, and magnetic resonance imaging.

RATIONALE AND OBJECTIVES: To evaluate the imaging capabilities of ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI) in monitoring interstitial cryotherapy and to compare them with visual control. METHODS: An experimental MR-compatible, vacuum-insulated and liquid nitrogen-cooled cryoprobe was inserted under in vitro conditions into a porcine liver, which was kept at a temperature of 37 +/- 1 degrees C, in a water bath with continuous stirring. The freezing procedure was controlled macroscopically, by US (Toshiba Sonolayer, 7.5-MHz linear array transducer), by CT (Siemens Somatom Plus, slice thickness 2-8 mm, 165-210 mA at 120 kV), and by MRI (Philips Gyroscan ACS-NT, FFE TR/TE/FA = 15/5.4/25 degrees, T1-SE 550/20, T2-TSE 1800/100) after the iceball reached its maximum size. RESULTS: The maximum iceball diameter around the probe tip was 12.0 mm by visual control, 12.4 mm by US, 12.7 mm by CT, and within 12.8 mm by spin echo sequences and 11 mm by gradient echo sequence. Due to the nearly signal-free appearance of the frozen tissue on MR images, the ice/tissue contrast on T1-weighted and gradient echo images was superior to T2-weighted images and CT images. Sonographically, the ice formation appeared as a hyperechoic sickle with nearly complete acoustic shadowing. CONCLUSION: Due to the better ice/tissue contrast, T1-weighted or gradient echo MR images were superior to CT and US in monitoring interstitial cryotherapy. Gradient echo sequences generally underestimated the ice diameter by 15%.     

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)).     

Experimental measurement and theoretical calculations on muscle and liver tissue during cryosurgery. II. A theory on freezing of tissue.

A theory concerning growth of an iceball using round cryoprobes is presented. The equation for heat conduction is solved for homogeneous tissue, and mathematical equations are presented for the radius of the iceball at any freezing time and temperature. The agreement between theory and freezing experiments at T = ?196 °C is good.     

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.     

Comparison of iceball diameter and temperature distribution achieved with 3-mm accuprobe cryoprobes in porcine and human liver tissue and human colorectal liver metastases in vitro

We aimed to assess the thermal profile and size of iceballs produced by Accuprobe cryoprobes in fresh porcine and human liver and human colorectal cancer liver metastases in vitro to allow better planning of cryosurgical treatment of liver metastases. Iceballs were produced by a 20-min single freeze cycle using 8-mm cryoprobes in pig liver in a waterbath at 37 degrees C (n = 8) and 3-mm cryoprobes in pig liver (n = 8), human liver (n = 3), and human colorectal cancer liver metastases (n = 8). The iceball diameters and the temperatures at different distances from the cryoprobe were measured. Mean iceball diameters produced by 8-mm cryoprobes in pig liver were 56.3 mm and varied from 38.7 to 39.6 mm for 3-mm cryoprobes in the different tissues used. There was no significant difference in iceball size in the different tissues. The diameter of the zone of -40 degrees C or less was approximately 44 mm using 8-mm cryoprobes in porcine liver and between 27 and 31 mm using 3-mm cryoprobes in the different tissues examined. The results may allow better preoperative planning of the cryosurgical treatment of liver metastases with Accuprobe cryoprobes.     

Ice recrystallization in a model system and in frozen muscle tissue

Recrystallization produces modifications on ice crystal sizes during storage and transport of frozen foods, reducing the advantages obtained by quick freezing and inducing physicochemical changes which alter their quality and shorten their shelf life. This process involves the growth of the larger crystals at the expense of the smaller ones, being the interfacial energy, the driving force of the phenomenon. In the present work recrystallization was analyzed using direct microscopic observation of ice crystals in a model solution (0.28 N NaCl) and indirect observation of frozen muscle tissue. The model solution allowed visualization of the interface behavior; from the analysis of the ice crystal frequency distributions, relationships between shape and size of the grains were established. A kinetic model based on the average system curvature was proposed obtaining a satisfactory fitness of the experimental data. Values of the kinetic constants determined at different temperatures allowed the estimation of the process activation energy. In muscle tissues isothermal freeze-substitution was used to observe the holes left by the ice in frozen semitendinous beef muscle stored at -5, -10, -15, and -20 degrees C during long periods of time. A different evolution of the mean ice crystal diameter was observed with respect to the model system. In meat samples, at long storage times, a limit diameter value was reached; this situation has been proved to be independent of temperature and initial size (freezing rate); a theoretical expression based on tissue characteristic parameters was proposed for its evaluation. Activation energy for recrystallization in muscle tissue was also determined, being comparable to values for protein denaturation and quality losses.     

Three-dimensional disorder of dipolar probes in a helical array. Application to muscle cross-bridges.

Fluorescence polarization and EPR experiments on azimuthally randomized helices bearing extrinsic (dipolar) probes yield information about the axial orientation and order of the probes. If the orientation of the probe on the structure bearing it is known and disorder is absent, the orientation of the structure may be ascertained. For cases where less probe orientation information is available and/or disorder is present, the available structural information is correspondingly reduced. Here we examine the available data on probes attached to cross-bridges in muscle fibers: four plausible cases of three-dimensional cross-bridge disorders are numerically modeled muscle in states of rigor and relaxation. In rigor, where the reported probe disorder is small (Thomas and Cooke, 1980), it was found that the cross-bridge disorder was also small. On the other hand, for the relaxed state where the probes are found to be completely disordered, the cross-bridges may have a considerable amount of order. This possibility is in concert with the results of x-ray diffraction, in which the presence of well-developed myosin-based layer lines indicates considerable order in relaxed muscle.     

A pig model of hepatic cryotherapy. In vivo temperature distribution during freezing and histopathological changes

We aimed to assess the temperature distribution in the cryolesion during hepatic cryotherapy and the association with postoperative histological changes to optimise the technique and allow better preoperative planning. Hepatic cryolesions were produced in 22 pigs following laparotomy using a CMS-cryosystem and 8mm-AccuProbe-Cryoprobes. The temperature was measured in 1 min intervals at different distances from the probe during freezing. The animals were treated in 5 groups: (i) single freezing of 20 min; (ii) double freezing of 20 min each; (iii) single freezing of 40 min; (iv) single freezing of 20 min (n=4), histology at 1 week p.o., and (v) single freezing of 20 min and Pringle manoeuvre; [(i)-(iii) and (v): histology at 24 h p.o.]. The mean diameter of the -38 degrees C isotherm, i.e., the zone of effective treatment for colorectal metastases was 37 mm for group (i) with a mean iceball diameter of 59 mm and about 46 mm for groups (ii, iii, and v) with mean iceball diameters of 78, 75, and 75 mm, respectively. At 7 days postoperatively secondary necrosis was seen in the largest central part of the lesion, wherever temperatures of -15 degrees C or lower were achieved during cryosurgery. Under the hypothesis that -38 degrees C is the effective temperature for the destruction of colorectal liver metastases, a lesion of 37-mm diameter may be effectively treated with a single 8mm-AccuProbe-Cryoprobe and a 20 min single freeze cycle and a lesion of 46 mm may be effectively treated when a double freeze-thaw cycle of 20 min each, a single freeze cycle of 40 min, or a 20 min single freeze cycle with additional Pringle manoeuvre is used, when it is perfectly placed in the lesion.     

Biomolecular sample considerations essential for optimal performance from cryogenic probes

For compounds dissolved in non-polar solvents, nuclear magnetic resonance spectroscopic investigations have benefited greatly from the advent of cryogenically cooled probes. Unfortunately the allure of significant increases in sensitivity may not be realized for compounds such as metabolites that are dissolved in solvents with high ionic-strengths such as solutions typically utilized for metabolomic or biomolecular investigations. In some cases there is little benefit from a cryogenically cooled probe over a conventional room temperature probe. Various sample preparation methods have been developed to minimize the detrimental effects of salt; for large numbers of metabolomic samples these preparation methods tend to be onerous and impractical. An alternative to manipulating the sample, is to utilize a probe that is designed to have a higher tolerance for solutions with high ionic-strengths. In order to acquire high-quality optimal data and choose the appropriate probe configuration (especially important for comparative quantitative investigations) the effects of salts and buffers on cryogenic probe performance must be understood. Herein we detail sample considerations for two cryogenic probes, a standard 5 mm and a narrow diameter 1.7 mm, in an effort to identify via integrals, intensities and noise levels the optimal choice for biomolecular investigations.     

Flow cytometry to assess biochemical pathways in heat-stressed Cronobacter spp. (formerly Enterobacter sakazakii)

Aims: Using a flow cytometry (FC)‐based approach in combination with four selected fluorescent probes, the biochemical pathway activated following the adaptation of Cronobacter spp. to lethal heat stress was investigated. This approach assessed the physiological changes induced in four strains of Cronobacter spp. Methods and Results: Using the commercially available live/dead viability assessment fluorescence probes, live, injured or dead bacterial cells were studied. Cellular respiration and membrane potential were evaluated using the dye‐labelled probe 3,3′‐dihexylocarbocyanine iodide, metabolic activity was evaluated using a fluorescein diacetate (FDA) probe, intracellular pH changes were measured using a carboxy‐fluorescein diacetate succinimidyl ester probe, and reactive oxygen species were measured using a hydroethidine fluorescent probe. Adaptation to lethal heat stress induced physiological changes that potentially improve the survival of Cronobacter spp. Conclusions: These data showed that in situ assessment of physiological behaviour of lethally stressed cells using multiparameter FC is a useful, rapid and sensitive tool to study and assess the viability and physiological state of Cronobacter cells. Significance and Impact of the Study:  This study shows that FC is a valuable tool in the study of physiological aspects of increased survival because of sublethal adaptation to heat.     

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.     

Effects of dead spermatozoa on motion characteristics and membrane integrity of live spermatozoa in fresh and cooled-stored equine semen

The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25 x 10(6) spermatozoa/ml with EZ-Mixin CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25 x 10(6) spermatozoa/ml and 10% seminal plasma from pooled ejaculates of the three stallions, in EZ-Mixin CST extender. Spermatozoa were killed in the pooled ejaculates by repeated freezing and thawing, then stored at -20 degrees C until warmed to 37 degrees C and mixed with aliquots of fresh spermatozoa to be cooled and stored in an Equitainer for 24h. Motion characteristics (% total motility (MOT), % progressive motility (PMOT), and mean curvilinear velocity (VCL)) for fresh and 24h cooled samples were determined using a computerized spermatozoal motion analyzer. The presence of up to 75% dead spermatozoa did not adversely affect MOT or PMOT of live spermatozoa in either fresh or cooled-stored semen. However, VCL and the percentage of membrane-intact spermatozoa were reduced compared to control samples when 75% (v/v) dead spermatozoa were added. Membrane integrity, as assessed by staining with carboxyfluoresein diacetate-propidium iodide, was highly correlated (r>0.8; P<0.001) with MOT and PMOT in both fresh and cooled-stored semen samples. Results of this study have application to the processing of both cooled and frozen equine semen.     

Schistosoma mansoni: peripheral and tissue eosinophilia in infected rats.

Peripheral eosinophilia is induced in Sprague-Dawley rats following infection with cercariae of Schistosoma mansoni. Beginning 3 weeks after infection, peripheral eosinophil levels rise above the baseline range; they reach peak values during the fifth week. Following a decline from peak values, peripheral eosinophil levels remain elevated and are observed to fluctuate for the next 5 months. The magnitudes of both the initial peak response at 5 weeks and the subsequent chronic level of peripheral eosinophils are dependent upon dose of cercariae. The initial peak response phase of peripheral eosinophilia coincides in time with the period of adult worm elimination (Weeks 4–6) in the schistosome-infected rat. Histological examination of the liver at 5 weeks after infection reveals eosinophil-rich inflammatory reactions associated with both live and dead worms residing in the portal blood vessels. Around live worms the inflammatory cells are localized in a perivascular arrangement; around dead worms these cells are in the vascular lumen in contact with destroyed worms. The chronic phase of peripheral eosinophilia is associated, in part, with inflammatory reactions surrounding eggs deposited in the liver by the few worm pairs which survive more than 6 weeks and remain within the liver. Histological examination during this period reveals granulomatous lesions within the liver surrounding eggs and dead worms. The granulomas are predominately monocytic (lymphocytes, macrophages) at 11 and 16 weeks. The initial peak response phase of peripheral eosinophilia appears to be a marker for tissue-localized reactions of eosinophils with worms. There are relationships between inflammatory reactions and survival of adult worms.     

Convection-Induced Biased Distribution of Actin Probes in Live Cells

Fluorescent markers that bind endogenous target proteins are frequently employed for quantitative live-cell imaging. To visualize the actin cytoskeleton in live cells, several actin-binding probes have been widely used. Among them, Lifeact is the most popular probe with ideal properties, including fast exchangeable binding kinetics. Because of its fast kinetics, Lifeact is generally believed to distribute evenly throughout cellular actin structures. In this study, however, we demonstrate misdistribution of Lifeact toward the rear of lamellipodia where actin filaments continuously move inward along the retrograde flow. Similarly, phalloidin showed biased misdistribution toward the rear of lamellipodia in live cells. We show evidence of convection-induced misdistribution of actin probes by both experimental data and physical models. Our findings warn about the potential error arising from the use of target-binding probes in quantitative live imaging.     

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry.

In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using 35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using 35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (less than 1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Ice crystals formed in tissue during cryosurgery. II. Electron microscopy

Tissues frozen by means of a cryosurgical probe have been examined by electron microscopy following techniques designed to preserve the ice crystal spaces.Ice crystals appeared similar whether tissues were quenched or not following cryosurgery and the various techniques of dehydration resulted in similar ice crystal architecture.Ice crystal spaces in the area deep to the freezing probe were intracellular both in epithelium and muscle although in the muscle zone some fibers contained large and others small crystal spaces. It is suggested that this might be due to variations in the local blood supply.At the periphery of the frozen area ice crystals were usually extracellular producing gross distortion of the cells which, however, retained intracellular structural integrity. These results are consistent with the belief of many workers that intracellular ice is lethal while extracellular ice is not, but no evidence of penetration of cell membrane by ice crystals was seen.     

Thermal expansion measurements of frozen biological tissues at cryogenic temperatures.

Thermal expansion data are essential for analyses of cryodestruction associated with thermal stresses during cryopreservation protocols as well as during cryosurgery. The present study tests a commonly used hypothesis that the thermal expansion of frozen tissues is similar to that of pure water ice crystals. This study further provides insight into the potential effect of the presence of cryoprotectants on thermal expansion. A new apparatus for thermal strain measurements of frozen biological tissues within a cryogenic temperature range is presented. Results are presented for fresh tissue samples taken from beef muscle, chicken muscle, rabbit muscle, rabbit bone, and pig liver. Pilot studies of the effect of cryoprotectants on thermal expansion are further presented for rabbit muscle immersed in dimethyl sulphoxide (2 mols/l) and glycerol (2 mols/l), and for pig liver perfused with dimethyl sulphoxide (2 mols/l). Thermal expansion of frozen soft biological tissues was found to be similar to that of water ice crystals in the absence of cryoprotectant. Thermal expansion of the rabbit bone was found to be about one half of that of frozen soft tissues. A significant reduction in the thermal expansion at higher temperatures was observed in the presence of cryoprotectants. A rapid change of thermal strain near -100 degrees C was also observed, which is likely to be associated with the glass transition process of the cryoprotectant solutions.     

Computerized tomographic imaging of cryosurgical iceball formation in brain

Computerized tomography (CT) was used to monitor the exact anatomical location and dimensions of the cryosurgical iceball within the brain. The gross and microscopic appearance of the tissue iceball was examined in both acute and chronic animals. Iceball formation was monitored in the brain of four dogs under a general anesthesia. The radiographic image of the iceball was that of a well-demarcated radiolucent sphere that disappeared upon thawing. The post-thaw contrast-enhanced CT scan revealed a zone of blood-brain barrier breakdown extending no more than 1 mm beyond the maximum diameter that the iceball had attained. Histological examination demonstrated a sharp transition from frankly necrotic brain within the iceball to the normal cytoarchitecture of the surrounding neuropil. The safety and efficacy of a neurosurgical ablative procedure depends on the precision with which it can be generated. The use of CT imaging to monitor the formation of the cryosurgical iceball offers the neurosurgeon a means to precisely control the size of the ablative lesion. Small deeply situated brain tumors can be incorporated into the iceball under direct CT observation, thereby ensuring the completeness of the cryoablation while minimizing damage to the surrounding brain.     

Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing

This study was designed to asses murine preantral follicle survival and growth, after cryopreservation of ovarian tissue by two different methodologies, solid-surface vitrification by the Cryologic vitrification method (CVM) and slow-rate freezing (SRF). Cryotreated tissue was stored in liquid nitrogen for 24h, and upon warming follicle viability was assessed by live/dead fluorescent probes, and by 7-day autotransplantation of both cryotreated tissue types to the left and right kidney capsule of the donor animals (n=16). The live/dead assay immediately upon tissue warming did not allow a distinction to be made in terms of follicle viability between the CVM and SRF cryoprocedure. In grafted tissue, follicular survival and growth was assessed by conventional histological examination and proliferating cell nuclear antigen immunohistochemistry. In each experimental group (control, CVM and SRF), follicles were classified according to developmental stage, and a comparison of the proportions of follicle stages between the three groups was executed by statistical analysis of variance. The fraction of primordial follicles in CVM and SRF grafts significantly decreased as compared to control tissue, whereas intermediary and primary follicles significantly increased. The proportion of secondary and antral follicles after SRF was significantly larger than after CVM, but did not differ significantly between CVM and control tissue. The observed massive follicle activation is a typical transplantation effect, but testifies to the survival of cryopreserved follicles. In both types of cryotreated tissue, growing follicles, including antral stage, were present in grafts from all recipient animals. The significantly more abundant further developed stages in SRF treated tissue, however, suggest that CVM treated tissue may have suffered a growth disadvantage. To our knowledge, this is the first time that the CVM technique has been utilized to vitrify preantral follicles.