Experimental measurements and theoretical calculations on muscle and liver tissue during cryosurgery. I. Experiments on freezing of rabbit tissue.

Experiments on freezing of muscle and liver tissue of 113 rabbits were performed. The diameter on the frozen surface, and the thickness and the mass of the iceball were measured for the live and dead, body-temperature, animal. Four continuously cooled and six massive probes (2.5–20-mm diameter) were used with liquid nitrogen as the cooling agent. The following conclusions can be drawn: (1) with use of round probe-tips, the iceball has approximately spherical symmetry. However, the depth of the frozen tissue is about 15% smaller than the lateral extension on the visible surface. (2) For continuously cooled probes the diameter of the iceball in the steady state is about five times as large as the probe diameter. The maximal iceball diameter for massive probes is two times larger than the probe diameter. (3) The different blood circulation of liver and muscle tissue has an influence of only 10% on the size of the iceball. For clinical applications this difference is of little importance. (4) For live tissue the iceball is about 15% smaller than for body-temperature dead tissue. Thus, the main heat-transport process in tissue is heat condition.     

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)).     

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.     

Long-term skin preservation by combined use of tissue culture and freezing techniques.

Simple but effective methods for shipping newly excised rabbit skin to a distant central laboratory for in vitro culture on a pigskin base, followed by freezing in a cryoprotective agent (DMSO) for frozen storage and subsequent reshipment to the originating laboratory while still frozen are described. At a suitable time the frozen tissue was rapidly thawed and transplanted to the autologous recipient rabbit. Of 12 cultures, seven indicated good to excellent cell growth and maturation on the host. The successful method combined the use of in vitro tissue culture and freezing to permit a central laboratory to grow the skin and ship it to the originating center for autografting at a convenient time.     

Effect of cold acclimation on bulk tissue electrical impedance: I. Measurements with birdsfoot trefoil at subfreezing temperatures

The resistive and reactive components of electrical impedance were measured for birdsfoot trefoil (Lotus corniculatus L.) stems at freezing temperatures to −8°C. As temperature decreased the specific resistance at frequencies between 49 hertz and 1.11 megahertz of stems from cold acclimated plants increased more rapidly than from nonacclimated plants. This temperature dependence of specific resistance could be characterized by an Arrhenius activation energy; cold acclimated stems had a larger Arrhenius activation energy than nonacclimated stems. The low frequency resistance is believed to characterize the extracellular region of the stems and the high frequency resistance is believed to characterize the intracellular region of the stems. Cold acclimation increased the intracellular but not the extracellular resistance at nonfreezing temperatures. Cold acclimated stems were not injured by freezing to −8°C and thawing, but nonacclimated stems were injured by freezing to temperatures between −2.2 and −5.6°C and thawing. Injury to nonacclimated stems at freezing temperatures below −2.2°C was indicated by a decrease in the ratio of resistance at 49 Hz to that at 1.11 megahertz.     

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.     

The cryopreservation of Chlamydomonas.

A cryophilic strain of the unicellular green alga Chlamydomonas, C. nivalis was found to be more resistant to the stresses both of freezing and thawing and of shrinkage and rehydration than was a mesophilic strain C. reinhardii. C. nivalis was found to have a higher degree of unsaturation of phospholipid fatty acids. Following freezing and thawing of C. reinhardii there was a direct correlation between reduction in cell viability and loss of membrane selective permeability. Activation of intracellular phospholipases occurred at an early stage of freezing injury. Attempts to cold harden C. reinhardii were unsuccessful. For C. reinhardii methanol was the only effective cryoprotectant for freezing to and thawing from ?196 °C and the effects of cooling rate upon cellular survival are presented.     

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.     

Slow freezing versus vitrification technique for human ovarian tissue cryopreservation: An evaluation of histological changes,WNT signaling pathway and apoptotic genes expression

This study compared slow freezing and vitrification of ovarian tissue by evaluation of histological changes, WNT signaling pathway and apoptotic genes expression. Ovarian tissue was obtained from women aging 27–38 years old. Ovarian cortex from each patient was divided into three pieces and randomly grouped as slow freezing, vitrification and control groups for investigation of WNT signaling gene expression and β-CATENIN presence as well as histological studies. The stromal structure of all ovaries were preserved. The number of secondary follicles decreased in vitrified group (P < 0.05). WNT-3, β-CATENIN, FZD-2 and GSK-3β expressions were significantly higher in slow frozen and vitrified groups, compared to control group (P < 0.05). On the contrary, AXIN1 expression in slow frozen samples were significantly lower than that of the vitrified and control group. The expression of apoptotic genes, excluding CASP3, was significantly decreased in slow-frozen samples (P < 0.05). Conversely, BAX:BCL-2 percentage significantly increased in vitrification versus slow freezing and control(P < 0.05). Follicles in slow frozen samples displayed nuclear and cytoplasmic β-CATENIN staining, while control and vitrification groups only showed β-CATENIN protein in the cytoplasm. The presented data show that slow freezing results in a better preservation regardless of the type of follicle. Therefore, it is concluded that slow freezing is still an ideal method for ovary cryopreservation.     

Influence of cold acclimation on membrane injury in frozen plant tissue

Cold-acclimated twigs of Amelanchier alnifolia Nutt. released less HCN at −4.5 C than nonacclimated twigs following slow freezing to −25 C or rapid freezing to −78 C. Cold-acclimated twigs frozen slowly to −25 C released more HCN than cold-acclimated twigs frozen only to −4.5 C. Cold-acclimated twigs frozen slowly to −25 C and then rapidly to −78 C released less HCN at −4.5 C than cold-acclimated twigs frozen rapidly to −78 C. In general, K⁺ efflux and the inability to reduce triphenyl tetrazolium chloride following freezing and thawing paralleled HCN release at −4.5 C. Because low K⁺ efflux and high triphenyl tetrazolium chloride reduction are known to depend upon membrane integrity, the increased K⁺ efflux and the decreased triphenyl tetrazolium chloride reduction following freezing and thawing provide indirect evidence that HCN release at −4.5 C is a measure of membrane damage in frozen cells.     

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.     

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%.     

Evaluation of a freeze-clamping technique designed for two- and three-dimensional metabolic studies of rat liver in vivo. Quenching efficiency and effect of clamping on tissue morphology

In order to apply a previously described freeze-clamping technique (B. Quistorff and B. Chance, 1980, Anal. Biochem.108, 237–248) in three-dimensional metabolic studies, it is necessary to clarify to which extent tissue morphology as well as metabolic state is preserved in the part of the freeze-clamped sample used for such studies. The present paper reports a comparative histological examination of rat liver, either freeze-clamped, applying the technique mentioned, or frozen without compression. It is demonstrated that neither the intra- nor the interlobular morphology of the central part of the sample is disturbed by the freeze-clamping process. In clamped as well as unclamped liver samples portal tracts could be identified, i.e., distinguished from efferent veins, in a depth below the capsule of 100–200 μm. Measurements of ATP, ADP, AMP, and P_i in the freeze-clamped sample at increasing distance from the surface indicate that for in situ freezing, significant metabolic changes did not occur until a depth of about 1 mm, while a delay of freezing of about 6 s seems to cause metabolic changes in the entire sample.     

Cryopreservation of human ovarian tissue: Comparison of rapid and conventional freezing

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (1 × 1–1.5 × 0.7–1 mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

Osmotic shrinkage as a factor in freezing injury in plant tissue cultures

Haplopappus gracilis and Acer saccharum tissue culture cells are extremely sensitive to freezing injury, and exhibit a decrease in survival from 98% at −1 C to 4% at −3 C (Haplopappus) and 92% at −3 C to 13% at −5 C (Acer) when suspended in distilled H₂O, seeded at −1 C, and then cooled by 0.1 C/minute. Similar results are obtained when cells are suspended in growth medium. The extent of shrinkage of cells during freezing can be duplicated by exposure of the cells to plasmolyzing solutions of nonpenetrating substances (Δ T_f = 1.86 vm). Solutions of sucrose and glycerol that produce extensive plasmolysis cause a decrease in survival within 3 to 5 minutes at room temperature, and the higher the molality to which the cell is exposed the greater the injury. Also, the rate of rehydration of the plasmolyzed cell and of the frozen cell affects its survival, with the slower rate being more beneficial. The close correlation between the decrease in survival at subzero temperatures and the decrease in survival when cells are placed in solutions having osmolalities, which could produce the same extent of shrinkage as these killing temperatures, suggests that this shrinkage is related to freezing injury in tissue culture cells.     

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.     

Intracellular freezing: effect of extracellular supercooling.

Human erythrocytes were frozen on the stage of a cryomicroscope at accurately controlled constant-cooling rates with varying degrees of extracellular supercooling. The formation of intracellular ice was detected by direct observation of the frozen cells through the microscope. A significant coupling effect was determined between the minimum cooling rate necessary to produce intracellular freezing and the extent of supercooling. Increased degrees of extracellular supercooling reduced the range of cooling rates for which water would freeze within the cell. Specific data points were obtained at ΔT_SC = 0, ?5, and ?12 °C for which the corresponding transition cooling rates were respectively ?845, ?800, and ?11 °C/min.An explanation for the occurrence of this phenomenon is presented based on the physiochemical processes that govern the freezing of a cell suspension.     

Comparative study of freezing time and temperature for effective Plodia interpunctella control using typical storage containers

Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) is a moth species that is able to feed on various vegetable commodities. Its control is economically critical for commercial food storing facilities such as warehouses. P. interpunctella causes quantitative and qualitative damage by eating important stored food crops such as dried Welsh onions (Allium fistulosum L.), and freezing treatment is a common method of control. To examine the effectiveness of freezing treatment, we changed the length of time of the conventional freezing method. The conventional method involves treatment below ?15 °C for 48 h, but we predicted that it would be effective with only 24 h freezing at ?25 °C. To test our theory, we conducted an experiment using three different frozen storage containers and assessed if the modified method was effective on the eggs and fourth instar larvae of P. interpunctella. Despite the temporary malfunctioning of one of the containers used in the experiment after incubation at 28 °C and 70% relative humidity for 10 days, the larval mortality rate was 100% and egg hatching rate was 0% in all samples, regardless of the treatment time. Further research is needed as this method is expected to decrease production costs and energy consumption and has the potential to be applied to other crops and pests.