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.     

Peripheral tissue freezing in cryosurgery

The recently formulated bioheat equation of Weinbaum and Jiji which accounts for the vascular ultrastructure and blood perfusion was applied to the freezing of peripheral tissue. Using quasi-steady approximation the temperature distribution in the two-phase tissue and the motion of the frozen front were determined. Results are in good agreement with Pennes' bioheat equation.     

Large volume freezing in experimental hepatic cryosurgery. Avoidance of bleeding in hepatic freezing by an improvement in the technique.

In experiments on the dog's liver, large volumes of tissue were frozen by pouring liquid nitrogen onto the liver surface. There were no complications and no death from the procedure. Safety was produced by covering the liver surface with one to three layers of dacron velour cloth impregnated with a water-soluble jelly. The protective action of the gel-cloth was due to splinting of the liver capsule and to a more uniform and slower rate of freezing. The tissue became necrotic and over several months was replaced by dense fibrous tissue and ultimately resorbed. Although bleeding has been prevented, the problem of limitation of depth penetration remains as a hindrance to utilization in the treatment of hepatic tumors.     

Numerical investigation of inspiratory airflow in a realistic model of the human tracheobronchial airways and a comparison with experimental results

In this article, the results of numerical simulations using computational fluid dynamics (CFD) and a comparison with experiments performed with phase Doppler anemometry are presented. The simulations and experiments were conducted in a realistic model of the human airways, which comprised the throat, trachea and tracheobronchial tree up to the fourth generation. A full inspiration/expiration breathing cycle was used with tidal volumes 0.5 and 1 L, which correspond to a sedentary regime and deep breath, respectively. The length of the entire breathing cycle was 4 s, with inspiration and expiration each lasting 2 s. As a boundary condition for the CFD simulations, experimentally obtained flow rate distribution in 10 terminal airways was used with zero pressure resistance at the throat inlet. CCM+ CFD code (Adapco) was used with an SST k-\(\upomega \) low-Reynolds Number RANS model. The total number of polyhedral control volumes was 2.6 million with a time step of 0.001 s. Comparisons were made at several points in eight cross sections selected according to experiments in the trachea and the left and right bronchi. The results agree well with experiments involving the oscillation (temporal relocation) of flow structures in the majority of the cross sections and individual local positions. Velocity field simulation in several cross sections shows a very unstable flow field, which originates in the tracheal laryngeal jet and propagates far downstream with the formation of separation zones in both left and right airways. The RANS simulation agrees with the experiments in almost all the cross sections and shows unstable local flow structures and a quantitatively acceptable solution for the time-averaged flow field.     

Current flow in skin frozen in experimental cryosurgery

Experiments were performed correlating tissue temperature and current flow during freezing canine skin. Current flow fell gradually to negligible levels as the tissue froze. Tissue temperature of ?31 °C correlated with current flow of 5 μA. Tissue temperature of ?55 °C correlated with zero current flow. The observations indicated that current flow was related to tissue temperature and that either or both could be used to predict tissue destruction in cryosurgery. However, tissue temperature measurement is the more accurate method of quantification.     

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.     

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.     

The process of freezing and the mechanism of damage during hepatic cryosurgery

Experiments were performed to correlate the structures of liver tissue frozen during cryosurgery, liver frozen at various constant cooling rates, and unfrozen, dried normal liver. The results show that during freezing of tissue ice forms and propagates along the vascular system, expanding during freezing at low cooling rates. This expansion occurs over most of the region frozen during cryosurgery and may be one of the mechanisms of damage to tissue during cryosurgery.     

Advanced hepatic tissue destruction in ablative cryosurgery: potentials of intermittent freezing and selective vascular inflow occlusion

Recent studies indicate that cryosurgery represents a promising approach to treat non-resectable liver tumors. To improve parenchymal tissue destruction, a variety of modifications of the freeze-thaw procedure have been suggested, including repetitive freezing and portal-triad cross-clamping. The aim of the present study was to analyze whether intermittent freezing by application of a double freeze-thaw procedure or selective vascular inflow occlusion are more effective than a single freeze-thaw cycle to achieve complete hepatic tissue destruction. Using a porcine model, intrahepatic cryolesions were induced by freezing the hepatic tissue for a total of 15 min (n=6, SF). Additional animals (n=6) underwent a double freeze-thaw cycle of 7.5 min each (DF). A third group of animals (n=6) was treated by a single 15-min freeze-thaw cycle during selective vascular inflow occlusion (VO-SF). Seven days after freezing, DF did not change the volume of the cryolesion (25.4+/-1.7 cm(3)) compared to SF (29.9+/-3.7 cm(3)), however, resulted in enhanced destruction of hepatocyte nuclear morphology (DF-score: 2.4+/-0.2 versus SF-score: 1.1+/-0.3; p<0.05) and attenuated leukocyte infiltration within the margin of the cryolesion (DF-score: 1.5+/-0.2 versus SF-score: 2.8+/-0.1; p<0.05). VO-SF was also effective to significantly enhance destruction of hepatocyte nuclear morphology (2.8+/-0.1; p<0.05 versus SF), but, additionally, markedly increased the volume of the cryolesions (43.3+/-5.3 cm(3); p<0.05 versus SF and DF). Interestingly, VO-SF further increased the number of apoptotic cells, while leukocyte infiltration (2.3+/-0.3) was not affected compared to that after SF-treatment. Thus, our data indicate that both DF and VO-SF are effective to enhance parenchymal cell destruction within the margin of the cryolesion. VO-SF additionally increases the volume of the lesion and may therefore be most attractive for successful clinical application.     

Stochastic finite element analysis of biological systems: comparison of a simple intervertebral disc model with experimental results

Statistical methods allow the effects of uncertainty to be incorporated into finite element models. This has potential benefits for the analysis of biological systems where natural variability can give rise to substantial uncertainty in both material and geometrical properties. In this study, a simple model of the intervertebral disc under compression was created and analysed as both a deterministic and a stochastic system. Factorial analysis was used to determine the important parameters to be included in the stochastic analysis. The predictions from the model were compared to experimental results from 21 sheep discs. The size and shape of the distribution of the axial deformations predicted by the model was consistent with the experimental results given that the number of model solutions far exceeded the number of experimental results. Stochastic models could be valuable in determining the range and most likely value of stress in a tissue or implant.     

The experimental study for efficacy and safety of pancreatic cryosurgery

Objective: This study was designed to basic information concerning the efficacy and safety of cryosurgery for pancreatic cancer. Fifteen healthy pigs were used to perform biochemical analysis and histological assessment. Methods: Following anesthesia and laparotomy, an argon–helium cryoprobe was inserted into the pancreas. The introduction of argon gas induced a rapid decrease in temperature to ?160 °C (Group I, 5 pigs) or ?110 °C (Group II, 5 pigs), respectively, resulting in ice-ball formation of 15–20 mm diameter after 5 min. Following freezing, helium gas was circulated in the probe tip to increase the temperature to 10–20 °C over 3 min to thaw. The freeze/thaw cycle was then repeated. Group III (3 pigs) had a cryoprobe inserted, but without freezing, and Group IV (2 pigs) included untreated or normal control animals. Levels of serum amylase (AMY), IL-6 and C-RP were measured prior to freezing and for 7 days following the procedure. All pigs were euthanized 7 days post-treatment and pancreases were examined histologically. Results: Neither hyperaemia, edema or hemorrhage were observed in the un-frozen parts of the pancreas. Histological assessment revealed a significant level of necrosis in the central and lateral regions of the tissue frozen within the ice-ball. All cellular ultrastructure was destroyed and only observable as a few of remaining nuclei with broken crests and degranulated mitochondria and rough endoplasmic reticulum. There was a significant increase of serum AMY levels for a brief period in both “deep frozen” and the “shallow frozen” groups. However, the AMY also increased in two pigs in the “normal control” group and one pig from the “inserted cryoprobe without freeze” control group. All experimental pigs appeared healthy until the sacrifice time. Conclusion: Cryosurgery is a safe and effective ablative procedure for pancreatic tissue resulting in minimal complications.     

Gastrointestinal absorption of americium-241 by orally exposed swine: comparison of experimental results with predictions of metabolic models

Two groups of 11-week-old swine (40 miniature and 40 domestic swine) received a single oral administration of 1.9 X 10(8) Bq (5.2 mCi) of 241Am citrate, and groups of eight animals, four of each type, were killed and sampled at 1, 2, 4, 8, 16, 24, 48, 72, and 96 h and 30 days later. Uptake and excretion patterns of the radioactivity appeared to occur in three phases: rapid uptake, rapid excretion, and then a slower excretion. All animals were systematically dissected, and the eviscerated carcasses were autoclaved for separation of bone and muscle. The predominant site of deposition was bone, and autoclaving had little effect on releasing 241Am from either bone or muscle. The maximum fractional gastrointestinal absorption of 1.1 X 10(-3) occurred 8 h after radionuclide administration. The tissue distribution data suggest partitions of 50, 20, and 30%, for bone, liver, and other soft tissues, respectively. Two metabolic models were evaluated: a modified Mewhinney-Griffith model and the ICRP 30 model to compare the biological data with model predictions. All models underestimated the actual early time data, but the fits to the experimental results were better at later times.     

VX hydrolysis by human serum paraoxonase 1: a comparison of experimental and computational results

Human Serum paraoxonase 1 (HuPON1) is an enzyme that has been shown to hydrolyze a variety of chemicals including the nerve agent VX. While wildtype HuPON1 does not exhibit sufficient activity against VX to be used as an in vivo countermeasure, it has been suggested that increasing HuPON1's organophosphorous hydrolase activity by one or two orders of magnitude would make the enzyme suitable for this purpose. The binding interaction between HuPON1 and VX has recently been modeled, but the mechanism for VX hydrolysis is still unknown. In this study, we created a transition state model for VX hydrolysis (VX(ts)) in water using quantum mechanical/molecular mechanical simulations, and docked the transition state model to 22 experimentally characterized HuPON1 variants using AutoDock Vina. The HuPON1-VX(ts) complexes were grouped by reaction mechanism using a novel clustering procedure. The average Vina interaction energies for different clusters were compared to the experimentally determined activities of HuPON1 variants to determine which computational procedures best predict how well HuPON1 variants will hydrolyze VX. The analysis showed that only conformations which have the attacking hydroxyl group of VX(ts) coordinated by the sidechain oxygen of D269 have a significant correlation with experimental results. The results from this study can be used for further characterization of how HuPON1 hydrolyzes VX and design of HuPON1 variants with increased activity against VX.     

Biomechanical analysis of pedalling for rehabilitation purposes: experimental results on two pathological subjects and comparison with non-pathological findings

In this paper the experimental results obtained by means of a prototype measuring device dedicated to the evaluation of the rehabilitation level of the lower limb are presented. The analysis of the experimental data collected on non-pathological subjects allows the identification of the characteristic meaning of the most significant parameters typical of healthy subjects. These data have been employed for a systematic comparison with the same parameters measured on two pathological subjects, in order to define quantitative indicators of the rehabilitation degree of the lower limbs and indicators of the "quality" of the movement.