David J. Swanlund

Investigation of the thermal and tissue injury behaviour in microwave thermal therapy using a porcine kidney model

Minimally invasive microwave thermal therapies are being developed for the treatment of small renal cell carcinomas (RCC, d<3 cm). This study assessed the thermal history and corresponding tissue injury patterns resulting from microwave treatment of the porcine renal cortex. Three groups of kidneys were evaluated: (1) in vitro treated, (2) in vivo with 2-h post-treatment perfusion (acute) and (3) in vivo with 7-day post-treatment perfusion (chronic). The kidneys were treated with an interstitial water-cooled microwave probe (Urologix, Plymouth, MN) that created a lesion centered in the renal cortex (50 W for 10 min). The thermal histories were recorded at 0.5 cm radial intervals from the probe axis for correlation with the histologic cellular and vascular injury. The kidneys showed a reproducible 2 cm chronic lesion with distinct histologic injury zones identified. The thermal histories at the edge of these zones were found using Lagrangian interpolation. The threshold thermal histories for microvascular injury and stasis appeared to be lower than that for renal epithelial cell injury. The Arrhenius kinetic injury models were fit to the thermal histories and injury data to determine the kinetic parameters (i.e. activation energy and frequency factor) for the thermal injury processes. The resultant activation energies are consistent in magnitude with those for thermally induced protein denaturation. A 3-D finite element thermal model based on the Pennes bioheat equation was developed and solved using ANSYS (V7.0). The real geometry of the kidneys studied and temperature dependent thermal properties were used in this model. The specific absorption rate (SAR) of the microwave probe required for the thermal modelling was experimentally determined. The results from the thermal modelling suggest that the complicated change of local renal blood perfusion with temperature and time during microwave thermal therapy can be predicted, although a first order kinetic model may be insufficient to capture blood flow changes. The local blood perfusion was found to be a complicated function of temperature and time. A non-linear model based on the degree of vascular stasis was introduced to predict the blood perfusion. In conclusion, interstitial microwave thermal therapy in the normal porcine kidney results in predictable thermal and tissue injury behaviour. Future work in human kidney tissue will be necessary to confirm the clinical significance of these results.

Cryopreservation of Equine Sperm: Optimal Cooling Rates in the Presence and Absence of Cryoprotective Agents Determined Using Differential Scanning Calorimetry

Abstract Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5°C/min and 20°C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 μm and a radius of 0.66 μm with an osmotically inactive cell volume (Vb) of 0.6Vo, where Vo is the isotonic cell volume. Sperm samples were collected using water-insoluble Vaseline in the artificial vagina and slow cooled at ≤0.3°C/min in an Equitainer-I from 37°C to 4°C. By fitting a model of water transport to the experimentally obtained DSC volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined best-fit parameters of water transport (at both 5°C/min and 20°C/min) in Kenney extender (absence of CPAs) are Lpg = 0.02 μm min−1 atm−1 and ELp = 32.7 kcal/mol with a goodness-of-fit parameter R2 = 0.96, and the best-fit parameters in the lactose-EDTA extender (the CPA medium) are Lpg[cpa] = 0.008 μm min−1 atm−1 and ELp[cpa] = 12.1 kcal/mol with R2 = 0.97. These parameters suggest that the optimal cooling rate for equine sperm is ∼29°C/min and is ∼60°C/min in the Kenney extender and in the lactose-EDTA extender. These rates are predicted assuming no intracellular ice formation occurs and that the ∼5% of initial osmotically active water volume trapped inside the cells at −30°C will form innocuous ice on further cooling. Numerical simulations also showed that in the lactose-EDTA extender, equine sperm trap ∼3.4% and ∼7.1% of the intracellular water when cooled at 20°C/min and 100°C/min, respectively. As an independent test of this prediction, the percentage of viable equine sperm was obtained after freezing at 6 different cooling rates (2°C/min, 20°C/min, 50°C/min, 70°C/min, 130°C/min, and 200°C/min) to −80°C in the CPA medium. Sperm viability was essentially constant between 20°C/min and 130°C/min.

Supraphysiological Thermal Injury in Dunning AT-1 Prostate Tumor Cells

To investigate the potential application of thermal therapy in the treatment of prostate cancer, the effects of supraphysiological temperatures (40-70 degrees C) for clinically relevant time periods (approximately 15 minutes) were experimentally studied on attached Dunning AT-1 rat prostate cancer cells using multiple assays. The membrane and reproductive machinery were the targets of injury selected for this study. In order to assess membrane injury, the leakage of calcein was measured dynamically, and the uptake of PI was measured postheating (1-3 hours). Clonogenicity was used as a measure of injury to the reproductive machinery 7 days post-injury after comparable thermal insults. Experimental results from all three assays show a broad trend of increasing injury with an increase in temperature and time of insult. Membrane injury, as measured by the fluorescent dye assays, does not correlate with clonogenic survival for many of the thermal histories investigated. In particular, the calcein assay at temperatures of < or = 40 degrees C led to measurable injury accumulation (dye leakage), which was considered sublethal, as shown by significant survival for comparable insult in the clonogenic assay. Additionally, the PI uptake assay used to measure injury post-thermal insult shows that membrane injury continues to accumulate after thermal insult at temperatures > or = 50 degrees C and may not always correlate with clonogenicity at hyperthermic temperatures such as 45 degrees C. Last, although the clonogenic assay yields the most accurate cell survival data, it is difficult to acquire these data at temperatures > or = 50 degrees C because the thermal transients in the experimental setup are significant as compared to the time scale of the experiment. To improve prediction and understanding of thermal injury in this prostate cancer cell line, a first-order rate process model of injury accumulation (the Arrhenius model) was fit to the experimental results. The activation energy (E) obtained using the Arrhenius model for an injury criterion of 30 percent for all three assays revealed that the mechanism of thermal injury measured is likely different for each of the three assays: clonogenics (526.39 kJ/mole), PI (244.8 kJ/mole), and calcein (81.33 kJ/mole). Moreover, the sensitivity of the rate of injury accumulation (d omega/dt) to temperature was highest for the clonogenic assay, lowest for calcein leakage, and intermediate for PI uptake, indicating the strong influence of E value on d omega/dt. Since the clonogenic assay is linked to the ultimate survival of the cell and accounts for all lethal mechanisms of cellular injury, the E and A values obtained from clonogenic study are the best values to apply to predict thermal injury in cells. For higher temperatures (> or = 50 degrees C) indicative of thermal therapies, the results of PI uptake can be used as a conservative estimate of cell death (underprediction). This is useful until better experimental protocols are available to account for thermal transients at high temperature to assess clonogenic ability. These results provide further insights into the mechanisms of thermal injury in single cell systems and may be useful for designing optimal protocols for clinical thermal therapy.

Cryosurgical changes in the porcine kidney: histologic analysis with thermal history correlation

Advances in minimally invasive renal cryosurgery have renewed interest in the relative contributions of direct cryothermic and secondary vascular injury-associated ischemic cell injury. Prior studies have evaluated renal cryolesions seven or more days post-ablation and postulated that vascular injury is the primary cell injury mechanism; however, the contributions of direct versus secondary cell injury are not morphologically distinguishable during the healing/repair stage of a cryolesion. While more optimal to evaluate this issue, minimal acute (< or = 3 days) post-ablation histologic data with thermal history correlation exists. This study evaluates three groups of porcine renal cryolesions: Group (1) in vitro non-perfused (n = 5); Group (2) in vivo 2-h post-ablation perfused (n = 5); and Group (3) in vivo 3-day post-ablation perfused (n = 6). The 3.4 mm argon-cooled cryoprobes thermal history included a 75 degrees C/min cooling rate, -130 degrees C end temperature, 60 degrees C/min thawing rate, and 15-min freeze time. An enthalpy-based mathematical model with a 2-D transient axisymmetric numerical solution with blood flow consideration was used to determine the thermal history within the ice ball. All three groups of cryolesions showed histologically similar central regions of complete cell death (CD) and transition zones of incomplete cell death (TZ). The CD had radii of 1.4, 1.1, and 1.0 cm in the non-perfused, 2-h and 3-day lesions, respectively. Capillary thrombosis was present in the 2-h perfused cryolesions with the addition of TZ arteriolar/venous thrombosis in the 3-day perfused lesions. Thermal modeling revealed the outer CD boundary in all three groups experienced similar thermal histories with an approximately -20 degrees C end temperature and 2 degrees C/min cooling and thawing rates. The presence of similar CD histology and in vitro/in vivo thermal histories in each group suggests that direct cryothermic cell injury, prior to or synchronous with vascular thrombosis, is a primary mediator of cell death in renal cryolesions.

TNF-α–based accentuation in cryoinjury—dose, delivery, and response

Cryosurgery is a minimally invasive cancer treatment using cryogenic temperatures. Intraoperative monitoring of iceball growth is an advantage of the treatment. However, whereas the iceball can be easily visualized, destruction within the iceball is incomplete and the means to monitor the “kill zone” are urgently needed. Recently, we have shown the ability of tumor necrosis factor-α (TNF-α) to enhance destruction within an iceball. To avoid systemic toxicity, we delivered TNF-α selectively to the tumor by a gold nanoparticle of 30-nm diameter (CYT-6091) tagged with TNF-α and thiol-derivatized polyethylene glycol. Using a dorsal skin fold chamber (DSFC) in a nude mouse, both normal skin and human prostate carcinoma (LNCaP Pro 5) were pretreated with soluble TNF-α (topically or i.v.) or CYT-6091 (i.v.) and frozen after 4 h. The cryolesion was assessed after 3 days by comparing histologic necrosis with perfusion defects. Hind limb tumors were also treated by visibly encompassing the tumor with an iceball and assessing gross changes over time. A 5-μg dose of soluble TNF-α or CYT-6091 increased the temperature threshold of necrosis in the tumor in the DSFC from −14.0 ± 1.6°C (n = 6) to 0.9 ± 1.5°C (n = 6) and −1.5 ± 3.7°C (n = 6), respectively. In hind limb tumors, the same dose resulted in significant tumor shrinkage and remission in 2 of 8 (for soluble TNF-α) and in 3 of 8 (for CYT-6091). The nanoparticle alone group without TNF-α increased the temperature threshold of necrosis to −7.0 ± 2.3°C in the tumor in the DSFC and more shrinkage of the tumor in the hind limb when compared with cryo alone treatment. Systemic toxicity was noted in all soluble TNF-α groups but none with CYT-6091. These results suggest that it is possible to destroy all of a tumor within an iceball by preincubation with TNF-α and systemic toxicity can be avoided by CYT-6091. [Mol Cancer Ther 2007;6(7):2039–47]

In Situ Thermal Denaturation of Proteins in Dunning AT-1 Prostate Cancer Cells: Implication for Hyperthermic Cell Injury

The in situ thermal protein denaturation and its correlation with direct hyperthermic cell injury in Dunning AT-1 prostate tumor cells were investigated in this study. The in situ thermal protein denaturation was studied using both Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The FTIR spectra at different temperatures show changes in protein secondary structure (from α helix to extended β sheet) during in situ thermal protein denaturation within AT-1 cells. Calorimetric studies using DSC show that endothermic heat release is associated with the in situ thermal protein denaturation. Furthermore, both the secondary structure changes detected by FTIR and the calorimetric changes detected by DSC were quantified and the kinetics of the overall in situ thermal protein denaturation was derived under different heating conditions. The onset temperature where the overall in situ thermal protein denaturation is first detectable was found to be scanning rate dependent (∼41°C at 2°C min−1 and ∼44°C at 5°C min−1). The kinetics of the overall in situ thermal protein denaturation was derived from both DSC and FTIR measurements and was fit using kinetic and statistical models. The kinetic data determined by FTIR and DSC under the same heating conditions match well with each other. The activation energy of the overall in situ thermal protein denaturation is found to be strongly dependent on the temperature range considered (the activation energy ranges from ∼110 kJ mol−1 between 44 and 90°C to ∼750 kJ mol−1 between 44 and 50°C). However, its dependence on heating rate is negligible. Several denaturation peaks, including a dominant one between ∼62 and 65°C, are identifiable from both the DSC and the FTIR results. To investigate directly the relationship between thermally induced cell injury and the in situ thermal protein denaturation, both acute (propidium iodide dye exclusion, assessed 3-h postthermal treatment) and chronic (clonogenics, assessed 7-day postthermal treatment) cell injury were quantified using AT-1 cells prepared under the same conditions as for the DSC protein studies. Comparisons of the results from the cell injury studies and the DSC protein denaturation studies show that the overall in situ thermal protein denaturation correlates well with both the acute and the chronic cell injury, which suggests that overall in situ thermal protein denaturation is an important mechanism of direct hyperthermic cell injury in AT-1 cells at the macromolecular level.

In vitro thermal therapy of AT-1 Dunning prostate tumours

To advance the utility of prostate thermal therapy, this study investigated the thermal thresholds (temperature-time) for prostate tissue destructionin vitro. The AT-1 Dunning prostate tumour model was chosen for the study. Three hundred micron thick sections were subjected to controlled temperature-time heating, which ranged from low (40°C, 15 min) to high thermal exposures (70°C, 2 min) (n = 6). After subsequent tissue culture at 37°C, the sections were evaluated for tissue injury at 3, 24 and 72 h by two independent methods: histology and dye uptake. A graded increase in injury was identified between the low and high thermal exposures. Maximum histologic injury occurred above 70°C, 1 min with >95% of the tissue area undergoing significant cell injury and coagulative necrosis. The control and 40°C, 15 min sections showed histologic evidence of apoptosis following 24 and 72 h in culture. Similar signs of apoptosis were minimal or absent at higher thermal histories. Vital-dye uptake quantitatively confirmed complete cell death after 70°C, 2 min. Using the dye data, Arrhenius analysis showed an apparent breakpoint at 50°C, with activation energies of 135.8 kcal/mole below and 4.7 kcal/mole above the threshold after 3 h in culture. These results can be used as a conservative benchmark for thermal injury in the cancerous prostate. Further characterization of the response to thermal therapy in an animal model and in human tissues will be important in establishing the efficacy of the procedure

Evaluation of thermal therapy in a prostate cancer model using a wet electrode radiofrequency probe.

PURPOSE To determine the temperature-time threshold of local cell death in vivo for thermal therapy in a prostate cancer animal model and to use this value as a benchmark to quantify global tissue injury. MATERIALS AND METHODS Two studies were designed in the Dunning AT-1 rat prostate tumor hind limb model. For both studies, a wet electrode radiofrequency (RF) probe was used to deliver 40 W of energy for 18 to 62 seconds after a 30-second infusion of hypertonic saline/Hypaque through the RF antenna. Thermal history measurements were obtained in tumors from at least two Fluoroptic probes placed radially 5 mm from the axis of a RF probe and 10 mm below the surface of the tissue. In study 1, the thermal history required for irreversible cell injury was experimentally determined by comparing the predicted injury accumulation (omega) with cell viability at the fluoroptic probe locations using an in vivo-in vitro assay. The omega value was calculated from the measured thermal histories using an Arrhenius damage model. In study 2, RF energy was applied for 40 seconds in all cases. At 1, 3, and 7 days after thermal therapy, triphenyltetrazolium chloride dye (TTC) and histologic analyses were performed to assess global tissue injury within a 5-mm radius from the axis of the RF probe. RESULTS Study 1 showed that cell survival dropped to 0 for 0.42 < omega < 0.7. This result was the basis for selection of 40 seconds of RF thermal therapy in study 2, which yielded omegaave = 0.5 in the tissue 5 mm from the probe axis. Both TTC and histology analysis showed that sham-treated tissue was not irreversibly injured. However, there was an inherent heterogeneity present in the tumor that accounted for as much as 15% necrosis in control or sham-treated tissue. In contrast, at 1, 3, and 7 days after therapy, significantly less enzyme activity was observed by TCC in thermally treated tissue compared with sham-treated tissue (35 v 85%; P < 0.001). Histologic analysis of thermally treated tissues revealed a gradual increase in the percent of coagulative necrosis (47%-70%) with a concomitant decrease in the percentage of shocked cells (53%-28%). At day 7, <3% viability was observed in treated tumors compared with 90% viability in sham-treated tissue. CONCLUSION The threshold of cellular injury in vivo corresponded to omega > 0.7 (> or =48 degrees C for 40 seconds). Global tissue injury could be conservatively predicted on the basis of local thermal histories during therapy.

Measurement and Prediction of Thermal Behavior and Acute Assessment of Injury in a Pig Model of Renal Cryosurgery

PURPOSE To analyze in vivo end temperatures and histologic injury in a standardized cryo-iceball using a porcine kidney model in order to establish the threshold temperature for tissue ablation. To evaluate the ability to predict end temperatures using a thermal finite element model. MATERIALS AND METHODS A single freeze/thaw cryolesion was created in five pig kidneys and the temperature history recorded. End temperature was calculated using a thermal finite element model. The threshold temperature for tissue injury was established by directly correlating end temperature and histologic injury. RESULTS Reproducible geometry and temperature profiles of the cryo-iceball were found. End temperature could be accurately predicted through thermal modeling, and correlation with histologic injury revealed a threshold temperature of -16.1 degrees C for complete tissue ablation. CONCLUSION Thermal modeling may accurately predict end temperature within a cryo-iceball. Provided threshold temperatures for tissue destruction are known, modeling may become a powerful tool in cryosurgery, improving the assessment of damage in normal and malignant tissue.

Cryothermic and Hyperthermic Treatments of Human Leiomyomata and Adjacent Myometrium and Their Implications for Laparoscopic Surgery

STUDY OBJECTIVE To evaluate the effects and feasibility of direct cryothermic and hyperthermic therapy on leiomyomata and adjacent myometrium, and to contribute to evidence-based treatment thresholds based on measurements of direct cell injury. DESIGN Experimental study (Canadian Task Force classification II-2). SETTING University hospital. SUBJECTS Leiomyoma and myometrium tissue from 10 women undergoing total abdominal hysterectomy with or without bilateral salpingo-oophorectomy. INTERVENTION In vitro cryothermic or hyperthermic therapy was performed with representative leiomyoma and myometrium tissue samples. Using a directional solidification stage to simulate cryothermic therapy, 10 leiomyoma and 6 myometrium specimens were cooled in vitro at a rate of -5 degrees C/minute to end temperatures of -20 degrees, -40 degrees, -60 degrees, and -80 degrees C with a 15-minute hold period and then rapidly thawed to 21 degrees C. Hyperthermic therapy was simulated using a preheated 45 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees, and 80 degrees C constant temperature copper heating block with a 10-minute treatment period. In conjunction with tissue culturing and control tissues, cell death was assessed with routine histology and viability dyes (ethidium homodimer/Hoechst). MEASUREMENTS AND MAIN RESULTS In cryothermic results, leiomyomata cell death (LCD) increased from 12% to 27% by histology and 26% to 38% by viability dye assay over the thermal range from -20 degrees to -80 degrees C, respectively. Myometrial cell death (MCD) increased from 10% to 12% and 4% to 20% for the same measurements, respectively. Whereas MCD appeared relatively stable from -40 degrees to -80 degrees C, it was significantly less than LCD over this range (p <0.05). For hyperthermic results, LCD increased from 17% to 88% by histology with progressive temperature increase from 45 degrees to 80 degrees C, respectively. The MCD showed a similar increase from 16% to 91% by histology over this temperature range. Hyperthermic histology and dye assay results were similar for LCD and MCD. CONCLUSIONS In comparison with myometrium, leiomyomata showed greater direct cryothermic and equal hyperthermic cell injury. Whereas cell death increased up to 70 degrees C and down to -80 degrees C, the interval increases in cell injury diminished with more extreme temperatures. In vivo studies of combined direct and ischemic vascular injury thresholds have yet to be performed, but direct LCD matrixes determined in this study will help provide guidelines for minimally invasive surgical techniques for the treatment of leiomyomata.