A parametric study of freezing injury in BPH1CAFTD-2 human prostate tumor cells.

Freeze injury in BPH1(CAFTD)-2 cells frozen/thawed in suspension was studied through a two-level four-parameter (2(4)) experimental design and analysis. The four parameters considered were end temperature, hold time, TNFalpha concentration, and thawing rate. Thermal parameter values chosen were based on the approximate thermal history cells would experience in the peripheral region of a cryosurgical iceball. Cell suspensions were frozen at a constant 10 degree C/min on a directional solidification stage and viability was assessed within 1 hr post-thaw using a dye exclusion assay. The parameters affecting cell survival were determined through calculation of the individual parameter effects (E) and interactions (I) according to factorial design guidelines; data set curvature (C) was also determined. Cell viability ranged from a maximum of 87.6 percent to a minimum of 17.6 percent indicating trends in cell survival were sensitive to the parameters chosen. Survival was affected by the following parameters in order: lowering the end temperature, increasing the hold time, adding TNFalpha, and reducing the thawing rate. In addition, all 2-way parameter interactions except TNFalpha hold time were statistically significant. Curvature analysis showed that cell viability at the midpoint of the data was nearly 20 percent lower than predicted based on linear interpolation. These results were verified and extended using analysis of variance (ANOVA). We conclude that cryoinjury in this tumor line can be influenced by multiple interacting thermal parameters, most importantly end-temperature and hold time, as well as the presence of the cytokine TNFalpha. Finally, although the cell type is tumorigenic results suggest the possibility of using freezing to control benign prostatic hyperplasia (BPH) in addition to cancer within the prostate.

Acute effect of vasectomy on the function of the rat epididymal epithelium and vas deferens.

Persistent infertility after apparently successful vasectomy reversal is common. One possible etiology is epididymal epithelial dysfunction resulting in improper sperm maturation after vasectomy reversal. The epididymal epithelium secretes a number of proteins that are thought to be required for the maturation of sperm. Ligation of the vas deferens during vasectomy may affect the synthesis of some of these proteins. In the present study, the function of the epididymal epithelium was assessed at early times after vasectomy (1, 4, and 7 days) by measuring the level of mRNA of 4 secreted proteins: Crisp-1, clusterin, osteopontin, and transferrin. In addition, the site of synthesis of these proteins was determined by immunocytochemistry. The results demonstrated that the expression of Crisp-1 and clusterin, representative epididymal secretory proteins, was largely unaffected by vasectomy. However, osteopontin mRNA increased in the vas deferens in response to vasectomy. Immunocytochemical localization of osteopontin suggested that both infiltrating immune cells and deferential luminal epithelium were responsible for this up-regulation. Transferrin expression was viewed as a marker for immune cells at the site of injury. However, both the caput epididymis and deferential epithelia were found to express transferrin, in addition to immune cells. In conclusion, there appear to be only minor changes in expression of genes encoding epididymal secretory proteins acutely after vasectomy, but, not surprisingly, there was evidence of an inflammatory response after vasectomy.

Assessing pH and oxygenation in cryotherapy-induced cytotoxicity and tissue response to freezing.

The microenvironmental pH and oxygenation is known to influence tumor cell response to heat, radiation, photodynamic and even chemotherapy. We have studied the previously untested influence of acidity and hypoxia on tumor and endothelial cell sensitivity to freezing. In addition, we have measured changes in oxygenation in vivo in murine FSaII fibrosarcomas after freeze injury. A low pH or low oxygenation environment was found to increase the sensitivity of tumor and endothelial cells to freezing at -20 degrees C or -40 degrees C in vitro. However, low pH and low oxygenation combined did not further increase cryosensitivity of the cells. In vivo, tumor oxygenation after freeze injury was studied immediately or 1-3 days after a standard freezing protocol was applied to FSaII tumors ranging from 250-500 mm3 grown in the rear-limb of C3H mice. Tumor oxygenation at the edge of the iceball was found to transiently increase 1-2 hours after freezing. At 1-3 days after freezing, a treatment that delayed FSaII tumor growth by approximately 1.5-fold, the mean tumor oxygenation was significantly increased by up to 2.5-fold from a control level of 5 mmHg partial pressure of oxygen (pO2), especially at the periphery of the tumor. We conclude that manipulation of pH or oxygenation has potential to increase the anti-tumor effects of minimally invasive cryosurgical techniques. Furthermore, the dynamic changes in oxygenation after freeze injury in vivo suggests value in combining cryotherapy with treatments dependent on oxygenation levels. Ultimately, these may be routes to more reliable treatment response with fewer recurrences.

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 alpha helix to extended beta 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 (approximately 41 degrees C at 2 degrees C min(-1) and approximately 44 degrees C at 5 degrees 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 approximately 110 kJ mol(-1) between 44 and 90 degrees C to approximately 750 kJ mol(-1) between 44 and 50 degrees C). However, its dependence on heating rate is negligible. Several denaturation peaks, including a dominant one between approximately 62 and 65 degrees 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.

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 cryoprobe's 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.

Sensitization of thermotolerant SCK cells to hyperthermia and freezing with reduction of intracellular pH: implications for cryosurgery.

BACKGROUND AND OBJECTIVES: During cryosurgery, cells frozen slowly at the outer part of the ice ball undergo severe dehydration and are subject to solute effects injury, which may be caused in part by protein denaturation. This study was undertaken to determine whether heat shock proteins (HSPs), the molecular chaperones that stabilize proteins against denaturation, have a protective effect on cells during slow freezing. In addition, we aimed to determine whether acidic conditions, similar to those found in many solid tumors, would effect this protection. METHODS: SCK cells were frozen at 5 degrees C/min to -10 degrees C or -20 degrees C before or after induction of thermotolerance, and at neutral or low pH conditions. Lethal damage was determined by clonogenics. RESULTS: Clonogenic survival was decreased by 50% in thermotolerant cells frozen to -10 degrees C after culture in acidic conditions (pH 6.6) compared with non-thermotolerant cells cultured at neutral pH. Induction of thermotolerance alone or low pH alone did not significantly sensitize SCK cells to freezing. All treatment groups were equally susceptible to killing when frozen to -20 degrees C. CONCLUSIONS: Our results show that induction of thermal tolerance does not protect SCK cells against subsequent freezing injury and that a low pH environment actually sensitizes these cells to freeze injury.

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.

Microvascular blood flow and stasis in transgenic sickle mice: utility of a dorsal skin fold chamber for intravital microscopy.

Vascular inflammation, secondary to ischemia-reperfusion injury, may play an essential role in vaso-occlusion in sickle cell disease (SCD). To investigate this hypothesis, dorsal skin fold chambers (DSFCs) were implanted on normal and transgenic sickle mice expressing human alpha and beta(s)/beta(s-Antilles) globin chains. Microvessels in the DSFC were visualized by intravital microscopy at baseline in ambient air and after exposure to hypoxia-reoxygenation. The mean venule diameter decreased 9% (P < 0.01) in sickle mice after hypoxia-reoxygenation but remained constant in normal mice. The mean RBC velocity and wall shear rate decreased 55% (P < 0.001) in sickle but not normal mice after hypoxia-reoxygenation. None of the venules in normal mice became static at any time during hypoxia-reoxygenation; however, after 1 hr of hypoxia and 1 hr of reoxygenation, 11.9% of the venules in sickle mice became static (P < 0.001). After 1 hr of hypoxia and 4 hr of reoxygenation, most of the stasis had resolved; only 3.6% of the subcutaneous venules in sickle mice remained static (P = 0.01). All of the venules were flowing again after 24 hr of reoxygenation. Vascular stasis could not be induced in the subcutaneous venules of sickle mice by tumor necrosis factor alpha (TNF-alpha). Leukocyte rolling flux and firm adhesion, manifestations of vascular inflammation, were significantly higher at baseline in sickle mice compared to normal (P < 0.01) and increased 3-fold in sickle (P < 0.01), but not in normal mice, after hypoxia-reoxygenation. Plugs of adherent leukocytes were seen at bifurcations at the beginning of static venules. Misshapen RBCs were also seen in subcutaneous venules.