Ultrastructural changes in the rat liver during Euro-Collins storage, compared with hypothermic in vitro ischemia

The ultrastructural alterations in liver tissue induced by in vitro ischemia at 4 degrees C under conditions commonly used for transplantation (Euro-Collins perfused and stored liver tissue) have been compared with changes due to hypothermic in vitro ischemia in non-perfused liver. It was found that the process of cell deterioration in non-perfused liver occurred very slowly; signs of irreversible damage appeared in sinusoidal lining cells before hepatocytes (after 24 and 96 h, respectively). Liver perfused with, and stored in Euro-Collins solution showed acceleration of the ischemical damage in both types of cell (irreversible damage to sinusoidal lining cells after 12 h and to hepatocytes after 52 h), compared with non-perfused liver. These findings indicate that the safe period for storage of rat liver in Euro-Collins before damage to the microcirculatory system is less than 12 h. It might also be questioned whether Euro-Collins treatment is the optimal procedure for tissue preservation before liver transplantation.     

Direct effects of altered temperature on renal structure and function

Although marked alterations in temperature often accompany ischemic, acute renal failure (ARF), the effects of altered temperature on renal structure and function have received little attention. In the present investigation, isolated rat kidneys perfused at 41 degrees C had extensive tubular damage and decreased function compared to kidneys perfused at 37 degrees C. In contrast, kidneys perfused at 30 degrees C had less tubular damage, and better function, than kidneys perfused at 37 degrees C. Increased temperature caused a 50% reduction in renal ATP (0.46 +/- 0.04 microM/100 mg tissue protein. 37 degrees C vs. 0.26 +/- 0.03 microM/100 mg tissue protein, 41 degrees C; p less than 0.05). The decreased ATP occurred despite reduced sodium reabsorption (129 +/- 8 microM/min/g, 37 degrees C vs. 65 +/- 12 microM/min/g, 41 degrees C, p less than 0.05) and normal renal oxygen consumption (QO2). These results suggest that increased temperature may cause an uncoupling of QO2 and sodium chloride transport, and an increase in nontransport mediated, basal metabolic rate may result in depleted cellular ATP levels and renal tubular cell death.     

Intravenous Shiga toxin 2 promotes enteritis and renal injury characterized by polymorphonuclear leukocyte infiltration and thrombosis in Dutch Belted rabbits

Enterohemorrhagic Escherichia coli (EHEC) infection causes hemolytic uremic syndrome, a leading cause of acute renal failure in children. Dutch Belted (DB) rabbits are susceptible to EHEC-induced disease. Using real-time quantitative RT-PCR we measured the renal mRNA expression of cytokines and fibrinolytic factors in DB rabbits challenged with intravenous Shiga toxin 2 (Stx2) (1200 ng/kg). Group 1 rabbits received an incremental dose during an 8-day period whereas Group 2 rabbits received a single dose. Group 1 rabbits developed mild disease. In contrast, Group 2 rabbits developed severe diarrhea, higher levels of circulating polymorphonuclear leukocytes, increased mean platelet volume, and increased fibrinogen levels. Group 2 rabbits developed polymorphonuclear leukocyte infiltration in the intestine and kidney as well as glomerular congestion, luminal constriction, and mesangial glomerulonephropathy. These renal lesions were associated with up-regulation of interleukin-8 (P<0.006), plasminogen activator inhibitor-1 (P<0.04), and tissue plasminogen activator (P<0.05). Circulating Stx2 promoted dose-dependent enteritis and renal injury characterized by inflammation and impaired fibrinolysis leading to thrombosis.     

Heat shock prevents simulated ischemia-induced apoptosis in renal tubular cells via a PKC-dependent mechanism

Heat shock produces cellular tolerance to a variety of adverse conditions; however, the protective effect of heat shock on renal cell ischemic injury remains unclear. Protein kinase C (PKC) has been implicated in the signaling mechanisms of acute preconditioning, yet it remains unknown whether PKC mediates heat shock-induced delayed preconditioning in renal cells. To study this, renal tubular cells (LLC-PK1) were exposed to thermal stress (43 degrees C) for 1 h and heat shock protein (HSP) 72 induction was confirmed by Western blot analysis. Cells were subjected to simulated ischemia 24 h after thermal stress, and the effect of heat shock (delayed preconditioning) on ischemia-induced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) and B cell lymphoma 2 (Bcl(2)) expression (Western) was determined. Subsequently, the effect of PKC inhibition on HSP72 induction and heat stress-induced ischemic tolerance was evaluated. Thermal stress induced HSP72 production, increased Bcl(2) expression, and prevented simulated ischemia-induced renal tubular cell apoptosis. PKC inhibition abolished thermal induction of HSP72 and prevented heat stress-induced ischemic tolerance. These data demonstrate that thermal stress protects renal tubular cells from simulated ischemia-induced apoptosis through a PKC-dependent mechanism.     

Beneficial effect of intracellular free high-mannose oligosaccharides on cryopreservation of mammalian cells and proteins

The cryoprotective effect of intracellular free high-mannose oligosaccharides (HMOS) on mammalian cells and proteins was examined by monitoring PC-12 cell viability and assaying protein kinase C (PKC)-epsilon activity. 1-Deoxymannojirimycin, an inhibitor of alpha-mannosidase, to cause an increase in intracellular free HMOS, significantly rescued PC-12 cells with 2-h freezing insult at -15 degrees C in a concentration (1-50mM)- and pretreatment time (48-72h)-dependent manner, as compared with unpretreated cells; full rescue from freezing injury was obtained with 1-deoxymannojirimycin at more than 25mM for 48-h pretreatment and more than 3mM for 72- and 96-h pretreatment. For PC-12 cells pretreated with 1-deoxymannojirimycin at 1mM for 72h, thawed cell viability after more than 8-w cryopreservation at -80 degrees C in 10% (v/v) dimethyl sulfoxide was much higher than that for cells without pretreatment. PKC-epsilon activity was well preserved after 16-h cryopreservation at -20 degrees C in the presence of mannose 9-N-acetylglucosamine 2 (Man9-GlcNAc2) (1 mM), an HMOS, while the activity was reduced to 15% without Man9-GlcNAc2. Collectively, the results of the present study suggest that intracellular free HMOS is a key molecule to protect mammalian cells and proteins from freezing injury; in other words, HMOS could be a new target for cryopreservation of mammalian cells and proteins.     

Comparative chondrogenesis of human cells in a 3D integrated experimental–computational mechanobiology model

We present an integrated experimental–computational mechanobiology model of chondrogenesis. The response of human articular chondrocytes to culture medium perfusion, versus perfusion associated with cyclic pressurisation, versus non-perfused culture, was compared in a pellet culture model, and multiphysic computation was used to quantify oxygen transport and flow dynamics in the various culture conditions. At 2 weeks of culture, the measured cell metabolic activity and the matrix content in collagen type II and aggrecan were greatest in the perfused+pressurised pellets. The main effects of perfusion alone, relative to static controls, were to suppress collagen type I and GAG contents, which were greatest in the non-perfused pellets. All pellets showed a peripheral layer of proliferating cells, which was thickest in the perfused pellets, and most pellets showed internal gradients in cell density and matrix composition. In perfused pellets, the computed lowest oxygen concentration was 0.075 mM (7.5% tension), the maximal oxygen flux was 477.5 nmol/m²/s and the maximal fluid shear stress, acting on the pellet surface, was 1.8 mPa (0.018 dyn/cm²). In the non-perfused pellets, the lowest oxygen concentration was 0.003 mM (0.3% tension) and the maximal oxygen flux was 102.4 nmol/m²/s. A local correlation was observed, between the gradients in pellet properties obtained from histology, and the oxygen fields calculated with multiphysic simulation. Our results show up-regulation of hyaline matrix protein production by human chondrocytes in response to perfusion associated with cyclic pressurisation. These results could be favourably exploited in tissue engineering applications.     

Cryoanalgesia: electrophysiology at different temperatures

Somatosensory evoked potentials (SEP) and sensory conduction velocity (SCV) were measured in rabbit sciatic nerves following graded cold lesioning. The SEP disappeared when injury was induced at temperatures below -60 degrees C, but returned on day 41+/-4 (mean+/-SD). SEP returned on day 56+/-11 days when the lesion was induced at 100 to -180 degrees C. The SEP latency was prolonged after creating lesions at -100 to -180 degrees C, compared with both the sham operated and the -20 degrees C groups. These experiments suggest the cryolesions produced at temperatures between -60 and -100 degrees C are most suitable for altering the electrophysiological conduction of the nerve, and may result in suitable post-operative analgesia.     

Thermal injury-plus-sepsis contributes to a substantial deletion of intestinal mesenteric lymph node CD4 T cell via apoptosis

Thermal injury (TI) with septic complications continues to be a serious clinical problem. One of the main concerns in such patients is immunosuppression related to functional derangements in intestinal CD4+ T lymphocytes. Extensive previous studies in thermal injury/septic patients and animal models of thermal injury/sepsis have shown decreased responsiveness of intestinal CD4+ T cells to antigen/mitogen. This hyporesponsiveness could significantly contribute to increase injured host susceptibility to pathogens including those translocating from host's gut lumen. Our previous studies indicated that while thermal injury or sepsis alone lead to suppressed proliferation and IL-2 production of intestinal CD4+ T cells, this study showed a substantial deletion via apoptosis of the Mesenteric Lymph Nodes (MLN) CD4+ T cells. Hence, thermal injury-plus-sepsis contributes not only to suppressed CD4+ T proliferation/IL-2 production but also to a substantial modulation of CD4+ T cell survivability. These findings allow us to conclude that while thermal injury alone can produce attenuated cell mediated responses without an overt change in CD4+ T cell survival, thermal injury with septic complications causes CD4+ T cell death and an irreversible loss of cell-mediated responses. The latter happening could be responsible for high morbidity and mortality in the injured host afflicted with thermal injury plus a critical infection.     

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.     

Cryopreservation of organs by vitrification: perspectives and recent advances

The cryopreservation of organs became an active area of research in the 1950s as a result of the rediscovery of the cryoprotective properties of glycerol by Polge, Smith, and Parkes in 1949. Over the ensuing four decades of research in this area, the advantages of vitrification, or ice-free cryopreservation, have become apparent. To date, experimental attempts to apply vitrification methods to vascularized whole organs have been confined almost entirely to the rabbit kidney. Using techniques available as of 1997, it was possible to vitrify blood vessels and smaller systems with reasonable success, but not whole organs. Beginning in 1998, a series of novel advances involving the control of cryoprotectant toxicity, nucleation, crystal growth, and chilling injury began to provide the tools needed to achieve success. Based on these new findings, we were first able to show that an 8.4M solution (VMP) designed to prevent chilling injury at -22 degrees C was entirely non-toxic to rabbit kidneys when perfused at -3 degrees C and permitted perfusion-cooling to -22 degrees C with only mild additional damage. We next investigated the ability of the kidney to tolerate a 9.3M solution known as M22, which does not devitrify when warmed from below -150 degrees C at 1 degrees C/min. When M22 was added and removed at -22 degrees C, it was sometimes [corrected] fatal, but when it was perfused for 25min at -22 degrees C and washed out simultaneously with warming, postoperative renal function recovered fully. When kidneys loaded with M22 at -22 degrees C were further cooled to an average intrarenal temperature of about -45 degrees C (about halfway through the putative temperature zone of increasing vulnerability to chilling injury), all kidneys supported life after transplantation and returned creatinine values to baseline, though after a higher transient creatinine peak. However, medullary, papillary, and pelvic biopsies taken from kidneys perfused with M22 for 25min at -22 degrees C were found to devitrify when vitrified and rewarmed at 20 degrees C/min in a differential scanning calorimeter. It remains to be determined whether this devitrification is seriously damaging and whether it can be suppressed by improving cryoprotectant distribution to more weakly perfused regions of the kidney or by rewarming at higher rates. In conclusion, although the goal of organ vitrification remains elusive, the prospects for success have never been more promising.     

Temperature dependence and GABA modulation of [3H]triazolam binding in the rat brain

The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Our major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of [3H]TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (Kd = 0.27 +/- 08 nM at 0 degree C; Kd = 1.96 +/- 0.85 nM at 37 degrees C) while the Bmax values remained unchanged (1220 +/- 176 fmoles/mg protein at 0 degree C and 1160 +/- 383 fmoles/mg protein at 37 degrees C). Saturation studies of [3H]TZ binding in the presence or absence of GABA (100 microM) showed a GABA-shift. At 0 degrees C the Kd values were (Kd = 0.24 +/- 0.03 nM/-GABA; Kd = 0.16 +/- 0.04/+GABA) and at 37 degrees C the Kd values were (Kd = 1.84 +/- 0.44 nM/-GABA; Kd = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, our findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists.     

Production of acetone and conversion of acetone to acetate in the perfused rat liver

The utilization of millimolar concentrations of [2-14C]acetone and the production of acetone from acetoacetate were studied in perfused livers from 48-h starved rats. We devised a procedure for determining, in a perfused liver system, the first-order rate constant for the decarboxylation of acetoacetate (0.29 +/- 0.09 h-1, S.E., n = 8). After perfusion of livers with [2-14C]acetone, labeled acetate was isolated from the perfusion medium and characterized as [1-14C]acetate. No radioactivity was found in lactate or 3-hydroxybutyrate. After 90 min of perfusion with [2-14C]acetone, the specific activity of acetate was 30 +/- 4% (n = 13) of the initial specific activity of acetone. We conclude that, in perfused livers from 2-day starved rats, acetone metabolism occurs for the most part via free acetate.     

Calcium antagonists and heat-induced hepatic injury

Several laboratories have demonstrated the value of the isolated perfused rat liver as a suitable model for heat-induced hepatic injury in vivo. Membrane changes caused by perfusion of rat livers at 42 degrees C for 90 min were similar to those induced by toxic chemicals or hypoxia. In an evaluation of several categories of drugs reported to reduce cell injury, calcium antagonists (nifedipine, dantrolene, and verapamil), were evaluated for their therapeutic potential for heat injury. Isolated rat livers were perfused at 42 degrees C for 90 min with and without calcium antagonists. Livers were also perfused at 37 degrees C. Potassium and transaminase leakage, bile production and ultrastructure were used to evaluate their responses. Neither of the three calcium antagonists significantly improved any of the functional parameters measured. However, dantrolene produced dilated or vesicular rough endoplasmic reticulum in the heated livers. These changes suggest selective intracellular action on endoplasmic reticulum of heated livers. Ring-shaped mitochondria and vesicular endoplasmic reticulum were observed in the heated, verapamil-treated livers, but these could not be quantitatively distinguished from controls. Nifedipine did not appear to alter intracellular membranes, but did increase bile production.     

Morphological changes of Pseudomonas pseudoalcaligenes in response to temperature selection

Adaptation to novel environments usually entails morphological changes. The cell morphology of six experimental populations of Pseudomonas pseudoalcaligenes and their common ancestor were examined with scanning electron microscopy (SEM). The six experimental populations were propagated under different temperatures for 10 months: three of them cultured at constant normal temperature (35 degrees C) forming the control group, and the other three cultured at incremental higher temperatures (from 41 degrees to 47 degrees C) as the HT group. SEM showed the deformed and elongated cells in the 6-h cultures of both ancestral and control populations at 45 degrees C, indicating that 45 degrees C is stressful for the ancestral and the control populations. In contrast, the HT populations retained normal cell shape in the 6-h cultures at both 35 degrees C and 45 degrees C. The mean cell volumes of control and HT populations increased 29% and 34%, respectively, relative to the ancestor at their respective thermal regimens, suggestion that the culturing conditions might favor larger cells.     

Melatonin prevents oxidative kidney damage in a rat model of thermal injury

Animal models of thermal trauma implicate oxygen radicals as causative agents in local wound response and distant organ injury following burn. This study was designed to determine the effect of melatonin treatment on levels of glutathione (GSH), malondialdehyde (MDA), protein oxidation (PO) and myeloperoxidase (MPO) activity in the kidney tissues of rats with thermal injury. Under ether anaesthesia, shaved dorsum of the rats was exposed to 90 degrees C bath for 10 s to induce burn injury. Rats were decapitated either 3 h or 24 h after burn injury. Melatonin was administered i.p. immediately after burn injury. In the 24-h burn group melatonin injections were repeated for two more occasions. In the sham group the same protocol was applied except that the dorsum was dipped in a 25 degrees C water bath for 10 s. Severe skin scald injury (30% of total body surface area) caused a significant decrease in GSH level, and significant increases in MDA and PO levels, and MPO activity at post-burn 3 and 24 hours. Treatment of rats with melatonin (10 mg/kg) significantly elevated the reduced GSH levels while it decreased MDA and PO levels as well as MPO activity.     

Comparison of the effect of 3- and 5-day hypothermic perfusion of dog kidneys on metabolism of tissue slices

Hypothermic perfusion effectively preserves the viability of kidneys for 3 days. Long-term preservation (5 days or greater) has not been consistently obtained. In this study, the differences between kidneys perfused for 3 and 5 days were compared by determining the "integrated-metabolic" capabilities of tissue slices incubated in vitro at 30 degrees C. The "integrated-metabolic" parameters determined include (1) respiration rates, (2) cell volume regulation [total tissue water (TTW) and saccharide permeable space], (3) rate of reaccumulation of K+ and pumping of Na+, (4) maintenance of ATP concentrations, and (5) mitochondrial functions. Conditions that result in high and low concentrations of ATP following perfusion of kidneys for 5 days were also compared for effects on tissue slice metabolism. The results indicate that energy metabolism in tissue slices is well preserved under all conditions and times of perfusion of kidneys. This includes average respiration rates (315 +/- 50, 275 +/- 35, and 255 +/- 45 mumol O2/hr/g dry wt at 0, 3, and 5 days, respectively, mitochondrial function [respiratory control ratio (RCR) = 4.6, 4.0, and 4.1 for 0, 3, and 5 days, respectively], and steady-state concentration of ATP in slices after incubation (4.0 +/- 1.45, 3.9 +/- 1.28, and 3.3 +/- 0.81 mumol/g/dry wt, for 0, 3, and 5 days, respectively). The primary differences between 3- and 5-day perfused kidneys were the capability of the slices to regulate cell volume and reaccumulate K+. Slices from kidneys perfused for 3 days maintained the TTW at 3.8 kg/kg dry wt, a value similar to that of control tissue slices. However, slices from 5-day perfused kidneys remained swollen (TTW = 4.6 kg/kg dry wt). Also, slices from the 5-day perfused kidney pumped K+ at less than one-half the rate found in slices from control or 3-day preserved kidneys. No significant differences were apparent in the permeability properties of the tissue slices from kidneys perfused for 3 and 5 days to radiolabeled saccharides. The defects in membrane-linked transport functions, resulting from long-term kidney perfusion, were reduced in kidneys containing a high concentration of ATP. The results suggest that one factor which may limit successful preservation of kidneys is the increased membrane permeability (to electrolytes) which is partially prevented by maintaining elevated concentrations of tissue ATP during perfusion.     

Effect of drug treatment on liver-slice function following 72-hour hypothermic perfusion

The viability of hypothermically perfused dog liver was evaluated with a tissue-slice technique. After being preserved for 72 hr, slices of liver were incubated at 30 degrees C for as long as 2 hr; then water content, K+/Na+ ratio, and ATP concentration were measured. Dog livers were assigned to the following experimental groups: Group 1 (no preservation; control); Group 2 (livers preserved for 72 hr); Group 3 (donor animals pretreated with 3.5 mg/kg of chlorpromazine (CPZ) and 20 mg/kg of methylprednisolone (MP), and livers preserved for 72 hr); Group 4 (livers pretreated with 2-deoxycoformycin (2-DOC), 50 mg/liter, and preserved for 72 hr); and Group 5 (combination of Group 3 and Group 4 treatments). Livers in Groups 2, 3, and 4 lost K+ during preservation, and the mean K+/Na+ ratio significantly decreased from a control value of 4.2 +/- 0.4 to 1.5-1.9 (P less than 0.05). Group 5 livers did not lose K+; mean K+/Na+ ratio was 3.9 +/- 0.5. Fresh livers (no preservation) rapidly reaccumulated K+ when the tissue slices were incubated for 2 hr at 30 degrees C; mean K+/Na+ ratio was 3.7 +/- 0.5. Tissue slices from Group 2 livers (72 hr preservation), and livers pretreated with CPZ-MP (Group 3) or pretreated with 2-DOC (Group 4) did not significantly reaccumulate K+ at 30 degrees C; mean K+/Na+ ratio was 1.7-2.1. Only slices prepared from liver pretreated with both CPZ-MP and 2-DOC reaccumulated K+; mean K+/Na+ ratio was 4.6 +/- 1.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein synthesis in perfused rat hearts after in vivo hyperthermia and in vitro cold ischemia

Isolated and perfused rat hearts can be maintained for up to 2.5 h with minimal synthesis of a stress protein with a relative mass (Mr) of 71 kilodaltons (SP71). Isolated hearts, subjected to 17 h of cold (4 degrees C) ischemia, upon perfusion (37 degrees C) synthesize a large amount of SP71. In the present study, the effect of in vivo hyperthermia on protein synthesis in isolated and perfused hearts was examined. Hearts were excised from rats subjected to a 15-min episode of hyperthermia (42 degrees C), either immediately (no recovery) or after 24 h of recovery. The excised hearts were perfused either immediately or after 17 h of cold ischemia. Hyperthermia (no recovery) increased [3H]leucine incorporation into SP71, while hyperthermia with a 24-h recovery did not increase incorporation into SP71 during perfusion (no ischemia). Hyperthermia (no recovery) increased the incorporation of [3H]leucine into SP71 seen after cold ischemia. Hyperthermia with a 24-h recovery decreased the incorporation of [3H]leucine into SP71 seen after cold ischemia. This reduction in synthesis of SP71 after 24-h recovery from hyperthermia could be caused by the accumulation of SP71 suppressing its own synthesis or a measure of protection (tolerance) induced by the hyperthermia.     

Effect of treatment schedule on the interaction of Cisplatin and radiation in human lung cancer cells

This study was designed to determine the effects of the treatment schedule on the interaction between cisplatin and radiation. Cells of a human squamous cell lung cancer cell line were treated with cisplatin and radiation using three treatment protocols: 1-h exposure to cisplatin immediately followed by irradiation (A), 4-day continuous exposure to cisplatin immediately followed by irradiation (B), and 1-h exposure to cisplatin followed by irradiation after a 4-day interval (C). The interactions were assessed by isobologram, cell cycle distribution and apoptosis. The combination resulted in a additive effect in every protocol. Cell cycle accumulation at G(2)/M phase before irradiation was observed in Protocols B and C, whereas no cell cycle shift in the limited time course was noted in Protocol A. Although a 4-day continuous exposure to cisplatin and a 1-h exposure to cisplatin followed by a 4-day interval before irradiation caused significantly increased apoptosis, an additional increase in apoptosis after irradiation was not observed in Protocols B and C, whereas Protocol A showed an additional increase. Despite a cell cycle shift favoring radiation sensitivity, the drug-radiation interactions in Protocols B and C were additive, possibly because of negative effects including induction of a durable G(2)/M-phase arrest and suppression of apoptosis by cisplatin.     

Percutaneous cryoablation of the kidney in a porcine model

Several reports have documented that renal cryoablation when used either via open or laparoscopic surgical techniques can be well tolerated in animal models. We sought to examine the feasibility of performing renal cryoablation percutaneously, under ultrasound guidance, in a porcine model. A total of 13 juvenile swine was treated. In each animal the lower pole of the left kidney was used as the target. Each animal had serum drawn for Hct, Wbc, CK, BUN, creatinine, and myoglobin pre- and postcryoablation and again at the time of sacrifice. Animals were sacrificed at 0-42 days postoperatively, and the treated renal units were harvested and submitted for histologic examination. The procedures were divided into three groups based on the extent of the freeze time. Group 1 (n = 4) was treated for 3-4 min, single freeze; Group 2 (n = 3), 6-7 min, single freeze; and Group 3 (n = 6), a double freeze-thaw cycle, each for 10 min. None of the animals in this experiment died or experienced significant clinical deteriorations at any time post-treatment. No significant differences in any of the measured serum markers were noted between pre- and post-treatment values. For animals in Groups 1 and 2 discrete cryolesions were created with sharp 1-mm borders and no perirenal reaction/damage to surrounding structures. For animals in Group 3 large areas of renal infarction/cryolesions were produced, with significant perirenal reaction in 5/6 animals and gross hydronephrosis/total renal loss in 2/6. Percutaneous renal cryoablation appears to be well tolerated in the porcine model which we used. Associated morbidity appears to be related to the extent of freezing, with a safety tolerance in the present study limited to target areas of approximately 3-5 cm3. These findings suggest that a pilot study of percutaneous renal cryoablation for patients with 3-4-cm exophytic lesions may be warranted.