ErbB2 Receptor Immunoreactivity in Prostate Cancer: Relationship to the Androgen Receptor,Disease Severity at Diagnosis and Disease Outcome

Background

ErbB2 is a member of the epidermal growth factor family of tyrosine kinases that is centrally involved in the pathogenesis of prostate cancer and several studies have reported that a high expression of this protein has prognostic value. In the present study, we have investigated whether tumour ErbB2 immunoreactivity (ErbB2-IR) has clinically useful prognostic value, i.e. that it provides additional prognostic information to that provided by routine clinical tests (Gleason score, tumour stage).

Methodology/Principal Findings

ErbB2-IR was measured in a well-characterised tissue microarray of tumour and non-malignant samples obtained at diagnosis. Additionally, mRNA levels of ErbB2-IR in the prostate were determined in the rat following manipulation of circulating androgen levels. Tumour ErbB2-IR was significantly associated with the downstream signalling molecule phosphorylated-Akt and with the cell proliferation marker Ki-67. The significant association of tumour ErbB2-IR with the Gleason score at diagnosis was lost when controlled for the association of both parameters with Ki-67. In the rat prostate, mRNA for ErbB2 was inversely associated with circulating androgen levels. There was no association between ErbB2-IR and the androgen receptor (AR)-IR in the tumours, but an interaction between the two parameters was seen with respect to their association with the tumour stage. Tumour ErbB2-IR was confirmed to be a prognostic marker for disease-specific survival, but it did not provide significant additive information to the Gleason score or to Ki-67.

Conclusions/Significance

It is concluded that tumour ErbB2-IR is of limited clinical value as a prognostic marker to aid treatment decisions, but could be of pathophysiological importance in prostate cancer.     

Effect of Mild Hypothermia on the Coagulation-Fibrinolysis System and Physiological Anticoagulants after Cardiopulmonary Resuscitation in a Porcine Model

The aim of this study was to evaluate the effect of mild hypothermia on the coagulation-fibrinolysis system and physiological anticoagulants after cardiopulmonary resuscitation (CPR). A total of 20 male Wuzhishan miniature pigs underwent 8 min of untreated ventricular fibrillation and CPR. Of these, 16 were successfully resuscitated and were randomized into the mild hypothermia group (MH, n = 8) or the control normothermia group (CN, n = 8). Mild hypothermia (33°C) was induced intravascularly, and this temperature was maintained for 12 h before pigs were actively rewarmed. The CN group received normothermic post-cardiac arrest (CA) care for 72 h. Four animals were in the sham operation group (SO). Blood samples were taken at baseline, and 0.5, 6, 12, 24, and 72 h after ROSC. Whole-body mild hypothermia impaired blood coagulation during cooling, but attenuated blood coagulation impairment at 72 h after ROSC. Mild hypothermia also increased serum levels of physiological anticoagulants, such as PRO C and AT-III during cooling and after rewarming, decreased EPCR and TFPI levels during cooling but not after rewarming, and inhibited fibrinolysis and platelet activation during cooling and after rewarming. Finally, mild hypothermia did not affect coagulation-fibrinolysis, physiological anticoagulants, or platelet activation during rewarming. Thus, our findings indicate that mild hypothermia exerted an anticoagulant effect during cooling, which may have inhibitory effects on microthrombus formation. Furthermore, mild hypothermia inhibited fibrinolysis and platelet activation during cooling and attenuated blood coagulation impairment after rewarming. Slow rewarming had no obvious adverse effects on blood coagulation.     

Myocardial gene expression profiling of rewarming shock in a rodent model of accidental hypothermia

Background

Severe accidental hypothermia represents a cardiovascular emergency associated with high mortality and poor recovery of cardiac function. The biochemical changes occurring within the heart during the development of hypothermia and subsequent resuscitation are not known.

Methods

By mRNA expression profiling, we have characterized gene expression changes occurring within the myocardium in an intact rat model of accidental hypothermia during cooling to a core temperature of 15 °C and subsequent rewarming to 37 °C. During the rewarming phase, these animals develop a profound low-output cardiac failure.

Results

Hypothermia induces expression of known mediators of thermotolerance, including heat-shock protein 70 and several factors involved in protection against apoptotic cell death. Upregulation of genes involved in autophagy and increased abundance of autophagosomal vesicles suggest involvement of autophagic degeneration in the development of myocardial dysfunction occurring during rewarming from hypothermia. Rewarming from hypothermia also induces expression of several pro-inflammatory genes involved in the nuclear factor kappa B (NFκB) signaling cascade.

Conclusions

Our data demonstrate that rewarming from hypothermia is associated with the induction of a cellular stress–response, including upregulation of autophagy and activation of pro-inflammatory signaling cascades. These data provide a framework for understanding the molecular changes that occur during induction of and rewarming from severe hypothermia, and identifies potential targets for cardioprotective interventions in resuscitation of victims of hypothermia.     

Effects of Mild Cold Shock (25°C) Followed by Warming Up at 37°C on the Cellular Stress Response

Temperature variations in cells, tissues and organs may occur in a number of circumstances. We report here that reducing temperature of cells in culture to 25°C for 5 days followed by a rewarming to 37°C affects cell biology and induces a cellular stress response. Cell proliferation was almost arrested during mild hypothermia and not restored upon returning to 37°C. The expression of cold shock genes, CIRBP and RBM3, was increased at 25°C and returned to basal level upon rewarming while that of heat shock protein HSP70 was inversely regulated. An activation of pro-apoptotic pathways was evidenced by FACS analysis and increased Bax/Bcl2 and BclX_S/L ratios. Concomitant increased expression of the autophagosome-associated protein LC3II and AKT phosphorylation suggested a simultaneous activation of autophagy and pro-survival pathways. However, a large proportion of cells were dying 24 hours after rewarming. The occurrence of DNA damage was evidenced by the increased phosphorylation of p53 and H2AX, a hallmark of DNA breaks. The latter process, as well as apoptosis, was strongly reduced by the radical oxygen species (ROS) scavenger, N-acetylcysteine, indicating a causal relationship between ROS, DNA damage and cell death during mild cold shock and rewarming. These data bring new insights into the potential deleterious effects of mild hypothermia and rewarming used in various research and therapeutical fields.     

Active core rewarming avoids bioelectrical impedance changes in postanesthetic patients

Background

Postoperative hypothermia is a common cause of complications in patients who underwent laparoscopic cholecystectomy. Hypothermia is known to elicit electrophysiological, biochemical, and cellular alterations thus leading to changes in the active and passive membrane properties. These changes might influence the bioelectrical impedance (BI). Our aim was to determine whether the BI depends on the core temperature.

Methods

We studied 60 patients (52 female and 8 male) age 40 to 80 years with an ASA I-II classification that had undergone laparoscopic cholecystectomy under balanced inhalation anesthesia. The experimental group (n = 30) received active core rewarming during the transanesthetic and postanesthesic periods. The control group (n = 30) received passive external rewarming. The BI was recorded by using a 4-contact electrode system to collect dual sets of measurements in the deltoid muscle. The body temperature, hemodynamic variables, respiratory rate, blood-gas levels, biochemical parameters, and shivering were also measured. The Mann-Whitney unpaired t -test was used to determine the differences in shivering between each group at each measurement period. Measurements of body temperature, hemodynamics variables, respiratory rate, and BI were analyzed using the two-way repeated-measures ANOVA.

Results

The gradual decrease in the body temperature was followed by the BI increase over time. The highest BI values (95 ± 11 Ω) appeared when the lowest values of the temperature (35.5 ± 0.5°C) were reached. The active core rewarming kept the body temperature within the physiological range (over 36.5°C). This effect was accompanied by low stable values (68 ± 3 Ω) of BI. A significant decrease over time in the hemodynamic values, respiratory rate, and shivering was seen in the active core-rewarming group when compared with the controls. The temporal course of shivering was different from those of body temperatue and BI. The control patients showed a significant increase in the serum-potassium levels, which were not seen in the active-core rewarming group.

Conclusions

The BI analysis changed as a function of the changes of core temperature and independently of the shivering. In addition, our results support the beneficial use of active core rewarming to prevent accidental hypothermia.     

Hypothermia and Postconditioning after Cardiopulmonary Resuscitation Reduce Cardiac Dysfunction by Modulating Inflammation,Apoptosis and Remodeling

Background

Mild therapeutic hypothermia following cardiac arrest is neuroprotective, but its effect on myocardial dysfunction that is a critical issue following resuscitation is not clear. This study sought to examine whether hypothermia and the combination of hypothermia and pharmacological postconditioning are cardioprotective in a model of cardiopulmonary resuscitation following acute myocardial ischemia.

Methodology/Principal Findings

Thirty pigs (28–34 kg) were subjected to cardiac arrest following left anterior descending coronary artery ischemia. After 7 minutes of ventricular fibrillation and 2 minutes of basic life support, advanced cardiac life support was started according to the current AHA guidelines. After successful return of spontaneous circulation (n = 21), coronary perfusion was reestablished after 60 minutes of occlusion, and animals were randomized to either normothermia at 38°C, hypothermia at 33°C or hypothermia at 33°C combined with sevoflurane (each group n = 7) for 24 hours. The effects on cardiac damage especially on inflammation, apoptosis, and remodeling were studied using cellular and molecular approaches. Five animals were sham operated. Animals treated with hypothermia had lower troponin T levels (p<0.01), reduced infarct size (34±7 versus 57±12%; p<0.05) and improved left ventricular function compared to normothermia (p<0.05). Hypothermia was associated with a reduction in: (i) immune cell infiltration, (ii) apoptosis, (iii) IL-1β and IL-6 mRNA up-regulation, and (iv) IL-1β protein expression (p<0.05). Moreover, decreased matrix metalloproteinase-9 activity was detected in the ischemic myocardium after treatment with mild hypothermia. Sevoflurane conferred additional protective effects although statistic significance was not reached.

Conclusions/Significance

Hypothermia reduced myocardial damage and dysfunction after cardiopulmonary resuscitation possible via a reduced rate of apoptosis and pro-inflammatory cytokine expression.     

Myocardial mechanical dysfunction and calcium overload following rewarming from experimental hypothermia in vivo

Rewarming patients from accidental hypothermia are regularly complicated with cardiovascular instability ranging from minor depression of cardiac output to fatal circulatory collapse also termed “rewarming shock”. Since altered Ca²⁺ handling may play a role in hypothermia-induced heart failure, we studied changes in Ca²⁺ homeostasis in in situ hearts following hypothermia and rewarming. A rat model designed for studies of the intact heart in a non-arrested state during hypothermia and rewarming was used. Rats were core cooled to 15 °C, maintained at 15 °C for 4 h and thereafter rewarmed. As time-matched controls, one group of animals was kept at 37 °C for 5 h. Total intracellular myocardial Ca²⁺ content ([Ca²⁺]_i) was measured using ⁴⁵Ca²⁺. Following rewarming we found a significant reduction of stroke volume and cardiac output compared to prehypothermic control values as well as to time-matched controls. Likewise, we found that hypothermia and rewarming resulted in a more than six-fold increase in [Ca²⁺]_i to 3.01 ± 0.43 μmol/g dry weight compared to 0.44 ± 0.05 μmol/g dry weight in normothemia control. These findings indicate that hypothermia-induced alterations in the Ca²⁺-handling result in Ca²⁺ overload during hypothermia, which may contribute to myocardial failure during and after rewarming.     

Adipose Hypothermia in Obesity and Its Association with Period Homolog 1, Insulin Sensitivity,and Inflammation in Fat

Visceral fat adiposity plays an important role in the development of metabolic syndrome. We reported previously the impact of human visceral fat adiposity on gene expression profile of peripheral blood cells. Genes related to circadian rhythm were highly associated with visceral fat area and period homolog 1 (PER1) showed the most significant negative correlation with visceral fat area. However, regulation of adipose Per1 remains poorly understood. The present study was designed to understand the regulation of Per1 in adipose tissues. Adipose Per1 mRNA levels of ob/ob mice were markedly low at 25 and 35 weeks of age. The levels of other core clock genes of white adipose tissues were also low in ob/ob mice at 25 and 35 weeks of age. Per1 mRNA was mainly expressed in the mature adipocyte fraction (MAF) and it was significantly low in MAF of ob/ob mice. To examine the possible mechanisms, 3T3-L1 adipocytes were treated with H₂O₂, tumor necrosis factor-α (TNF-α), S100A8, and lipopolysaccharide (LPS). However, no significant changes in Per1 mRNA level were observed by these agents. Exposure of cultured 3T3-L1 adipocytes to low temperature (33°C) decreased Per1 and catalase, and increased monocyte chemoattractant protein-1 (Mcp-1) mRNA levels. Hypothermia also worsened insulin-mediated Akt phosphorylation in 3T3-L1 adipocytes. Finally, telemetric analysis showed low temperature of adipose tissues in ob/ob mice. In obesity, adipose hypothermia seems to accelerate adipocyte dysfunction.     

Whole-body temperature gradients under surface,perfusion, and combined surface/perfusion hypothermia

Using various methods of hypothermia and halothane-diethyl ether azeotrope anesthesia whole-body temperature gradients were evaluated in 20 adult mongrel dogs. Simultaneous measurements were taken of brain, rectal, esophageal, pharyngeal, liver, jugular vein, shoulder muscle, thigh muscle, and subcutaneous temperatures during (i) surface, (ii) perfusion (slow and rapid cooling), and (iii) combined surface/perfusion methods of hypothermia. Throughout cooling and rewarming core temperature gradients averaged 1.2 °C and during circulatory arrest core temperatures decreased an average of 0.3 °C under pure surface hypothermia. Animals, thermoregulated by extracorporeal methods only, developed larger core temperature gradients during cooling and a significant increase (average = 3.1 °C) was noted in core temperatures during circulatory arrest. This pattern was particularly pronounced during rapid perfusion cooling. Hypothermia induction by combined surface/perfusion, in contrast to pure perfusion methods, resulted in smaller gradients without remarkable increase in core temperature (average = 1.3 °C) during the arrest period. These findings when correlated with the shorter total operating time and ease of operative management and resuscitation lead us to the conclusion that combined surface/ perfusion hypothermia techniques have certain advantages over either pure surface or pure perfusion techniques alone.     

Hypothermia in Preterm Infants in the First Hours after Birth: Occurrence,Course and Risk Factors

BackgroundHypothermia is associated with increased morbidity and mortality rates. Preterm infants frequently have hypothermia when they are admitted to the NICU, but there is no data on the occurrence of hypothermia during the first hours after admission.ObjectiveTo investigate the occurrence of hypothermia in preterm infants in the first three hours of admission and to identify risk factors.MethodsInfants < 32 weeks of gestation included in a randomized trial with admission temperature as primary outcome were retrospectively analyzed for the occurrence of hypothermia (< 36.5°C) in the first three hours after admission. Risk factors were identified using linear regression analysis and logistic regression.ResultsIn total 80 infants were included with a median (IQR) gestational age at birth of 29 (27–30) weeks. In 93% of the infants hypothermia occurred in the first three hours after admission. The median (IQR) duration of hypothermia was 101 (34–162) minutes, of which 24 (7–52) minutes the hypothermia was mild, 45 (4–111) minutes moderate, severe hypothermia hardly occurred. Gestational age and the occurrence of hypothermia at birth were independent risk factors for the occurrence of moderate and severe hypothermia and significantly correlated with duration of hypothermia.ConclusionsHypothermia occurred often and for a long period in preterm infants in the first three hours of life, low gestational age and admission temperature were independent risk factors     

Intermittent Hypothermia Is Neuroprotective in an in vitro Model of Ischemic Stroke

Objective: To investigate whether the intermittent hypothermia (IH) protects neurons against ischemic insult and the potential molecular targets using an in vitro ischemic model of oxygen glucose deprivation (OGD).Methods: Fetal rat cortical neurons isolated from Day E18 rat embryos were subjected to 90-min OGD and hypothermia treatments during reoxygenation before examining the changes in microscopic morphology, cell viability, microtubule- associated protein 2 (MAP-2) release, intracellular pH value and calcium, reactive oxygen species (ROS) generation, mitochondrial membrane potential (△Ψm) and neuronal death using cell counting kit (CCK-8), enzyme-linked immunosorbent assay (ELISA), BCECF AM, Fluo-3 AM, DCFH-DA and dihydroethidium (DHE), JC-1 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively.Results: 90-min OGD induced morphologic abnormalities, cell viability decline, MAP-2 release, intracellular acidosis, calcium overload, increased ROS generation, △Ψm decrease and cell death in primary neurons, which was partially inhibited by continuous hypothermia (CH) and intermittent hypothermia (IH). Interestingly, 6-h CH was insufficient to reduce intracellular calcium overload and stabilize mitochondrial membrane potential (△Ψm), while 12-h CH was effective in reversing the above changes. All IH treatments (6×1 h, 4×1.5 h or 3×2 h) effectively attenuated intracellular free calcium overload, inhibited ROS production, stabilized mitochondrial membrane potential (△Ψm) and reduced delayed cell death in OGD-treated cells. However, only IH intervals longer than 1.5 h appeared to be effective in preventing cell viability loss and intracellular pH decline.Conclusion: Both CH and IH were neuroprotective in an in vitro model of ischemic stroke, and in spite of shorter hypothermia duration, IH could provide a comparable neuroprotection to CH.     

Fever-Range Hyperthermia vs. Hypothermia Effect on Cancer Cell Viability,Proliferation and HSP90 Expression

Purpose

The current study examines the effect of fever-range hyperthermia and mild hypothermia on human cancer cells focusing on cell viability, proliferation and HSP90 expression.

Materials and Methods

A549 and H1299 lung carcinoma, MCF7 breast adenocarcinoma, U87MG and T98G glioblastoma, DU145 and PC3 prostate carcinoma and MRC5 normal fetal lung fibroblasts cell lines were studied. After 3-day exposure to 34°C, 37°C and 40°C, cell viability was determined. Cell proliferation (ki67 index), apoptosis (Caspase 9) and HSP90 expression was studied by confocal microscopy.

Results

Viability/proliferation experiments demonstrated that MRC5 fibroblasts were extremely sensitive to hyperthermia, while they were the most resistant to hypothermia. T98G and A549 were thermo-tolerant, the remaining being thermo-sensitive to a varying degree. Nonetheless, as a universal effect, hypothermia reduced viability/proliferation in all cell lines. Hyperthermia sharply induced Caspase 9 in the U87MG most thermo-sensitive cell line. In T98G and A549 thermo-tolerant cell lines, the levels of Caspase 9 declined. Moreover, hyperthermia strongly induced the HSP90 levels in T98G, whilst a sharp decrease was recorded in the thermo-sensitive PC3 and U87MG cell lines. Hyperthermia sensitized thermo-sensitive cancer cell lines to cisplatin and temozolomide, whilst its sensitizing effect was diminished in thermo-tolerant cell lines.

Conclusions

The existence of thermo-tolerant and thermo-sensitive cancer cell lines was confirmed, which further encourages research to classify human tumor thermic predilection for patient stratification in clinical trials. Of interest, mild hypothermia had a universal suppressing effect on cancer cell proliferation, further supporting the radio-sensitization hypothesis through reduction of oxygen and metabolic demands.     

ThermoSpots to Detect Hypothermia in Children with Severe Acute Malnutrition

Introduction

Hypothermia is a risk factor for increased mortality in children with severe acute malnutrition (SAM). Yet frequent temperature measurement remains unfeasible in under-resourced units in developing countries. ThermoSpot is a continuous temperature monitoring sticker designed originally for neonates. When applied to skin, its liquid crystals are designed to turn black with hypothermia and remain green with normothermia.

Aims

To (i) estimate the diagnostic accuracy of ThermoSpots for detecting WHO-defined hypothermia (core temperature <35.5°C or peripheral temperature <35.0°C) in children with SAM and (ii) determine their acceptability amongst mothers.

Methods

Children with SAM in a malnutrition unit in Malawi were enrolled during March-July 2010. The sensitivity and specificity of ThermoSpots were calculated by comparing the device colour against ‘gold standard’ rectal temperatures taken on admission and follow up peripheral temperatures taken until discharge. Guardians completed a questionnaire to assess acceptability.

Results

Hypothermia was uncommon amongst the 162 children enrolled. ThermoSpot successfully detected the one rectal temperature and two peripheral temperatures recorded that met the WHO definition of hypothermia. Overall, 3/846 (0.35%) temperature measurements were in the WHO-defined hypothermia range. Interpreting the brown transition colour (between black and green) as hypothermia improved sensitivities. For milder hypothermia definitions, sensitivities declined (<35.4°C, 50.0%; <35.9°C, 39.2%). Specificity was consistently above 94%. From questionnaires, 40/43 (93%) mothers reported they were 90–100% happy with the device overall. Free-text answers revealed themes of “Skin Rashes”, “User-satisfaction” and “Empowerment".

Conclusion

Although hypothermia was uncommon in this study, ThermoSpots successfully detected these episodes in malnourished children and were acceptable to mothers. Research in settings where hypothermia is common is needed to determine performance with certainty. Instructing users to act when the device’s transition colour appears could improve accuracy. If reliable, ThermoSpots may offer simple, acceptable and continuous temperature measurement for high-burden areas and reduce the workload of over-stretched staff.     

Glucose, glycogen, and insulin responses in the hypothermic rat

The rat appears to be unable to utilize glucose during hypothermia. The objective of this study was to examine carbohydrate homeostasis during induction, hypothermia, and rewarming phases. Groups of normothermic animals were euthanized to serve as time controls for comparison. Hypothermia (15 degrees C) was produced by exposure to helox (80% helium:20% oxygen) at 0 +/- 1 degree C. Hyperglycemia was noted during the induction process (169 +/- 8 in control vs 326 +/- 49 mg/dl). Serum glucose increased further during 4 hr of hypothermia, but following rewarming (Tre of 33 +/- 1 degrees C) was reduced (153 +/- 16 mg/dl) significantly (P less than 0.05). Serum insulin was depressed during hypothermic induction (from 48 +/- 4 in controls to 19 +/- 3 microU/ml in hypothermic rats) and increased only slightly during the arousal process, remaining significantly lower than in normothermic subjects. Initial hepatic, skeletal muscle, and cardiac glycogen concentrations were reduced 34, 68, and 75%, respectively, during hypothermic induction. While liver glycogen decreased further during 4 hr of hypothermia, skeletal and cardiac stores increased markedly. During rewarming, hepatic glycogen was markedly decreased, while skeletal and cardiac stores were maintained. These data suggest that hyperglycemia in the hypothermic rat can be accounted for by glycogenolysis and hypoinsulinemia. In addition, this study indicates repletion of skeletal and cardiac muscle glycogen during maintained hypothermia and sparing of muscle glycogen during rewarming.     

Effects of hypothermia on energy metabolism in rat and Richardson's ground squirrel hearts

Belke, Darrell D., Lawrence C. H. Wang, and Gary D. Lopaschuk. Effects of hypothermia on energy metabolism in rat and Richardson's ground squirrel hearts. J. Appl.Physiol. 82(4): 1210-1218, 1997.Glycolysis,glucose oxidation, palmitate oxidation, and cardiac function weremeasured in isolated working hearts from ground squirrels and ratssubjected to a hypothermia-rewarming protocol. Hearts wereperfused initially for 30 min at 37°C, followed by 2 h ofhypothermic perfusion at 15°C, after which hearts were rewarmed to37°C and further perfused for 30 min. Functional recovery in groundsquirrel hearts was greater than in rat hearts after rewarming.Hypothermia-rewarming had a similar general effect on the variousmetabolic pathways in both species. Despite these similarities, totalenergy substrate metabolic rates were greater in rat than groundsquirrel hearts during hypothermia despite a lower level of work beingperformed by the rat hearts, indicating that rat hearts are lessefficient than ground squirrel hearts during hypothermia.After rewarming, energy substrate metabolism recovered completely inboth species, although cardiac work remained depressed in rat hearts.The difference in functional recovery between rat and ground squirrelhearts after rewarming cannot be explained by general differences inenergy substrate metabolism during hypothermia or after rewarming.

     

Tissue-specific extravasation of albumin-bound Evans blue in hypothermic and rewarmed rats

The effects of hypothermia and rewarming on endothelial integrity were examined in intestines, kidney, heart, gastrocnemius muscle, liver, spleen, and brain by measuring albumin-bound Evans blue loss from the vasculature. Ten groups of twelve rats, normothermic with no pentobarbital, normothermic sampled at 2, 3, or 4 h after pentobarbital, hypothermic to 20, 25, or 30 degrees C, and rewarmed from 20, 25, or 30 degrees C, were cooled in copper coils through which water circulated. Hypothermic rats were cooled to the desired core temperature and maintained there for 1 h; rewarmed rats were cooled to the same core temperatures, maintained there for 1 h, and then rewarmed. Following Evans blue administration, animals were euthanized with methoxyflurane, tissues removed, and Evans blue extracted. Because hypothermia and rewarming significantly decrease blood flow, organ-specific flow rates for hypothermic and rewarmed tissues were used to predict extravasation. Hypothermia decreased extravasation in tissues with continuous endothelium (brain, muscle) and increased it in tissues with discontinuous endothelium (liver, lung, spleen). All tissues exhibited significant (p < 0.05) differences from normothermic controls. These differences are attributed to a combination of anesthesia, flow, and (or) change in endothelial permeability, suggesting that appropriate choice of organ and temperature would facilitate testing pharmacological means of promoting return to normal perfusion.     

Hypothermia Protects and Prolongs the Tolerance Time of Retinal Ganglion Cells against Ischemia

Purpose

Hypothermia has been shown to be neuroprotective in the therapy of ischemic stroke in the brain. To date no studies exist on the level of the inner retina and it is unclear if hypothermia would prolong the ischemic tolerance time of retinal ganglion cells, which are decisive in many ischemic retinopathies.

Methods

Bovine eyes were enucleated and stored either at 21°C or 37°C for 100 or 340 minutes, respectively. Afterwards the globes were dissected, the retina was prepared and either the spontaneous ganglion cell responses were measured or the retina was incubated as an organotypic culture for additional 24 hours. After incubation the retina was either processed for histology (H&E and DAPI staining) or real-time PCR (Thy-1 expression) was performed.

Results

Hypothermia prolonged ganglion cell survival up to 340 minutes under ischemic conditions. In contrast to eyes kept at 37°C the eyes stored at 21°C still showed spontaneous ganglion cell spiking (56.8% versus 0%), a 5.8 fold higher Thy-1 mRNA expression (not significant, but a trend) and a preserved retinal structure after 340 minutes of ischemia.

Conclusion

Hypothermia protects retinal ganglion cells against ischemia and prolongs their ischemic tolerance time.     

A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2)

The gene expression changes produced by moderate hypothermia are not fully known, but appear to differ in important ways from those produced by heat shock. We examined the gene expression changes produced by moderate hypothermia and tested the hypothesis that rewarming after hypothermia approximates a heat-shock response. Six sets of human HepG2 hepatocytes were subjected to moderate hypothermia (31°C for 16 h), a conventional in vitro heat shock (43°C for 30 min) or control conditions (37°C), then harvested immediately or allowed to recover for 3 h at 37°C. Expression analysis was performed with Affymetrix U133A gene chips, using analysis of variance-based techniques. Moderate hypothermia led to distinct time-dependent expression changes, as did heat shock. Hypothermia initially caused statistically significant, greater than or equal to twofold changes in expression (relative to controls) of 409 sequences (143 increased and 266 decreased), whereas heat shock affected 71 (35 increased and 36 decreased). After 3 h of recovery, 192 sequences (83 increased, 109 decreased) were affected by hypothermia and 231 (146 increased, 85 decreased) by heat shock. Expression of many heat shock proteins was decreased by hypothermia but significantly increased after rewarming. A comparison of sequences affected by thermal stress without regard to the magnitude of change revealed that the overlap between heat and cold stress was greater after 3 h of recovery than immediately following thermal stress. Thus, while some overlap occurs (particularly after rewarming), moderate hypothermia produces extensive, time-dependent gene expression changes in HepG2 cells that differ in important ways from those induced by heat shock.     

Cardiovascular effects of levosimendan during rewarming from hypothermia in rat

BackgroundPrevious research aimed at ameliorating hypothermia-induced cardiac dysfunction has shown that inotropic drugs, that stimulate the cAMP, – PKA pathway via the sarcolemmal β-receptor, have a decreased inotropic effect during hypothermia. We therefore wanted to test whether levosimendan, a calcium sensitizer and dose-dependent phosphodiesterase 3 (PDE3) inhibitor, is able to elevate stroke volume during rewarming from experimental hypothermia.MethodsA rat model designed for circulatory studies during experimental hypothermia (4 h at 15 °C) and rewarming was used. The following three groups were included: (1) A normothermic group receiving levosimendan, (2) a hypothermic group receiving levosimendan the last hour of stable hypothermia and during rewarming, and (3) a hypothermic placebo control group. Hemodynamic variables were monitored using a Millar conductance catheter in the left ventricle (LV), and a pressure transducer connected to the left femoral artery. In order to investigate the level of PKA stimulation by PDE3 inhibition, myocardial Ser23/24-cTnI phosphorylation was measured using Western-blot.ResultsAfter rewarming, stroke volume (SV), cardiac output (CO) and preload recruitable stroke work (PRSW) were restored to within pre-hypothermic values in the levosimendan-treated animals. Compared to the placebo group after rewarming, SV, CO, PRSW, as well as levels of Ser23/24-cTnI phosphorylation, were significantly higher in the levosimendan-treated animals.ConclusionThe present data shows that levosimendan ameliorates hypothermia-induced systolic dysfunction by elevating SV during rewarming from 15 °C. Inotropic treatment during rewarming from hypothermia in the present rat model is therefore better achieved through calcium sensitizing and PDE3 inhibition, than β-receptor stimulation.