Platonin, a cyanine photosensitizing dye, causes attenuation of circulatory shock, hypercoagulable state, and tissue ischemia during heat stroke

The aim of this study was to investigate the therapeutic effect of platonin, a cyanine photosensitizing dye as well as an inhibitor of proinflammatory cytokines, in an animal model of heat stroke. Anesthetized rats, immediately after the onset of heat stroke, were divided into two major groups and given the following: normal saline (1 mL per kg body weight) intravenously, or platonin (12.5-50 microg/mL per kg body weight) intravenously. They were exposed to ambient temperature of 43 degrees C to induce heat stroke. Another group of rats was exposed to room temperature (26 degrees C) and used as normothermic controls. Their physiologic and biochemical parameters were continuously monitored. When the vehicle-treated rats underwent heat exposure, their survival time values were found to be 18 to 22 min. Resuscitation with intravenous doses of platonin, but not normal saline, immediately at the onset of heat stroke, significantly improved survival during heat stroke (41-147 min). All heat-stressed animals displayed systemic inflammation and activated coagulation, evidenced by increased tumor necrosis factor-alpha, prothrombin time, activated partial thromboplastin time, fibrinogen degradation products, and D-dimer, and decreased platelet count and protein C. Biochemical markers evidenced cellular ischemia and injury/dysfunction: plasma levels of blood urea nitrogen, creatinine, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and alkaline phosphatase, and striatal levels of partial pressure of oxygen, local cerebral blood flow, glycerol, glutamate, and lactate/pyruvate were all elevated during heat stroke. The systemic inflammation, hypercoagulable state, and cerebral ischemia and injury during heat stroke were all significantly suppressed by platonin. The data demonstrate that platonin therapy may resuscitate heat stroke victims by reducing circulatory shock, systemic inflammation, hypercoagulable state, and tissue ischemia and injury.     

Brain cooling causes attenuation of cerebral oxidative stress, systemic inflammation, activated coagulation, and tissue ischemia/injury during heatstroke

The purpose of the present study was to assess the therapeutic effect of hypothermic retrograde jugular vein flush (HRJVF) on heatstroke. HRJVF was accomplished by infusion of 4 degrees C isotonic sodium chloride solution via the external jugular vein (1.7 mL/100 g of body weight over 5 min). Immediately after the onset of heatstroke, anesthetized rats were divided into 2 major groups and given the following: 36 degrees C or 4 degrees C isotonic sodium chloride solution, i.v. They were exposed to ambient temperature of 43 degrees C to induce heatstroke. Another group of rats was exposed to room temperature (24 degrees C) and used as normothermic controls. When the 36 degrees C saline-treated rats underwent heat exposure, their survival time values were found to be 23 to 28 min. Immediately after the onset of heatstroke, resuscitation with an i.v. dose of 4 degrees C saline significantly improved survival during heatstroke (208-252 min). All heat-stressed animals displayed systemic inflammation and activated coagulation, evidenced by increased tumor necrosis factor alpha, prothrombin time, activated partial thromboplastin time, and d-dimer, and decreased platelet count and protein C. Biochemical markers evidenced cellular ischemia and injury/dysfunction: plasma levels of blood urea nitrogen, creatinine, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and alkaline phosphatase; and striatal levels of glycerol, glutamate, and lactate/pyruvate; dihydroxy benzoic acid, lipid peroxidation, oxidized-form glutathione reduced-form glutathione, dopamine, and serotonin were all elevated during heatstroke. Core and brain temperatures and intracranial pressure were also increased during heatstroke. In contrast, the values of mean arterial pressure, cerebral perfusion pressure, and striatal levels of local blood flow, partial pressure of oxygen, superoxide dismutase, catalase, glutathione peroxidase, and glutathions reductase activities were all significantly lower during heatstroke. The circulatory dysfunction, systemic inflammation, hypercoagulable state, and cerebral oxidative stress, ischemia, and damage during heatstroke were all significantly suppressed by HRJVF. These findings demonstrate that brain cooling caused by HRJVF therapy may resuscitate persons who had a stroke by attenuating cerebral oxidative stress, systemic inflammation, activated coagulation, and tissue ischemia/injury during heatstroke.     

Human umbilical cord blood-derived CD34+ cells cause attenuation of multiorgan dysfunction during experimental heatstroke

Multiorgan dysfunction ensuing from severe heatstroke includes hypotension, hepatic and renal failure, hypercoagulable state, activated inflammation, and cerebral ischemia and injury. We attempted to assess whether human umbilical cord blood-derived CD34+ cell therapy improves survival during experimental heatstroke by attenuating multiorgan dysfunction. Anesthetized rats, immediately after the onset of heatstroke, were divided into 2 major groups and given CD34- or CD34+ cells (1 x 10(5)-5 x 10(5)/mL/kg body weight) i.v. They were exposed to ambient temperature of 43 degrees C to induce heatstroke. Another group of rats were exposed to room temperature (26 degrees C) and used as normothermic controls. Hypotension, hepatic and renal failure (evidenced by increased serum urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels in plasma), hypercoagulable state (evidenced by increased prothrombin time, activated partial thromboplastin time, and D-dimer, and decreased platelet count and protein C in plasma), activated inflammation (evidence by increased TNF-alpha levels in serum), and cerebral dysfunction (evidenced by intracranial hypertension, cerebral hypoperfusion and hypoxia, and cerebral ischemia and injury) were monitored. When the CD34- cell-treated or untreated rats underwent heat stress, their survival time values were found to be 19 to 23 min. Resuscitation with CD34+ cells significantly improved survival time (duration, 63-291 min). As compared with normothermic controls, all CD34- cell-treated heatstroke animals displayed hypotension, hepatic and renal failure, hypercoagulable state, activated inflammation, and cerebral ischemia and injury. However, CD34+ cell therapy significantly caused attenuation of all the above-mentioned heatstroke reactions. In addition, the levels of IL-10 in plasma and glial cell line-derived neurotrophic factors in brain were all significantly increased after CD34+ cell therapy during heatstroke. Our data indicate that CD34+ cell therapy may resuscitate persons who had a heatstroke by reducing multiorgan dysfunction or failure.     

Inhibition of coagulation and inflammation by activated protein C or antithrombin reduces intestinal ischemia/reperfusion injury in rats

OBJECTIVE: To examine whether administration of activated protein C or antithrombin reduces local splanchnic derangement of coagulation and inflammation and attenuates intestinal dysfunction and injury following intestinal ischemia/reperfusion. DESIGN: Randomized prospective animal study. SETTING: University research institute. SUBJECTS: Adult male Wistar rats, weighing 300-325 g (n = 72). INTERVENTIONS: Rats were subjected to superior mesenteric artery occlusion consisting of 20 or 40 mins of ischemia and 3 hrs of reperfusion. A randomized intravenous administration of vehicle (0.9% NaCl), heparin, antithrombin, or activated protein C was performed during ischemia, 15 mins before reperfusion. Coagulation and fibrinolysis variables obtained from portal blood were correlated with mucosal fibrin deposition (determined by anti-rat fibrin antibody staining), intestinal function (glucose/water clearance), and intestinal injury (histologic evaluation by Park/Chiu score). MEASUREMENTS AND MAIN RESULTS: Activated protein C- or antithrombin-treated animals demonstrated less ischemia/reperfusion-induced intestinal dysfunction and histologic changes compared with control animals, whereas intravenous administration of heparin only showed less histologic derangement. Activated protein C- or antithrombin-treated animals showed less thrombin generation, fibrin degradation products, and fibrin deposition compared with control animals, as confirmed by histologic examination, whereas heparin administration showed only a limited reduction of portal fibrin degradation product concentrations. Furthermore, activated protein C or antithrombin administration markedly inhibited the inflammatory response, as reflected by reduced interleukin-6 plasma concentrations to baseline values, whereas heparin had no effect. CONCLUSIONS: Administration of activated protein C or antithrombin inhibited local and systemic derangement of coagulation and inflammation following intestinal ischemia/reperfusion, diminished mucosal fibrin deposition, and attenuated ischemia/reperfusion-induced intestinal injury. These observations suggest that activated protein C or antithrombin reduces ischemia/reperfusion-induced intestinal injury, both through their anticoagulant and anti-inflammatory effects.     

Hypothermic retrograde jugular perfusion reduces brain damage in rats with heatstroke

OBJECTIVE: To determine whether direct retrograde ice saline infusion in the jugular vein without cardiopulmonary bypass protects rat brains after heatstroke. DESIGN: Randomized, controlled, prospective study. SETTING: University physiology research laboratory. SUBJECTS: Sprague-Dawley rats (270-320 g, males). INTERVENTIONS: Rats were randomized into three groups and given a) no resuscitation after onset of heat stroke (HS, n = 8); b) ice saline infusion in the femoral vein after onset of heat stroke (HS + F, n = 8); or c) retrograde ice saline infusion in the external jugular vein after onset of heat stroke (HS + J, n = 8). Rats were exposed to an ambient temperature of 43 degrees C after vessel cannulation. Their mean arterial pressure, heart rate, colonic temperature, and brain temperature were continuously recorded. Survival time and brain pathology were checked. MEASUREMENTS AND MAIN RESULTS: Although colonic temperature decreased 0.8-1.0 degrees C 15 mins after heatstroke in all groups, no treatment-related changes in colonic temperature were noted in any group. However, significant changes were observed in brain temperature. Fifteen minutes after heatstroke, brain temperature was 37.6 +/- 0.4 degrees C, 36.1 +/- 0.4 degrees C, and 33.6 +/- 0.8 degrees C in HS, HS + F, and HS + J, respectively. Survival time was 16.1 +/- 2.1, 33.0 +/- 3.8, and >120 mins in these groups, respectively. Neuron damage score was significantly lower in HS + J and without lateralization. CONCLUSIONS: We successfully demonstrated that direct retrograde hypothermic perfusion via the jugular vein without cardiopulmonary bypass protected the brain after heat stroke. This technique cooled the brain but did not significantly interfere with body temperature.     

Platonin, a cyanine photosensitizing dye, is effective for attenuation of heatstroke in rats

The present study was performed to assess the prophylactic effect of platonin, a cyanine photosensitizing dye and an inhibitor of proinflammatory cytokines, in an animal model of heatstroke. Anesthetized rats were immediately divided into 2 major groups after the start of heat stress and administered either isotonic sodium chloride solution (dose, 1 mL/kg of body weight i.v.) or platonin (dose, 12.5-50 microg/mL per kilogram of body weight i.v.). They were exposed to ambient temperature of 43 degrees C to induce heatstroke. Another group of rats were exposed to room temperature (26 degrees C) and used as normothermic controls. Their physiological and biochemical parameters were continuously monitored. When the isotonic sodium chloride solution-pretreated rats underwent heat stress, their survival time values were found to be from 20 to 24 min. Pretreatment with intravenous doses of platonin (12.5-50 microg/mL per kilogram of body weight) immediately after the start of heat exposure significantly improved survival time during heatstroke (duration, 63-185 min). As compared with normothermic controls, all vehicle-pretreated heatstroke animals displayed higher levels of creatinine, serum urea nitrogen, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, tumor necrosis factor alpha, prothrombin time, activated partial thromboplastin time and D-dimer in the plasma, cellular ischemia and injury markers in striatum, and intracranial pressure. In contrast, all vehicle-pretreated heatstroke animals had lower levels of mean arterial pressure, cerebral perfusion pressure, cerebral blood flow, brain Po2, and platelet count and protein C in the plasma. Immediately after the start of heat exposure, the previous administration of platonin significantly improved survival time by reducing the systemic inflammation, hypercoagulable state, and tissue ischemia and damage during heatstroke. The results demonstrate that platonin is effective for attenuation of heatstroke reactions.     

Resuscitation from experimental heatstroke by estrogen therapy

OBJECTIVE: We investigated the effect of estrogen therapy on inflammatory responses, cardiovascular functions, and survival in a rat model of heatstroke. DESIGN: Controlled, prospective study. SETTING: Hospital medical research laboratory. SUBJECTS: Sprague-Dawley rats (280-312 g of body weight, males and females). INTERVENTIONS: Four major groups of anesthetized rats were designated for experiments: a) vehicle-treated male rats; b) vehicle- or premarin-treated estrus female rats; c) vehicle- or premarin-treated ovariectomized rats; and d) vehicle- or premarin-treated leuprolide-treated rats. All animals were exposed to heat stress (ambient temperature 43 degrees C for 70 mins) and then allowed to recover at room temperature (24 degrees C). Their survival time (interval between the onset of heatstroke and animal death) and physiologic and biochemical variables were monitored. Vehicle (normal saline 1 mL/kg of body weight, intravenously) or premarin (1 mg/mL/kg of body weight, intravenously) was administered 70 mins after initiation of heat stress. Ovariectomy or leuprolide (100 mug/kg/day, subcutaneously) injection was conducted 4 wks before the start of heat stress experiments. Another group of rats were exposed to 24 degrees C and used as normothermic controls. MEASUREMENTS AND MAIN RESULTS: Compared with the estrus female rats, the ovariectomized rats, the leuprolide-treated rats, and male rats all had lower levels of plasma estradiol and lower survival time values. However, after an intravenous dose of premarin, both the plasma estradiol and survival time values were significantly increased. Compared with the normothermic controls, the vehicle-treated male and ovariectomized rats all displayed higher levels of serum tumor necrosis factor-alpha, which could be suppressed by premarin therapy. In contrast, the serum levels of IL-10 in these groups were significantly elevated by premarin during heatstroke. Furthermore, the heatstroke-induced hyperthermia, arterial hypotension, intracranial hypertension, and cerebral hypoperfusion, hypoxia, and ischemia were significantly attenuated by premarin therapy in ovariectomized rats. CONCLUSIONS: We successfully demonstrated that estrogen replacement may improve survival during heatstroke by ameliorating inflammatory responses and cardiovascular dysfunction.     

Effects of hypothermia for a short period on histologic outcome and extracellular glutamate concentration during and after cardiac arrest in rats

OBJECTIVE: To evaluate the therapeutic effects of hypothermia for a short period (20 mins, 31 degrees C) using a cardiac arrest model (5 mins) in rats. DESIGN: Prospective animal study. SETTING: Experimental laboratory in a university hospital. SUBJECTS: Male Wistar rats (n = 42). INTERVENTION: Direct current (DC) potential and extracellular glutamate concentrations (microdialysis) were monitored in the hippocampal region. Histologic observation was performed 7 days later. MEASUREMENTS AND MAIN RESULTS: No animal died or showed severe complications as a result of hypothermia for a short period. In nontreated animals (group F), extracellular glutamate concentration simultaneously increased with the onset of membrane depolarization and continued to increase during the reperfusion period (maximum, 212% +/- 40% of the pre-ischemia level) until the onset of DC recovery. In animals in which hypothermia was initiated before the onset of ischemia (group A), extracellular glutamate concentration did not increase during the ischemia period. When hypothermia was initiated at the onset of resuscitation (group B), the glutamate concentration immediately decreased. In animals in which hypothermia was initiated at 4.9 +/- 1.3 mins (immediately after DC recovery, group C), 10 mins (group D), and 20 mins (group E) after the onset of resuscitation, changes in extracellular glutamate concentration were the same as those in nontreated animals. The percentage of injured neurons was significantly attenuated (compared with group F, 82% +/- 10%) when hypothermia was initiated before DC recovery (group A, 5% +/- 3%; group B, 29% +/- 22%) or immediately after DC recovery (group C, 58% +/- 18%, 9.9 +/- 1.3 mins after the onset of ischemia). CONCLUSIONS: Hypothermia for a short period decreased glutamate concentration when it was initiated before DC recovery and attenuated neuronal damage when it was initiated before or immediately after DC recovery. The therapeutic time window for hypothermia for a short period is about 10 mins after the onset of ischemia.     

Resuscitation from experimental heatstroke by transplantation of human umbilical cord blood cells

OBJECTIVE: Human umbilical cord blood cells (HUCBCs) are effective in the treatment of conventional stroke in experimental models. In the study described herein, we administered HUCBCs into the femoral vein or directly into the cerebral ventricular system and assessed their effects on circulatory shock, cerebral ischemia, and damage during heatstroke. DESIGN: Controlled, prospective study. SETTING: Hospital medical research laboratory. SUBJECTS: Sprague-Dawley rats (287 +/- 16 g body weight, males). INTERVENTIONS: Anesthetized rats, immediately after the onset of heatstroke, were divided into four major groups and given the following: a) normal saline or AIM-V medium intravenously (0.3 mL) or intracerebroventricularly (10 microL); b) peripheral blood mononuclear cells (5 x 10 in 0.3 mL AIM-V medium, intravenously, or 5 x 10 in 10 microL AIM-V medium, intracerebroventricularly); or c) HUCBCs (5 x 10 in 0.3 mL AIM-V medium, intravenously, or 5 x 10 in 10 microL AIM-V medium, intracerebroventricularly). Another group of rats, under urethane anesthesia, were exposed to room temperature (26 degrees C) and used as normothermic controls. Urethane-anesthetized animals were exposed to an ambient temperature of 43 degrees C to induce heatstroke. Their physiologic and biochemical parameters were continuously monitored. MEASUREMENTS AND MAIN RESULTS: When the vehicle-treated rats underwent heat exposure, their survival time values were found to be 21-23 mins. Resuscitation with intravenous or intracerebroventricular doses of HUCBCs, but not peripheral blood mononuclear cells, immediately at the onset of heatstroke significantly improved survival during heatstroke (61-148 mins). As compared with values for normothermic controls, the vehicle-treated heatstroke rats had lower mean arterial pressure, cerebral blood flow, and brain PO2 values but higher intracranial pressure and cerebral ischemia values and more injury markers. The circulatory shock, intracranial hypertension, cerebral hypoperfusion and hypoxia, increment of cerebral ischemia, and damage markers during heatstroke were all significantly attenuated by intravenous or intracerebroventricular delivery of HUCBCs but not peripheral blood mononuclear cells. CONCLUSIONS: We successfully demonstrate that HUCBC therapy may resuscitate heatstroke victims by reducing circulatory shock and cerebral ischemic injury; central delivery of HUCBCs seems superior to systemic delivery of HUCBCs in resuscitating patients with heatstroke.     

Heat shock pretreatment may protect against heatstroke-induced circulatory shock and cerebral ischemia by reducing oxidative stress and energy depletion

The mechanisms underlying the protective effects of heat shock pretreatment on heatstroke remain unclear. Here we attempted to ascertain whether the possible occurrence of oxidative stress and energy depletion exhibited during heatstroke can be reduced by heat shock preconditioning. In the present study, colonic temperature, mean arterial pressure, heart rate, striatal levels of heat shock protein 72 (HSP72), local Po2, brain temperature, cerebral blood flow, cellular ischemia and damage markers, dihydroxybenzoic acid (DHBA), lipid peroxidation, glutathione, glutathione peroxidase and reductase activities, and ATP were assayed in normothermic control rats and in heatstroke rats with or without preconditioning 16 or 96 h before initiation of heatstroke. Heatstroke was induced by exposing the anesthetized rats to a high ambient temperature (Ta = 43 degrees C) until the moment at which MAP decreased from its peak level. Sublethal heat shock pretreatment 16 h before initiation of heatstroke, in addition to increasing striatal HSP72 levels, conferred significant protection against heatstroke-induced arterial hypotension, striatal ischemia and damage, increment of hydroxyl radical formation, lipid peroxidation, glutathione oxidation, and decrement of glutathione peroxidase activity and ATP. However, at 96 h after heat shock, when striatal HSP72 expression returned to basal levels, the above responses that occurred during onset of heatstroke were indistinguishable between the two groups. These results suggest that heat shock pretreatment induces HSP72 overexpression in striatum and confers protection against heatstroke-induced striatal ischemia and damage by reducing oxidative stress and energy depletion.     

Hypothermia attenuates circulatory shock and cerebral ischemia in experimental heatstroke

We tested the hypothesis in a rat model that body cooling suppresses circulatory shock and cerebral ischemia in heatstroke. Animals under urethane anesthesia were exposed to water blanket temperature (Tblanket) of 42 degrees C until mean arterial pressure (MAP) and local cerebral blood flow (CBF) in the hippocampus began to decrease from their peak levels, which was arbitrarily defined as the onset of heatstroke. Control rats were exposed to 26 degrees C. Extracellular concentrations of glutamate, glycerol, lactate, and lactate/pyruvate in the hippocampus were assessed by microdialysis methods. Cooling was accomplished by decreasing Tblanket from 42 degrees C to 16 degrees C. The values of MAP and CBF after the onset of heat stroke in heatstroke rats received no cooling were all significantly lower than those in control rats. However, the neuronal damage score and extracellular levels of ischemia and damage markers in the hippocampus were greater. Cooling immediately after the onset of heatstroke reduced the heatstroke-induced circulatory shock, cerebral ischemia, neuronal damage, and surge of tissue ischemia and damage markers in the hippocampus, and resulted in prolongation of survival time. Delaying the onset of cooling reduced the therapeutic efficiency. The results suggest that body cooling attenuates circulatory shock and cerebral ischemia insults in heatstroke.     

Gabexate mesilate, a synthetic protease inhibitor, reduces ischemia/reperfusion injury of rat liver by inhibiting leukocyte activation.

OBJECTIVE: To investigate whether gabexate mesilate, a synthetic protease inhibitor with anticoagulant properties, prevents hepatic damage by inhibiting leukocyte activation, we examined its effect on ischemia/reperfusion injury of rat liver in which activated leukocytes play a critical role. DESIGN: Prospective, randomized, controlled study. SETTING: Research laboratory at a university medical center. SUBJECTS: Male Wistar rats weighing 220 to 280 g. INTERVENTIONS: Hepatic damage was evaluated by changes in bile flow and serum transaminase concentrations after ischemia/ reperfusion. Rats received continuous intravenous infusions of gabexate mesilate (10 mg/kg/hr) or intravenous administration of an inactive derivative of activated factor X (Xa), a selective inhibitor of thrombin generation (3 mg/kg), immediately before the induction of ischemia in the median and left lobes of the liver. To determine whether gabexate mesilate inhibits leukocyte activation, we examined the effects of gabexate mesilate on hepatic concentrations of tumor necrosis factor-alpha and rat interleukin-8 and on hepatic myeloperoxidase activity after ischemia/reperfusion. MEASUREMENTS AND MAIN RESULTS: Hepatic dysfunction, observed after 60 mins of ischemia/reperfusion, showed a reduction in bile flow. The ischemia/reperfusion-induced decrease in bile flow was prevented by administration of gabexate mesilate. Serum transaminase concentrations increased after hepatic ischemia/reperfusion, peaking 12 hrs after reperfusion. Gabexate mesilate significantly inhibited the ischemia/reperfusion-induced increase in serum transaminase levels seen 12 hrs after reperfusion. Although an inactive derivative of factor Xa inhibited the increases in serum levels of fibrin and fibrinogen degradation products 6 hrs after reperfusion, it did not prevent ischemia/ reperfusion-induced liver injury. Hepatic levels of tumor necrosis factor-alpha, rat interleukin-8, and myeloperoxidase were significantly increased after ischemia/reperfusion. These increases were significantly inhibited by gabexate mesilate but unaffected by an inactive derivative of factor Xa. CONCLUSION: Gabexate mesilate reduced ischemia/reperfusion-induced hepatic injury not by inhibiting coagulation, but by inhibiting leukocyte activation.     

Mild or moderate hypothermia but not increased oxygen breathing prolongs survival during lethal uncontrolled hemorrhagic shock in rats, with monitoring of visceral dysoxia.

OBJECTIVE: To test the hypotheses that during lethal uncontrolled hemorrhagic shock (UHS) in rats compared with normothermia and room air breathing: a) mild hypothermia would prolong survival time as well as moderate hypothermia; b) oxygen breathing would prolong survival further; and c) hypothermia and oxygen would mitigate visceral ischemia (dysoxia) during UHS. DESIGN: Prospective, randomized, controlled laboratory animal study. SETTING: Animal research facility. SUBJECTS: Male Sprague-Dawley rats. INTERVENTION: Fifty-four rats were lightly anesthetized with halothane during spontaneous breathing. UHS was induced by blood withdrawal of 3 mL/100 g over 15 mins, followed by 75% tail amputation with topical application of heparin. Five minutes after tail cut, rats were randomly divided into nine groups (6 rats each) with three rectal temperature levels (38 degrees C [100.4 degrees F; normothermia] vs. 34 degrees C [93.2 degrees F; mild hypothermia] vs. 30 degrees C [86 degrees F; moderate hypothermia]) by surface cooling; each with 3 FIO2 levels (0.25 vs. 0.5 vs. 1.0). Rats were observed without fluid resuscitation until death (apnea and pulselessness). Visceral ischemia was monitored by observing liver and gut surface PCO2. MEASUREMENTS AND MAIN RESULTS: Mean survival time, which was 51 mins in the control group with normothermia and FIO2 of 0.25, was more than doubled with hypothermia, to 119 mins in the combined mild hypothermia groups (p < .05) and to 132 mins in the combined moderate hypothermia groups (p < .05; NS for moderate vs. mild hypothermia). FIO2 had no statistically significant effect on survival time. Increases in visceral surface PCO2 correlated with hypotension (r2 = .22 for intestine and .40 for liver). Transiently, increased FIO2, not hypothermia, mitigated visceral ischemia. CONCLUSIONS: Both mild and moderate hypothermia prolonged survival time during untreated, lethal UHS in rats. Increased FIO2 had no effect on survival. The effects of hypothermia and increased FIO2 during UHS on viscera, the ability to be resuscitated, and outcome should be explored further.     

Characterization of neuroprotective effects of biphalin, an opioid receptor agonist, in a model of focal brain ischemia

Approximately 795,000 people experience a new or recurrent stroke in the United States annually. The purpose of this study was to assess the protective effect of a nonselective opioid receptor agonist, biphalin, in brain edema and infarct damage by using both in vitro and in vivo models of stroke. In an in vivo model of ischemia, biphalin significantly decreased edema (66.6 and 58.3%) and infarct (52.2 and 56.4%) ratios in mouse transient (60-min occlusion/24-h reperfusion) and permanent (6 h) middle cerebral artery occlusion models, respectively. Biphalin administration also showed decreased neurodegeneration in hippocampal, cortical, and striatal brain tissue after ischemia, evidenced by reduced Fluoro-Jade C staining. In addition, biphalin improved neurological function after stroke injury evidenced by neurological score and locomotor activity evaluation. Biphalin significantly decreased penumbral expression of Na(+), K(+), 2Cl(-) cotransporter (NKCC) and the translocation of the conventional isoforms of protein kinase C (PKC). It also reversed the activation of PKC-induced cell volume increase during ischemia in primary neuronal cell cultures exposed to 1 h of oxygen glucose deprivation. These data suggest that opioid receptor activation provides neuroprotection during stroke, and a possible explanation of this mechanism could be the inhibition of NKCC function via the regulation of PKC-dependent cell signaling.     

Protein C -1641 AA is associated with decreased survival and more organ dysfunction in severe sepsis

OBJECTIVE: Protein C contains an A/G polymorphism at position -1641 and a C/T polymorphism at -1654 associated with risk of deep venous thrombosis. We tested the hypothesis that these polymorphisms are associated with altered outcome in patients having severe sepsis, in which protein C is a central molecule. DESIGN: Prospective cohorts, gene-association study. SETTING: Tertiary care medical/surgical intensive care unit. PATIENTS: We first recruited a derivation cohort of patients having severe sepsis (n = 62). A second replication cohort was similarly defined but larger (n = 402). We tested for biological plausibility in a third cohort of post-cardiopulmonary bypass patients (n = 61). INTERVENTIONS: Patients were genotyped at protein C -1641 and -1654. MEASUREMENTS AND MAIN RESULTS: The primary outcome variable was survival in cohorts 1 and 2 and postoperative serum interleukin-6 concentration in cohort 3. Severity of individual organ dysfunctions and systemic inflammation were secondary outcome variables. In the first derivation cohort, the protein C -1641 AA genotype was associated with decreased 28-day survival (p < .05). This finding was confirmed in the much larger replication cohort of patients having severe sepsis (p = .028). In addition, the protein C -1641 AA genotype was associated with significantly more organ dysfunction and more clinical evidence of systemic inflammation (p < .05). Furthermore, the -1641 AA genotype was associated with increased serum interleukin-6 at 4 and 24 hrs after cardiopulmonary bypass (p = .024). There was no association of -1654 A/G with phenotype in any cohort. CONCLUSIONS: Protein C -1641 AA genotype is associated with decreased survival, more organ dysfunction, and more systemic inflammation in patients having severe sepsis and with increased interleukin-6 levels after cardiopulmonary bypass surgery.     

D-lactate is not a reliable marker of gut ischemia-reperfusion in a rat model of supraceliac aortic clamping

OBJECTIVE: D-lactate is the dextrorotatory form of L-lactate. L-lactate is the isomer routinely tested in clinical practice to assess cell hypoxemia. D-lactate has been recently proposed as a specific marker of gut ischemia-reperfusion (IR), particularly after surgery for ruptured aortic aneurysms. We sought to assess D-lactate as a reliable marker of gut IR in a rat model of supraceliac aortic clamping. DESIGN: Prospective, randomized trial. SETTING: Animal research center. SUBJECTS: Male Wistar rats. INTERVENTIONS: After general anesthesia, rats were randomized into two groups (n = 8 in each). The IR group underwent a laparotomy, aortic clamping for 40 mins, and 1 hr of reperfusion. The control group underwent the same procedure, except for aortic clamping. MEASUREMENTS AND MAIN RESULTS: The following variables were tested after 1 hr of reperfusion (IR group) or after the equivalent time (control group): 1) tissue and cell insult via ileum morphometry and electron microscopy, serum glutamic transaminases (serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase), pH, and L-lactate; 2) systemic inflammatory response via tumor necrosis factor-alpha; and 3) D-lactate levels. After IR, mucous membrane thickness and villi height decreased significantly, respectively by 30% and 45%, and electron-microscopic examination showed typical IR mucous membrane cell insult. IR also caused lactic acidosis (pH = 7.16 +/- 0.05 vs. 7.31 +/- 0.02, p < .01; L-lactate = 7.1 +/- 1.6 vs. 1.6 +/- 0.4 mmol/L, p = .001) and increased blood levels of transaminases. Concurrently, the inflammatory response was characterized by an increase in tumor necrosis factor-alpha (213 +/- 129 vs. 47 +/- 32 pg/mL, p < .05). However, blood levels of D-lactate never increased after IR. CONCLUSIONS: D-lactate is not a reliable marker of gut IR in our model of supraceliac aortic clamping in rats.     

Hyperbaric oxygen reduces infarct volume in rats by increasing oxygen supply to the ischemic periphery

OBJECTIVE: Hyperbaric oxygen (HBO) increases oxygen supply to anoxic areas. To examine the therapeutic effect of HBO on ischemic stroke, we measured infarct volume as well as cerebral blood flow (CBF), oxygen supply, and lipid peroxidation in the ischemic periphery. DESIGN: Prospective experimental study in rats. SETTING: Experimental laboratory in a university teaching hospital. SUBJECTS: Thirty-eight adult rats. INTERVENTION: The rats were anesthetized (1% halothane) and intubated. Focal ischemia was induced by ligating the right middle cerebral and right common carotid arteries. Nineteen animals were exposed to 2 hrs of HBO (100% oxygen, 3 atmospheres absolute), initiated 10 mins after the onset of ischemia. The remaining animals were kept at ambient pressure and used as controls. MEASUREMENTS AND MAIN RESULTS: At the initiation of ischemia, CBF measured by a laser-Doppler flow probe placed in the ischemic periphery was reduced to 47%+/-11% and 51%+/-15% of normal levels in animals exposed or not to HBO, respectively. These altered values were not affected further by administration of HBO and remained stable throughout a 2-hr observation period. Arterial oxygen pressure and content were significantly increased to 1571+/-130 torr (209.41+/-17.32 kPa; p < .0001) and 1.03+/-0.04 mmol/dL (p < 0.0001), respectively, in HBO-treated animals compared with nontreated animals (139+/-14 torr [18.53+/-1.87 kPa] and 0.86+/-0.04 mmol/dL, respectively). The calculated increase in the oxygen supply to the ischemic periphery was 20%. The infarct volume of HBO-treated animals measured 24 hrs after the onset of focal cerebral ischemia was significantly reduced by 18% (HBO-treated, 132+/-13 mm3 vs. nontreated, 161+/-29 mm3; p = .02). Lipid peroxidation was unchanged after 120 mins of HBO administration in the cerebral cortex where the laser-Doppler flow probe was placed. CONCLUSIONS: HBO at 3 atmospheres absolute reduced infarct volume by increasing oxygen supply to the ischemic periphery without aggravating lipid peroxidation, suggesting that HBO can be useful in treating stroke victims.     

Hydroxyethyl starch produces attenuation of circulatory shock and cerebral ischemia during heatstroke

We hypothesized that hydroxyethyl starch (HES), which maintains colloid osmotic pressure and potentially "seals" capillary leaks, would ameliorate circulatory shock and cerebral ischemia during heatstroke in a rat model. Animals under urethane anesthesia were exposed to high ambient temperature (Ta) of 42 degrees C until mean arterial pressure and local cerebral blood flow in the striatum began to decrease from peak level, which was arbitrarily defined as the onset of heatstroke. Control rats were exposed to 24 degrees C. In rats treated with 1 mL/kg, 11 mL/kg, or 22 mL/kg of normal saline (NS) immediately after the onset of heatstroke, the values for survival time (interval between the initiation of heatstroke and animal death) were found to be 21 +/- 2, 36 +/- 9, or 92 +/- 7 min, respectively. Intravenous administration of 11 mL/kg of HES (about 5 times the volume-expanding effect of 11 mL/kg of NS), but not 2 mL/kg of HES (about the same volume-expanding effect as 11 mL/kg NS), significantly increased the survival time from the control values of 36 +/- 9 min to new values of 181 +/- 13 min. In NS (11 mL/kg)-treated or HES (2 mL/kg)-treated rats after heatstroke onset, the values for mean arterial pressure, stroke volume, total peripheral resistance, cerebral blood flow, blood pH, Paco2, Pao2, and brain Po2 were significantly lower than those of rats kept at Ta 24 degrees C. In contrast, the values for colonic temperature and the extracellular concentrations of glutamate, glycerol, and lactate/pyruvate ratio obtained in striatum were significantly higher than those of controls. The heatstroke-induced arterial hypotension, decreased stroke volume and total peripheral resistance, decreased blood pH and Pao2, decreased brain Po2, and increased levels of striatal glutamate, glycerol, and lactate/pyruvate ratio in NS-treated rats were all attenuated significantly by increasing the volume expansion with 11 mL/kg of HES administered immediately at the onset of heatstroke. Our data suggest that HES therapy seems superior to NS treatment during heatstroke. The benefit of HES therapy during heatstroke might have something to do with volume expansion rather than capillary permeability.     

Peritoneal lavage with activated protein C alters compartmentalized coagulation and fibrinolysis and improves survival in polymicrobial peritonitis

OBJECTIVE: During peritonitis, intra-abdominal fibrin entraps bacteria and hampers their elimination. Systemic administration of anticoagulant activated protein C improves survival in patients with severe sepsis, but its precise mode of action is unclear. This study in polymicrobial peritonitis assessed the effects of local activated protein C administration in peritoneal lavage fluid on coagulation, fibrinolysis, and survival. DESIGN: Prospective, randomized study. SETTING: University-based research laboratory. SUBJECTS: C57BL/6 mice. INTERVENTIONS: Twenty-four hours after induction of peritonitis by cecal ligation and puncture, mice underwent peritoneal lavage with activated protein C (1.0 microg/mL) or saline. Peritoneal lavage fluid, blood, and lungs were sampled after 24, 48, or 72 hrs (n = 8/group/time point). For survival analysis, maximum observation was 96 hrs (n = 22/group). Clotting time, tissue factor expression, thrombin-antithrombin complexes, fibrin degradation products (D-dimers), plasminogen activator, and plasminogen activator inhibitor were used to assess coagulation and fibrinolysis responses. MEASUREMENTS AND MAIN RESULTS: Activated protein C lavage reduced abdominal bacterial load, abdominal and pulmonary clotting times, D-dimers (p < .05 vs. saline), pulmonary tissue factor expression, and fibrin depositions, without clear effects on systemic thrombin generation. Activated protein C lavage decreased plasma and abdominal tissue plasminogen activator levels with increased inhibitor plasminogen activator inhibitor-1 levels (p < .05) but had reverse effects on pulmonary fibrinolysis. Survival improved from 55% (saline) to 80% after intra-abdominal activated protein C administration (p = .03). CONCLUSIONS: Peritoneal lavage with activated protein C may rebalance coagulation and fibrinolysis within compartments and improve survival in polymicrobial peritonitis.     

Mild or moderate hypothermia, but not increased oxygen breathing, increases long-term survival after uncontrolled hemorrhagic shock in rats

OBJECTIVE: To test the hypotheses that, for uncontrolled hemorrhagic shock (UHS) in rats, mild hypothermia, compared with normothermia, would increase long-term survival as well as moderate hypothermia, oxygen breathing would increase survival further, and hypothermia and oxygen would mitigate visceral ischemia (dysoxia) during UHS. DESIGN: Prospective, randomized study. SETTING: Animal research laboratory. SUBJECTS: A total of 54 male Sprague-Dawley rats. INTERVENTIONS: Under light anesthesia and spontaneous breathing, rats underwent UHS phase I of 75 mins, with initial withdrawal of 3 mL/100 g of blood over 15 mins, followed by UHS via tail amputation and limited fluid resuscitation to maintain mean arterial pressure at > or =40 mm Hg; resuscitation phase II of 60 mins (from 75 mins to 135 mins) with hemostasis and aggressive fluid resuscitation to normalize hemodynamics; and observation phase III to 72 hrs. Rats were randomly divided into nine groups (n = 6 each) with three rectal temperature levels (38 degrees C [normothermia] vs. 34 degrees C [mild hypothermia] vs. 30 degrees C [moderate hypothermia]) by surface cooling; each with 3 FIO2 levels (0.25 vs. 0.5 vs. 1.0). MEASUREMENTS AND MAIN RESULTS: Hypothermia increased blood pressure compared with normothermia. Increased FIO2 had no effect on blood pressure. Additional blood loss from the tail cut was small, with no differences among groups. Hypothermia and FIO2 of 0.5 decreased visceral hypoxia, as measured by the difference between visceral (liver and jejunum) surface Pco2 and PaCO2 during UHS. Compared with normothermia, mild hypothermia increased the survival time and rate as well as moderate hypothermia (p < .01 by life table), without a significant difference between mild and moderate hypothermia. Increased FIO2 had no effect on survival time or rate. CONCLUSIONS: After severe UHS and resuscitation in rats, mild hypothermia during UHS, compared with normothermia, increases blood pressure, survival time and 72-hr survival rate as well as moderate hypothermia. Mild hypothermia is clinically more feasible and safer than moderate hypothermia. Increased FIO2 seems to have no significant effect on outcome.