Methylene blue and epinephrine: a synergetic association for anaphylactic shock treatment.

BACKGROUND: Severe hypotension resulting from anaphylactic shock may be refractory to epinephrine and impair cerebral oxygenation and metabolism contributing to anaphylactic shock morbidity and mortality. Refractoriness to epinephrine could be corrected by nitric oxide pathway inhibitors such as methylene blue. OBJECTIVES: To compare the systemic and regional (brain and skeletal muscle) effects of epinephrine and methylene blue given alone or in combination in a rat model of anaphylactic shock. DESIGN: Prospective laboratory study. SETTING: University laboratory. SUBJECTS: Male Brown-Norway rats (n = 60). INTERVENTIONS: After sensitization and induction of anaphylactic shock by ovalbumin, animals received either vehicle (ovalbumin group) or a 3-mg/kg methylene blue bolus (methylene blue group) or epinephrine (epinephrine group) or both (methylene blue-epinephrine group). Sensitized control rats received only vehicle and no ovalbumin (control group). MEASUREMENT AND MAIN RESULTS: Mean arterial pressure, cardiac output, cerebral blood flow, skeletal muscular oxygen partial pressure, cerebral oxygen partial pressure, skeletal muscular, and cerebral interstitial lactate/pyruvate ratio were measured. Cleaved caspase 3 and hypoxia-inducible factor-1α expression were analyzed in the cerebral cortex by Western blot. Without treatment, rats died rapidly within 15 mins from a decrease in cardiac output and mean arterial pressure, whereas treated rats survived until the end of the experiment. Methylene blue alone extended survival time but without significant improvement of hemodynamic variables and tissue perfusion and did not prevent neuronal injury. Epinephrine restored partially systemic hemodynamic variables and cerebral perfusion preventing glutamate-induced excitotoxicity. Compared with epinephrine alone, the methylene blue-epinephrine association avoided neuronal excitotoxicity and had an additive effect both on hemodynamic variables and for prevention of brain ischemia. Neither treatment could significantly restore cardiac output or prevent muscular compartment ischemia and microvascular leakage. CONCLUSIONS: Anaphylactic shock is associated with severe impairment of cerebral blood flow despite correction of arterial hypotension. Epinephrine must still be considered as the first-line vasoconstrictive agent to treat anaphylactic shock. The epinephrine-methylene blue association was the most effective treatment to prevent cerebral ischemia and could be used in anaphylactic shock refractory to epinephrine.

Activated protein C improves lipopolysaccharide-induced cardiovascular dysfunction by decreasing tissular inflammation and oxidative stress.

BACKGROUND: Recombinant human activated Protein C (APC) is used as an adjunctive therapeutic treatment in septic shock. APC seemingly acts on coagulation-inflammation interaction but also by decreasing proinflammatory gene activity, thus inhibiting subsequent production of proinflammatory cytokines, NO and NO-induced mediators, reactive oxygen species production and leukocyte-endothelium interaction. The hemodynamic effects of APC on arterial pressure and cardiac function are now well established in animal models. However, the specific effects of APC on heart and vessels have never been studied. OBJECTIVES: To investigate the potential protective properties of therapeutic ranges of APC on a rat endotoxic shock model in terms of anti-inflammatory and cytoprotective pathways. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. INTERVENTIONS: Rats were exposed to lipopolysaccharide (LPS) (10 mg/Kg intravenous). Endotoxic shock was treated with infusion of saline with or without APC (33 microg/kg/h) during 4 hrs. Hemodynamic parameters were continuously assessed and measurements of muscle oxygen partial pressures, NO and superoxide anion (O2(-)) by spin trapping, of NF-kappaB, metalloproteinase-9 (MMP-9) and inducible NO synthase (iNOS) by Western blotting, as well as leukocyte infiltration and MMP-9 activity were performed at both the heart and aorta level (tissue). MAIN RESULTS: APC partially prevented the reduction of blood pressure induced by LPS and improved both vascular hyporeactivity and myocardial performance. This was associated with a decreased up-regulation of NF-kappaB, iNOS and MMP-9. LPS-induced tissue increases in NO and O2(-) production were decreased by APC. Furthermore, APC decreased tissue leukocyte infiltration/activation as assessed by a decrease in myeloperoxidase and matrix metalloproteinase 9 activity. CONCLUSIONS: These data suggest that APC improves cardiovascular function: 1) by modulating the endotoxin induced-proinflammatory/prooxidant state, 2) by decreasing endothelial/leukocyte interaction and 3) by favoring stabilization of the extracellular matrix.

Effects of the TREM-1 pathway modulation during mesenteric ischemia-reperfusion in rats.

OBJECTIVES: The triggering receptor expressed on myeloid cells (TREM)-1, a receptor expressed on the surface of neutrophils and monocytes/macrophages, synergizes with the Toll-like receptors in amplifying the inflammatory response mediated by microbial components. Because the pathogenesis of ischemia-reperfusion-induced gastrointestinal tissue injury and multiple organ failure implies leukocyte activation and bacterial translocation, we hypothesized that the TREM-1 pathway modulation would prove beneficial in this setting. DESIGN: Animal study. SETTING: Research laboratory. SUBJECTS: Adult male Wistar rats (250-300 g). INTERVENTIONS: Rats were subjected to intestinal ischemia-reperfusion induced by occlusion of the superior mesenteric artery during 60 mins and reperfused for 180 mins. At the time of reperfusion, animals were administered with LP17 (a synthetic TREM-1 inhibitor), a control peptide, or a vehicle (normal saline). Plasma concentrations of tumor necrosis factor-alpha, interleukin-6, and soluble TREM-1 were measured by enzyme-linked immunosorbent assay. Hepatic activation of the transcriptional factor nuclear factor-kappaB was assessed by electrophoretic mobility shift assay. Hepatic oxidant-antioxidant balance was estimated by measurement of lipid peroxidation and catalase activity. Ileal mucosal permeability was estimated by fluorescein dextran-4 clearance and bacterial translocation by mesenteric lymph nodes culture. MEASUREMENTS AND MAIN RESULTS: Ischemia-reperfusion was associated with cardiovascular collapse, lactic acidosis, and systemic and hepatic inflammatory response that were partly prevented by LP17 administration. Liver lipid peroxidation and catalase depletion were attenuated by LP17. Ischemia-reperfusion induced a marked increase in ileal mucosal permeability and an associated bacterial translocation that was also prevented by TREM-1 modulation. LP17 delayed mortality. CONCLUSIONS: The modulation of the TREM-1 pathway by the means of a synthetic peptide may be useful during acute mesenteric ischemia.

Myocardial lactate deprivation is associated with decreased cardiovascular performance, decreased myocardial energetics, and early death in endotoxic shock.

OBJECTIVE: We examined whether lactate availability is a limiting factor for heart function during endotoxic shock, and whether lactate deprivation thus induces heart energy depletion, thereby altering cardiovascular performance. The study goals were to determine whether muscle lactate production is linked to beta(2)-stimulation and to ascertain the effects of systemic lactate deprivation on hemodynamics, lactate metabolism, heart energetics, and outcome in a lethal model of rat's endotoxic shock. INTERVENTIONS: We modulated the adrenergic pathway in skeletal muscle using microdialysis with ICI-118551, a selective beta(2)-blocker. Muscle lactate formation in endotoxic shock was further inhibited by intravenous infusion of ICI-118551 or dichloroacetate (DCA), an activator of pyruvate dehydrogenase (DCA) and their combination. RESULTS: Muscle lactate formation was decreased by ICI-118551. During endotoxic shock both ICI-118151 and DCA decreased circulating and heart lactate concentrations in parallel with a decrease in tissue ATP content. The combination ICI-118551-DCA resulted in early cardiovascular collapse and death. The addition of molar lactate to ICI-1185111 plus DCA blunted the effects of ICI-118551+DCA on hemodynamics. Survival was markedly less with ICI-118551 than with endotoxin alone. CONCLUSION: Systemic lactate deprivation is detrimental to myocardial energetics, cardiovascular performance, and outcome.

Constitutive nitric oxide synthase inhibition combined with histamine and serotonin receptor blockade improves the initial ovalbumin-induced arterial hypotension but decreases the survival time in brown norway rats anaphylactic shock.

Anaphylactic shock accidents after allergen exposure are frequent. After immunization with ovalbumin (OVA), a common dietary constituent, we evaluated the efficacy of pretreatment with histamine-receptor or serotonin-receptor blockers administered alone or in combination with a nitric oxide synthase inhibitor (L-NAME) on OVA-induced anaphylactic shock in Brown Norway rats. Animals were allocated to the following groups (n = 6 each): control (0.9% saline); diphenydramine (15 mg kg(-1)); cimetidine (20 mg kg(-1)); diphenydramine + cimetidine; dihydroergotamine (50 microg kg(-1)); diphenydramine + cimetidine + dihydroergotamine; L-NAME (100 mg/kg) alone or associated with diphenydramine, cimetidine, diphenydramine + cimetidine, dihydroergotamine, or diphenydramine + cimetidine + dihydroergotamine. Mean arterial blood pressure (MABP), heart rate (HR), and survival time were monitored for 60 min following treatment. The shock was initiated with i.v. OVA. The MABP drop after i.v. OVA was worsened by diphenydramine and was modestly attenuated by cimetidine, dihydroergotamine, or both together. L-NAME potentiated slightly the effects of cimetidine and dihydroergotamine by lessening the initial MABP decrease, but this transient effect was not sufficient to prevent the final collapse or to improve survival time. Decreased vasodilatory (prostaglandins E2), increased vasoconstrictory (thromboxane B2) prostaglandins, and unchanged leukotriene C4 concentrations were contributory to the overall hemodynamic changes. Thus, the combined blockade of vasodilator mediators (histamine, serotonin, and nitric oxide) slowed the MABP drop in anaphylactic shock, but did not improve survival. More studies are needed to understand these discordant effects.

Vascular ATP-sensitive potassium channels are over-expressed and partially regulated by nitric oxide in experimental septic shock.

PURPOSE: To study the activation and expression of vascular (aorta and small mesenteric arteries) potassium channels during septic shock with or without modulation of the NO pathway. METHODS: Septic shock was induced in rats by peritonitis. Selective inhibitors of vascular K(ATP) (PNU-37883A) or BK(Ca) [iberiotoxin (IbTX)] channels were used to demonstrate their involvement in vascular hyporeactivity. Vascular response to phenylephrine was measured on aorta and small mesenteric arteries mounted on a wire myograph. Vascular expression of potassium channels was studied by PCR and Western blot, in the presence or absence of 1400W, an inducible NO synthase (iNOS) inhibitor. Aortic activation of the transcriptional factor nuclear factor-kappaB (NF-κB) was assessed by electrophoretic mobility shift assay. RESULTS: Arterial pressure as well as in vivo and ex vivo vascular reactivity were reduced by sepsis and improved by PNU-37883A but not by IbTX. Sepsis was associated with an up-regulation of mRNA and protein expression of vascular K(ATP) channels, while expression of vascular BK(Ca) channels remained unchanged. Selective iNOS inhibition blunted the sepsis-induced increase in aortic NO, decreased NF-κB activation, and down-regulated vascular K(ATP) channel expression. CONCLUSIONS: Vascular K(ATP) but not BK(Ca) channels are activated, over-expressed, and partially regulated by NO via NF-κB activation during septic shock. Their selective inhibition restores arterial pressure and vascular reactivity and decreases lactate concentration. The present data suggest that selective vascular K(ATP) channel inhibitors offer potential therapeutic perspectives for septic shock.

Effects of the TREM 1 pathway modulation during hemorrhagic shock in rats.

The triggering receptor expressed on myeloid cells (TREM) 1, a receptor expressed on the surface of neutrophils and monocytes/macrophages, synergizes with the Toll-like receptors in amplifying the inflammatory response mediated by microbial components. Because the pathogenesis of severe blood loss-induced excessive inflammation and multiple organ failure implies leukocyte activation and bacterial translocation, we hypothesized that the TREM-1 pathway modulation would prove beneficial in this setting. Wistar rats were subjected to a 1-h period of hemorrhagic shock and then reperfused with shed blood and ringer lactate for 1 h. At the time of reperfusion, animals were administered with LP17 (a synthetic soluble TREM-1 decoy receptor), a control peptide, or a vehicle (isotonic sodium chloride solution). Plasma concentration of TNF-alpha, IL-6, and soluble TREM-1 were measured by enzyme-linked immunosorbent assay. Lung permeability was assessed by the weight-dry ratio and fluorescein isothiocyanate-labeled albumin lung-blood ratio. Organ dysfunction was appreciated by measuring plasma aspartate aminotransferase and urea concentrations. Bacterial translocation was estimated by blood, mesenteric lymph nodes, and spleens culture. Hemorrhagic shock associated with cardiovascular collapse, lactic acidosis, systemic inflammatory response, and organ dysfunction that was partly prevented by LP17 administration. Hemorrhagic shock induced a marked increase in lung permeability that was also prevented by TREM-1 modulation. Finally, LP17 improved survival. Thus, the early modulation of the TREM-1 pathway by means of a synthetic peptide may be useful during severe hemorrhagic shock in rats in preventing organ dysfunction and improving survival.

Increased aerobic glycolysis through beta2 stimulation is a common mechanism involved in lactate formation during shock states.

During septic shock, muscle produces lactate by way of an exaggerated NaK-adenosine triphosphatase (ATPase)-stimulated aerobic glycolysis associated with epinephrine stimulation possibly through beta2 adrenoreceptor involvement. It therefore seems logical that a proportion of hyperlactatemia in low cardiac output states would be also related to this mechanism. Thus, in low-flow and normal-to-high-flow models of shock, we investigate (1) whether muscle produces lactate and (2) whether muscle lactate production is linked to beta2 adrenergic stimulation and Na+K+-ATPase. We locally modulated the adrenergic pathway and Na+K+-ATPase activity in male Wistar rats' skeletal muscle using microdialysis with nonselective and selective beta blockers and ouabain in different models of rodent shock (endotoxin, peritonitis, and hemorrhage). Blood flow at the probe site was evaluated by ethanol clearance. We measured the difference between muscle lactate and blood lactate concentration, with a positive gradient indicating muscle lactate or pyruvate production. Epinephrine levels were elevated in all shock groups. All models were associated with hypotension and marked hyperlactatemia. Muscle lactate concentrations were consistently higher than arterial levels, with a mean gradient of 2.5+/-0.3 in endotoxic shock, 2.1+/-0.2 mM in peritonitis group, and 0.9+/-0.2 mM in hemorrhagic shock (P<0.05 for all groups). Muscle pyruvate concentrations were also always higher than arterial levels, with a mean gradient of 260+/-40 microM in endotoxic shock, 210+/-30 microM in peritonitis group, and 90+/-10 microM in hemorrhagic shock (P<0.05 for all groups). Despite a decrease in blood flow, lactate formation was decreased by all the pharmacological agents studied irrespective of shock mechanism. This demonstrates that lactate production during shock states is related, at least in part, to increased NaK-ATPase activity under beta2 stimulation. In shock state associated with a reduced or maintained blood flow, an important proportion of muscle lactate release is regulated by a beta2 receptor stimulation and not secondary to a reduced oxygen availability.

Comparative effects of early versus delayed use of norepinephrine in resuscitated endotoxic shock.

OBJECTIVE: To assess hemodynamic, tissue oxygenation, and tissue perfusion changes by comparing traditional therapy (fluid resuscitation followed by vasopressor treatment) and alternative therapy (early vasopressor treatment) in a hyperkinetic and sedated model of endotoxic shock. DESIGN: Prospective controlled experimental study. SETTING: Animal research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Rats were anesthetized, mechanically ventilated, paralyzed, and instrumented to measure mean arterial pressure, heart rate, pulse pressure variation, aortic and mesenteric blood flow, muscle and liver tissue oxygen pressure, blood gas, and lactate. Rats were randomly divided into five groups (n = 7): endotoxin alone (Endo), endotoxin plus norepinephrine (Endo/NE), endotoxin plus fluid therapy alone (ENDO/Fl), endotoxin plus fluid therapy plus late catecholamine (Endo/Fl/Late NE), and endotoxin plus fluid therapy plus simultaneous norepinephrine administration (Endo/Fl/Early NE). MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure increased to baseline values only in the catecholamine-treated group (p < .05). In ENDO/Fl, Endo/Fl/Late NE, and Endo/Fl/Early NE, aortic blood flow was maintained. Mesenteric blood flow was maintained at baseline values only in the catecholamine-treated groups. Mesenteric/aortic blood flow ratio was higher in the early catecholamine group (p < .05). Endo and ENDO/Fl were associated with a marked decrease in liver PO2, which was maintained in catecholamine-treated groups (p < .01). Plasma lactate was lower in the Endo/Fl/Early NE group. Volume resuscitation was higher in Endo and Endo/Fl/Late NE groups with 28 +/- 6 and 27 +/- 4 mL, respectively, when compared with the Endo/Fl/Early NE group (19 +/- 3 mL) (p < .05). CONCLUSIONS: The use of norepinephrine was associated with improved mean arterial pressure, sustained aortic and mesenteric blood flow, and better tissue oxygenation when compared with fluid resuscitation alone, irrespective of time of administration. The early use of norepinephrine plus volume expansion was associated with a higher proportion of blood flow redistributed to the mesenteric area, lower lactate levels, and less infused volume. Thus, the early use of norepinephrine is safe and may decrease the need for volume resuscitation.