Heat stress is associated with decreased lactic acidemia in rat sepsis

Abstract

Elevated plasma lactate has been shown to correlate with mortality in patients with septic shock. Heat stress prior to sepsis has resulted in reduction in acute lung injury and mortality. We investigated whether heat stress resulted in decreased plasma lactate concentration and protected the lung by decreasing the inflammatory response to sepsis. < 0.05) and were not significantly different when compared with control rats. Septic rats with or without heat pretreatment had significantly higher myeloperoxidase activity in the lung than did control groups. Heat pretreatment did not prevent neutrophil infiltration or inflammatory mediator production in the lung. Prior heat stress ameliorates lactic acidemia in rat sepsis. Heat stress did not attenuate the pulmonary inflammatory process. The mechanism of heat-induced protection from lactic acidemia in sepsis needs to be further explored.

Background:

Elevated plasma lactate has been shown to correlate with mortality in patients with septic shock. Heat stress prior to sepsis has resulted in reduction in acute lung injury and mortality. We investigated whether heat stress resulted in decreased plasma lactate concentration and protected the lung by decreasing the inflammatory response to sepsis.

Results:

< 0.05) and were not significantly different when compared with control rats. Septic rats with or without heat pretreatment had significantly higher myeloperoxidase activity in the lung than did control groups. Heat pretreatment did not prevent neutrophil infiltration or inflammatory mediator production in the lung.

Conclusion:

Prior heat stress ameliorates lactic acidemia in rat sepsis. Heat stress did not attenuate the pulmonary inflammatory process. The mechanism of heat-induced protection from lactic acidemia in sepsis needs to be further explored.

Introduction

].

].

]. The mechanism of cardiopulmonary protection by heat in sepsis is unknown but may be related to a decrease in proinflammatory cytokine production.

The objectives of this study were to determine whether heat stress resulted in decreased plasma lactate concentration and protected the lung by decreasing the inflammatory response to sepsis. Our hypotheses were that heat exposure prior to sepsis results in attenuation of lactic acidemia and that heat-exposed rats have less sepsis-induced lung injury from suppression of inflammatory response. We showed that heat stress ameliorates lactic acidemia in rat sepsis but does not attenuate the pulmonary inflammatory process.

Heat stress groups

Rats were placed in a small heating chamber, which was previously warmed to 42°C. After 40–45min, the rats' temperatures reached 41°C, at which time the rats were removed from the chamber and allowed to cool passively. The temperature remained above 41°C for 10–15 min. Rats were then returned to their cages and allowed free access to food and water.

Normothermia groups

Rats were placed at room temperature for 45min in the same chamber that was previously used to heat the rats. Rats were then returned to their cages and allowed free access to food and water.

Cecal ligation and perforation (groups 1 and 2)

]. Briefly, a 2-cm midline incision was made and the cecum was isolated. Stool was milked back from the ascending colon into the cecum. The cecum was ligated just inferior to the ileocecal valve to maintain the continuity between ileum and colon. The antimesenteric surface was punctured twice using an 18-gauge needle. The cecum was returned to the abdomen and the abdomen was closed in two layers. Rats were then given 0.9% saline (5 ml/kg body weight) by intraperitoneal injection, and 0.1 ml buprenorphine subcutaneously.

Sham cecal ligation and perforation (groups 3 and 4)

A 2-cm midline incision was made and cecum was isolated. It was returned to the abdomen without ligation or perforation. The abdomen was closed in two layers. Rats were then given 0.9% saline (5 ml/kg body weight) by intraperitoneal injection, and 0.1 ml buprenorphine subcutaneously.

Day 3

Arterial blood was obtained for blood gas, lactate and determination of cytokines. The rats were then killed and their cecums were inspected for evidence of ischemia. The pulmonary artery was cannulated, abdominal aorta was transected, and cold phosphate-buffered saline was infused until the wash fluid from the severed aorta was clear. The trachea was then cannulated and the lungs were lavaged with 50ml warmed phosphate-buffered saline (37°C). The lavage fluid was then centrifuged and the supernatant was saved at -70°C until further analysis.

Tumor necrosis factor-α, interleukin-1β, and macrophage inflammatory protein-2 assays

All assays were performed using commercially available kits (Biosource International, Camarillo, CA, USA). Previously coated microtiter plates containing the primary antibodies specific for rat TNF-α, IL-1β, and MIP-2 were used. After the addition of standard concentrations of the respective cytokine and unknown samples of plasma or lavage fluid, secondary cytokine specific antibodies were placed into these wells. After removing excess antibody, streptavidin-peroxidase was added, which binds to the secondary biotinylated antibody. After washing the plate, a substrate solution was added, which binds to streptavidin–peroxidase to produce a color. The intensity of this color is proportional to the TNF-α, IL-1β, or MIP-2 concentrations, respectively. The sensitivities of these assays are less than 4 pg/ml, less than 20 pg/ml and less than 1 pg/ml for TNF-α, IL-1β, and MIP-2, respectively.

Myeloperoxidase assays

-dianisidine dihydrochloride (Sigma Chemical, St Louis, Missouri, USA) and 0.0005% hydrogen peroxide. Myeloperoxidase activity in each sample (in triplicate) was determined by measuring the change in absorbency per minute at 460 nm using a spectrophotometer (Shimadzu, Wood Dale, Illinois, USA) capable of performing a kinetic scan over 120s. The protein content in 100 μ l of sample supernatant was determined (bicinchoninic acid kit Sigma). Myeloperoxidase activity was expressed as change in optical density/min/g/l protein.

assays

assays are 3.6 and 3.0 pg/ml, respectively.

Statistics

analysis using a Student Neuman–Keuhl test when appropriate. Values are expressed as mean ± standard error of the mean.

Survival

Among rats with CLP, two rats in the heated group and one rat in the unheated group died before sample collection. All rats receiving a sham operation survived regardless of administration of heat.

Cecum

Rats with CLP had ischemia of the cecum. The cecums of sham rats appeared normal.

Plasma lactate

). Pretreatment with heat resulted in prevention of lactic acidemia in septic rats (group 1). Plasma lactate concentrations in group 1 were similar to those in control rats (groups 3 and 4).

Arterial blood gases

).

Pulmonary myeloperoxidase activity

).

Cytokines and eicosanoids in plasma and lung lavage

).

≤ 0.05, versus all other groups. CLP, cecal ligation and perforation.

≤ 0.05, versus sham rats. MPO, myeloperoxidase; CLP, cecal ligation and perforation.

Blood gas values for various groups

, arterial oxygen tension.

Eicosanoid concentrations in the lavage fluid

Values are expressed as mean ± standard error of the mean. CLP, cecal ligation and perforation; PG, prostaglandin; TX, thromboxane.

Discussion

]. In the present rat model of endotoxemia, hyperlactatemia could have resulted from shock, metabolic dysfunction, sepsis-induced liver damage, and/or ARDS. Heat stress caused attenuation of an increase in serum lactate in rats with CLP. The mechanism of prevention of lactic acidemia by heat stress in sepsis is unclear, but it may be related to decreased lactate production or increased lactate clearance. Decreased lactate production could be a result of either increased oxygen delivery or improved mitochondrial oxygen utilization. A lower lactic acid level in the heat-stressed septic rats could also reflect improved organ perfusion, and therefore increased lactate clearance and utilization.

]. In the present study, septic rats had inflammation and acute lung injury as shown by sequestration of neutrophils in the lung indicated by pulmonary myeloperoxidase activity. The myeloperoxidase activity in the lungs was similar in septic rats with or without prior heat stress. Attenuation of increase in lactic acid in the heated, septic group does not appear to be related to decreased pulmonary inflammation by heat stress.

]. Longer periods of heat exposure or higher temperatures than used in the present study might be more effective in attenuation of the pulmonary inflammatory response in sepsis.

In conclusion, we observed that heat stress ameliorated lactic acidemia in a rat model of sepsis, possibly by preserving the organ perfusion, improving oxygen delivery or preventing cellular dysfunction. We were unable to demonstrate attenuation of acute lung injury by heat stress in endotoxemia. Further studies are required to determine the optimal heat stress conditions for cardiopulmonary protection of endotoxemic rats and regulation of the inflammatory response.

Keywords

• acute lung injury
• heat stress
• lactate
• septic shock
• tumor necrosis factor