Study of thermo-regulation as a worsening marker of experimental sepsis in an animal model *

Objective: to analyze variations in body temperature and in plasma nitrate and lactate concentrations in rats submitted to the experimental sepsis model. Method: a total of 40 rats divided equally into five groups. The induction of endotoxemia was performed with intravenous administration of lipopolysaccharide, 0.5 mg/Kg, 1.5 mg/Kg, 3.0 mg/Kg, and 10 mg/Kg, respectively. The control group received 0.5 mL of saline solution. The experiment lasted six hours, with evaluations performed at 0 (baseline data), 2nd, 4th, and 6thhours. Results: The animals that received doses up to 3.0 mg/kg showed a significant increase in body temperature compared to the group with 10 mg/kg, which showed a decrease in these values. The increase in plasma nitrate and lactate concentrations in the groups with lipopolysaccharide was significantly higher than in the group that received the saline solution and was correlated with the increase in body temperature. Conclusion: the variations in body temperature observed in this study showed the dose-dependent effect of lipopolysaccharide and were correlated with the increase in the concentrations of nitrate and plasma lactate biomarkers. The implications of this study are the importance of monitoring body temperature, together with the assessment of these pathophysiological markers, which suggest worsening in the prognosis of sepsis.


Introduction
Despite the large number of studies available in the literature, limitations are still found in the understanding of the pathophysiological mechanisms, which result in high rates of sepsis-related morbidity and mortality in Intensive Care Units (ICUs) (1) . The clinical course of the disease can lead to a worsening of the prognosis, when changes occur to the stages of severe sepsis and septic shock (2) . This change represents a mortality rate ranging from 10% to 40% (3)(4) .
Among the clinical manifestations presented in the disease, body temperature is an important cardinal sign about the health conditions, whose strict control of thermo-regulation can increase the chances of survival of the patients (5) . However, the mechanisms that result in an ineffective thermo-regulation during the most severe stages of sepsis, mainly related to hypothermia, remain misunderstood (1,5) .
NO formation occurs endogenously from L-arginine catabolism, resulting in the formation of L-citrulline and NO through enzymatic reaction of the NO synthase (NOS) enzyme (10)(11) . Among the NOS isoforms produced in the body, inducible NO (iNOS) participates in the immune response and can be produced through external stimuli, such as in the presence of lipopolysaccharide (LPS) and pro-inflammatory cytokines (10)(11) .
In addition to the increase in NO during the stages of sepsis, plasma lactate can also be found in high concentrations. This increase can be considered a marker of tissue hypoperfusion when found in concentrations ˃1.0 mmol/L (2) . The elevation of plasma lactate results from the production of energy by anaerobic glycolytic (12) , mainly observed in septic shock. Although these two biomarkers show a significant increase in the course of the disease, only lactate is used as a predictor of severity in the clinical practice.
Thermo-regulation has been extensively investigated in experimental models of sepsis and septic shock (13)(14) , showing that the same inflammatory agent can induce both fever and hypothermia (14) . However, the mediators that participate in hypothermia are still misunderstood (15) . It is believed that NO can influence the control of body temperature.
A number of studies in animals have shown different effects of NO on temperature, whether in situations where donors or inhibitors of its synthesis are administered (16)(17) . In a study with an animal model submitted to endotoxemia (a condition similar to sepsis), it was identified that NO acts as a pyretic mediator of fever. The study showed that the pharmacological administration of NO synthesis inhibitors resulted in a decrease in body temperature during the febrile response (16) . In contrast, the reduction of febrile states was also observed when administering NO donors in the lateral cerebral ventricle of rabbits, revealing a stimulus in the antipyretic activity in the central nervous system (17) .
A number of studies involving the measurement of NO production during sepsis in humans are rare; however, in general, they evidence a small increase in this production (18) . It is suggested that this increase may be correlated with the decrease in body temperature in patients with septic shock (19) .
With regard to lactate, as well as NO, the relation between the concentrations of this pathophysiological marker and body temperature in sepsis is little discussed in the literature. In the clinical context, high lactate concentrations serve as a global parameter to identify metabolic impairment in critically ill patients (12,20) .
During the nursing practice, body temperature control is used as a reference of the patient's pathophysiological conditions. The increase or decrease in temperature signals situations that require immediate interventions, with a focus on preserving homeostasis.
Therefore, the monitoring of the vital signs allows the nursing team to early identify organic changes suggestive of sepsis and/or other complications (21) . In this scenario, In addition, they were exposed to a 12/12 hour light-  120; and 240 µM).
In the data analysis, One-Way ANOVA was used, of significance adopted for all the tests was 0.05 (5%).

Results
The administration of LPS at a concentration In humans, changes in body temperature identified in sepsis are also related to fever and/or hypothermia, www.eerp.usp.br/rlae 7 Souza ALT, Batalhão ME, Cárnio EC.
which are characteristic signs for the screening and diagnosis of the disease (2) . It is suggested that fever is a frequent manifestation in sepsis. On the other hand, hypothermia is common in septic shock, being interpreted as a clinical worsening of the patient's prognosis, increasing the chances of death (5,(23)(24) .
Although there are different attempts to reproduce sepsis and septic shock in animal models, it is important to interpret the results with caution, since rats have different responses than humans (24) . In cases of infection, humans usually have a fever and in some cases there may be a decrease in temperature, whereas rodents usually have a reduction in body temperature in the face of a significant infection (24)(25) .
So far, the understanding of the participation of NO in the regulation of body temperature leads to different interpretations: some studies indicate that the inhibition of NO synthesis with the use of L-NAME (inhibitor of NO synthesis) injected into the peritoneum prevented fever in animals submitted to LPS administration (26) , suggesting that NO may act as a pyretic mediator of fever. On the other hand, it is also shown that the increase in NO concentrations in animals submitted to endotoxemia resulted in hypothermia (27) . In addition, the administration of NO donors in the intracerebroventricular region reduced fever in rabbits (17) , suggesting a central antipyretic effect.
In a study with humans carried out by researchers who work in our laboratory, a correlation was observed between the decrease in body temperature and the elevation of plasma NO concentrations in septic shock situations, which was not observed during sepsis (19) .
However, we observed a positive correlation in the groups of animals that received lower doses of LPS, with body temperature values directly related to NO concentrations. This difference between the groups of animals that received different doses can be attributed to a greater resistance of experimental animals to LPS.
The correlation observed between NO doses and body temperature in endotoxemia, with lower doses of LPS, may suggest the action of NO, with its bactericidal property (28) , associated with increased body temperature, as a way of defending the body. Thus, the increase in temperature may be linked to greater activity of the immune system, with the production of prostaglandin E2 (PGE2) and consequently an increase in body temperature.
On the other hand, knowing the harmful effect of NO in high concentrations, the inverse correlation observed in a study with humans (19) during septic shock can be indicative of failure in the body's response capacity, associated with increased oxidative stress and consequently hypothermia.

Considering the different responses between
humans and animals, the results of our study suggest that, probably, the higher concentration of NO in humans would result in a decrease in body temperature, since the opposite effect occurs in animals. This hypothesis has been investigated in studies with human beings, and the correlation between the increase in nitrate concentrations and the decrease in body temperature in septic shock has been confirmed (19) .
It should be noted that the intense decrease in body temperature observed in the group that received the highest dose of LPS (10 mg/kg), accompanied by the significant increase in nitrate concentrations, shows that the higher the dose administered, the lower the body temperature, confirming the notes found in the literature (29) .  (30) .
Since it is a mediator of difficult dosage in the clinical environment, the evaluation of NO is often restricted to scientific research.
In the stages of sepsis and in situations of endotoxemia, there is an increase in anaerobic metabolism and lactate production, which in turn alter the functioning of the immune cells (31) . The increase in this production may result in the negative regulation of glycolytic enzymes, specifically hexokinase and phosphofrutokinase, both in immune cells (32) and in a variety of tissues (33) . Thus, considering the importance of aerobic glycolysis for the functioning of the immune cells in activity, the negative regulation of these enzymes under the influence of lactate implies the functional impairment of these cells (6) .
Recent studies have shown that decreased lactate production has resulted in improved animal survival (34)(35)(36) , while high lactate concentrations in peritoneal dialysis solutions inhibited LPS-induced maturation of dendritic cells (10 ng/mL) (37) . Lactate treatment also increased the production of genes associated with M2 (VEGF and Arg1) and markers (Fizz1, Mgl1, and Mgl2) (38) . M2 is an immunosuppressive phenotype derived from the macrophages found in the late stages of sepsis; its increase may result in critical dysfunction in the immune system (38) .
In the adaptive immune system, the presence of high concentrations of lactate in the synovial fluid and in the joints of patients with rheumatoid arthritis, played a signaling role for the localization of T cells at the site of inflammation (32) . When carrying out in vitro experiments, the study authors point out that extracellular sodium lactate and lactic acid block the motility of CD4+ and CD8+ T cells, respectively (32) .
As in the animal models, the increase in lactate concentrations in sepsis and septic shock in humans is interpreted as a poor prognosis. This increase has an impact on the reduction of the survival chances (20) and signals dysfunctions in the immune system (31)(32) . In this context, our results reinforce the importance of monitoring lactate in experimental and clinical research studies, since it is an easy variable to measure and makes it possible to understand its behavior in endotoxemia and/or sepsis.
The data obtained in our study, showing the elevation of lactate concentrations after the administration of LPS, are in accordance with the evidence in the literature (13,39) .
This increase appears to have an immuno-modulatory effect leading to changes in thermo-regulation. However, it is necessary to expand the number of studies to explain the effect of lactate on body temperature. We believe that there is certain potential to consider body temperature assessments, associated with plasma NO and lactate concentrations, as a way to assess a change in septic patient prognosis.

Conclusion
This study showed that the animals submitted to experimental sepsis showed ineffective thermo-regulation, according to the dose of LPS administered. The animals that received higher doses of LPS had a significantly lower temperature in relation to the other endotoxemic groups, which showed an increase in temperature. This behavior was accompanied by an increase in plasma NO and lactate concentrations. It was also identified that fever was correlated with high concentrations of plasma NO and lactate, important pathophysiological mediators observed during endotoxemia. The study has as its implications for Nursing the importance of monitoring body temperature, together with the assessment of these pathophysiological markers, which suggest a worsening in the prognosis of sepsis.